Nanomaterial-assisted CRISPR gene-engineering – A hallmark for triple-negative breast cancer therapeutics advancement

Triple-negative breast cancer (TNBC) is the most violent class of tumor and accounts for 20–24% of total breast carcinoma, in which frequently rare mutation occurs in high frequency. The poor prognosis, recurrence, and metastasis in the brain, heart, liver and lungs decline the lifespan of patients by about 21 months, emphasizing the need for advanced treatment. Recently, the adaptive immunity mechanism of archaea and bacteria, called clustered regularly interspaced short palindromic repeats (CRISPR) combined with nanotechnology, has been utilized as a potent gene manipulating tool with an extensive clinical application in cancer genomics due to its easeful usage and cost-effectiveness. However, CRISPR/Cas are arguably the efficient technology that can be made efficient via organic material-assisted approaches. Despite the efficacy of the CRISPR/Cas@nano complex, problems regarding successful delivery, biodegradability, and toxicity remain to render its medical implications. Therefore, this review is different in focus from past reviews by (i) detailing all possible genetic mechanisms of TNBC occurrence; (ii) available treatments and gene therapies for TNBC; (iii) overview of the delivery system and utilization of CRISPR-nano complex in TNBC, and (iv) recent advances and related toxicity of CRISPR-nano complex towards clinical trials for TNBC.


Introduction
Breast-related carcinoma (BC) is one of the potential sources of tumour-dependent death in females accounting for an expected 28% of new tumour cases [1][2][3]. It is an extremely diverse disease, and several signalling biomolecules and cascades facilitate its instigation and evolution [4,5]. According to genetic expression investigation, multiple subtypes of BC have been recognized based on the allelic expression profile and deficiency of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) [6]. Although ER-positive disease accounts for approximately 70-75%, triple-negative breast carcinoma (TNBC) appraises 24% of all newly BC conveyed cases [7,8], and 33% of patients embark relapse [9] . However, 30% of BC deaths occur because of TNBC destructive pathology and metastasis [10,11]. It has the most rapid and aggressive metastasis, and progression compared to other forms of BC [3], and generally occurs in premenopausal women, who hardly survive longer than two years [12,13]. Additionally, TNBC has been conveyed to be more usual in either germline-based (20-25%), or somatic (5-10%) deficit in BRCA DNA repair associated (BRCA) gene [14], which shelters TP53 mutation that deactivates p53 protein [3]. Nearly 48-66% of BRCA1 deficit carriers nurture TNBC, a much higher incidence ratio than non-BRCA1 deficit individuals [3,14]. Nevertheless, limited numbers of treatment alternatives are accessible due to the absence of expression receptors and lack of 49]. In this context, nanomaterials such as metal oxide, silicon, silica, plasmonic, lipids, and protein-based nanocarriers are becoming imperative clinical tools, yielding therapeutic precision and analytical functionality that would not be accomplished through large-scale methods [50]. It deals with various techniques for diagnosing, imaging, controlling, and delivering several types of payloads to the desired targeted site [39]. It supports the distribution of CRISPR/Cas at high efficiency and lesser toxicity that would overwhelm physiological obstacles [47]. The encapsulation via nano-based cargo precludes the susceptible CRISPR complex from deterioration intervened by proteases and nucleases in biological fluids matrix [51]. Since nucleic acid has a negative charge, besides the Cas9 nuclease's immense size (%160 kDa), due to this nanocarriers are designed with positive charges to enhance the packaging capacity and effective communication with cells [52][53][54], or festooned with cell-penetrating peptides (CPPs) [30,55] to ease the plasma membrane penetration. However, its successful clinical application is a tremendous task due to low in vivo efficacy of nanocarriers [41].
This review first describes the molecular mechanism, possible genetic involvement, signalling pathways of TNBC occurrence, and the pivotal role of proteome and transcriptome in TNBC. Then, discuss the milestones of all ongoing treatments/therapies of TNBC, which were overcome via CRISPR/Cas gene engineering. We also discuss diverse CRISPR/ Cas systems and nano-derived vehicles, including novel delivery, gene expression, and recent advances together with their implication in clinical trials. Nonetheless, the aim is to determine an inclusive summary of CRISPR/nano-based clinical trials, and breakthrough preclinical models of TNBC while considering future exertion. Although the review focuses on cancer, as many of the same principles apply to a host of other diseases, it may prove helpful to readers in a broad range of disciplines.

Triple-negative breast cancer
The triple-negative breast cancer (TNBC) is the most severe and highly violent type of breast carcinoma where none of the receptors (ER and PR) and HER2 protein are involved and frequently mutated in younger patients [56,57]. It shows aggressive progression, high mitotic index, metastasis rate/probability, histological level, and relapse [13]. It has heterogeneous immunohisto-molecular bases that are divided into many sub-classes such as mesenchymal, basal-like immune-suppressed (ER-/PR-/HER2-/IMlow), basal-like immune activated (ER-/PR-/HER2-/IMþ), mesenchymal stem-like, luminal/androgen receptor (HER2-/PR-/ARþ), metaplastic (0.2-5%), and immunomodulatory [13,58,59]. Regardless of this valuable classification, we have restricted knowledge about its pathogenesis due to the intense nature of heterogeneity [7], the mutation in various genes, transcription factors, proteome, and signalling pathways that make it utmost complex in nature for effective treatment.

Genetic mechanism of TNBC
The TNBC occurs due to the copy number change [60], polymorphism, missense mutation [3], transversions, frame-shift or non-sense mutation, and deletion [61] in the coding, enhancer, or promoter region of proto-oncogenes that code a mutated proto-oncoproteins which exhibited reduced tumour protective function [62,63]. However, basic knowledge of gene's product, namely "protein" structure and conformation, is essential to understand the complex genetic mechanism of TNBC. Proteins are made-up of amino acids chained by peptide bonds to each other in a linear fashion, while the carbon atom is located in the central part bound at four sites; two with amino acids, one with aminoacyl group, and the other with carboxyl group. All proteins have two significant domains; C-terminal or carboxyl-terminal (COOH) and N-terminal or amino-terminal (NH 2 ). Usually, the C-terminal of the protein has a site for the DNA-binding, once mutation occurs in the C-terminal of protein loses its integrity, stability, recruitment of other factors, and chromatin binding [64,65] that can be studied via VIPUR (Fig. 1). The VIPUR scores successfully analyse any mutation in the conformation of protein structure and function. Its 0.8-10 score shows non-functionality/poor folding or mutation in protein, while lower VIPUR scores around 0.1-0.5 indicate more inclination toward wild-type protein structure [66,67]. Moreover, methylated CG repeats or CpG island presented on the proto-oncogene become frequently mutated and highly conserved over the evolutionary time scales [68]. Generally, in CG residues, cytosine (C) is deaminated into thymine (T) and guanine (G) into adenine (A). For instance, the TP53 gene is about 74.4% responsible for TNBC in females globally [69][70][71]. Its gene product "p53" protein showed 90% missense mutation from CpG sites that span 190 diverse codons in the TP53 gene exon that code the DNA-binding region of the p53 protein [68]. As a result, p53 losses its function, no or poor DNA binding, and restricted transcription [69,72,73] caused enhancement in tumour-infiltrating lymphocytes in the stroma [73], epithelial-mesenchymal transition, high rate of tumorigenesis, cellular invasion/migration, drug resistance, rapid cell division in TNBC cell lines [74,75], and large-sized tumour growth in females [76]. Similarly, the R330 frame-shift mutation in the coding region at the C-terminus of the GATA3 gene leads to variations in epithelial to mesenchymal tissues causing overexpression and abnormal regulation of breast cell growth and progression. At the genetic level, the mutation in a single allele of the GATA3 gene induces about 25% of its genomic redistribution and accumulation in TNBC [77].
The MUC1-C could crosstalk with receptor tyrosine kinases as WEE1 that regulate G2/M/S phase checkpoint, DNA damage responses, and alter retinoblastoma (RB) gene function via its three active oncogenic cytoplasmic motifs such as YHPM, YTNP, and CQC motifs. The YHPM motif contains a specific binding sequence for phosphoinositide 3-kinase (PI3K), while PI3K phosphorylation activates serine/threonine kinase (AKT) protein that phosphorylates and deactivates glycogen synthase kinase (GSK) 3β and initiate WNT effector β-catenin pathway [112]. Concurrently, tyrosine at the YTNP site of MUC1-C phosphorylated and initiated MUC1-C to SOS linking via interacting growth factor receptor-bound protein 2 (GRB2). Genomic crosstalk of MUC1-C induces RAS → MEK → ERK signalling pathway that propagates signals for TNBC cells mutation, irregular cell division, and proliferation. Additionally, the PIK3CA/PI3K signalling pathway is around 40% aberrated in TNBC, where the extracellular matrix stimulation and phosphoinositide 3-kinase (PI3K) generate phosphatidylinositol 3,4,5-trisphosphate PI(3,4,5) P 3 at the site of the plasma membrane that systematically transformed into phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 ). The PI(3,4,5)P 3 and PI(3,4)P 2 activate diverse kinases, such as 3-phosphoinositide dependent protein kinase-1 (PDK1 or PDPK1) and serum and glucocorticoid kinase 3 (SGK3), that recruit AKT thereby upregulation of Inositol polyphosphate 4-phosphatase type II (INPP4B) consort with Rab7 in late endosome/lysosome compartment and dephosphorylate PI(3,4)P 2 into phosphatidylinositol 3-phosphate (PI(3)P)-effector (HRS) by triggering class-I PI3Kα via PTEN to block EGFR expression, AKT/PI3K signalling, and augment lysosomal GSK3β degradation that inhibits TNBC proliferation and relapse [112]. Similarly, mTORC1 and mTORC2 upregulation through PI3K prompt RICTOR and GATOR2 that induced violent tumorigenesis in mammary gland, while its pharmacological inhibition by Hippo pathway could be idyllic elucidation (Fig. 2). The hippo signalling pathway is composed of tetra-core, large tumour suppressor 1/2 (LATS1/LATS2), and mammalian Ste20-like kinases 1/2 (MST1/MST2), activated by lack of nutrients, extracellular matrix stiffness, and greater cell density. The FRMD6 and neurofibromatosis 2 (NF2) proteins act over a regulatory sequence of Hippo by phosphorylating tetra-core complex, whereas STING-associated vasculopathy of infantile-onset (SAVI) protein contribute to systemizing MST1 and MST2 [113]. Upon activation, hippo phosphorylates Yes association protein (YAP)/TAZ (also known as WW domain-containing transcription regulator 1) complex at serine on 127 positions of YAP and subjected to subsequent uibiqutinization to prevent further dimerization of YAP/mTOR multiple oncogenic pathways in vivo patient-derived xenografts [59]. Thus, the genomic abnormality in PI3K/AKT/mTOR pathway is one of the major issues commonly found in TNBC together with various sub-class of breast carcinoma, causative to tumour progression and tolerance against prevailing treatments [29]. In recent times, targeting the AKT/mTOR signalling pathway is a practical approach for the therapy of TNBC, but its feature de novo signalling and action mechanism remain to be elucidated.
The TNBC is the most critical kind of breast melanoma to treat because of its impassiveness against the latest clinical therapies, poor prognosis, high rate of metastasis, and type conversion, such as aberrant BRCA1 mammary carcinoma is primarily ERα positive but, as they grow, turn into negative or more basal-like TNBC. Furthermore, inherited BRCA1 carriers have 87% cumulative risk of developing ovarian/breast tumours throughout their lifespan [91]. Additionally, BRCA1 defected breast express TP53 mutation in TNBC patients. Mechanistically, germline deficiency of BRCA1 or BRCA2 induces multiple tumour proliferation genes such as TP53 or TP53BP1 and NOTCH1 that further activate signalling cascade, ultimately provoking epithelial to mesenchymal transmutation (EMT), and TNBC cell proliferation. For example, the most frequent p53 missense mutation in the 175 codon substitutes histidine with arginine, which creates a steric deterrent that decreases zinc binding affinity around 100-1000 folds, thus causing misfolding of p53 protein [3]. The free-state misfolding of p53 inactivates complementary cell cycle checkpoint via Chk1 and WEE1 and regulates EMT, further initiating TNBC cell division [3,114]. Subsequently, p53 interacts with aurora B via the TGF-β pathway while crosslinking with leucine-zipper and sterile-α-motif kinase (ZAK) and CSF1R to trigger EM transmutation in breast tissues. Aurora B has prime importance in terms of mitosis regulation, whereas ZAK kinase regulates the p38 MAPK pathway [95].
Hypothetically, extracellular matrix (EMC), and integrins are the critical players in chemo-immuno resistance during tumorigenesis [11,115,116]. The integrins are heterodimer receptors of the cellular membrane, supposed to rheostat cytoskeletal association, cellular linkage, relocation, and signal transduction through launching focal linkage multiplexes that aid mechanical associations to transfer signals between the intracellular compartment and ECM of the interrelating cells [116]. Thus, the ECM possesses an extremely dynamic erection that is frequently refashioned by cells through improved reassembly, synthesis, chemical adjustment, and degradation [115]. Generally, tumours cells generate an excess amount of ECM like fibronectin 1 (FN1), fibroblasts, and collagen. For example, overexpression of ECM enzymes, lysyl oxidase (LOX) and matrix metalloproteases, enhance renovation of lysine deposits in collagen prolyl and elastin into extremely responsive aldehydes resulting in cross-linking and equilibrium of ECM's proteins (type I collagen and elastin) that induce TNBC cells motility, invasion and cell adhesion [11].
In addition, transcriptome reprogramming is also one of the perilous features of TNBC, where mutant driver oncogenes or tumour suppressor proteome expression depends on its enhancer or promoter region to initiate tumorigenesis, development, and metastasis [4]. In addition, changes in the TNBC gene expression profile can be due to cis-element alteration in noncoding regions where transcriptional factors or other regulatory elements exit [117]. Enhancers are non-transcribe regions of the human genome that possess cis-regulatory elements, thereby promoting transcription of the target gene [118]. Generally, it is characterized by acetylation renowned as histone modifications and coactivators association. Super-enhancers are a diverse class of drivers that express a specific set of products that describe cellular individuality [117]. Recently, super-enhancers exhibited a critical character in the overexpression of TNBC driver genes. For example, bromo and extra-terminal domain (BET) proteins and their variants such as BRD2, BRD3, BRD4, and BRDT are evolving as therapeutic markers for TNBC. The BRD4 and p300 bind with its proximal super-enhancer region enriched in H3K27ac and regulate cell-specifying oncogenic transcriptional plans. However, the direct substitution of BET inhibitors with thienotriazolodiazepine (JQ1) enhances acetyl-lysine binding to dislocate BET proteins from chromatin, consequential selective transcriptional reactions in addition to high anti-proliferative efficiency. The discernment of BET obstruction commences from the localization of BETs to super-enhancers that impede anti-tumour impacts in preclinical and clinical tribunals [94]. Epigenomic profiling has been conducted in several solid carcinomas to classify subclass of specific super-enhancers. However, inclusive knowledge related to functional importance in TNBC carcinogenesis of super-enhancers has not been fully established yet [4,118].
The fundamental factor behind the demise of TNBC patients is neurons-based metastasis and EMT. The EMT is devoted to the commencement of tumour metastasis in TNBC that cause impairment in cellular adhesion junctions. However, EMT has retained a highly complex genetic mechanism elicited via discrepant intracellular and extracellular transcriptomes and proteomes signalling that foment carcinoma cells to decamp their prime location [165]. Subsequent eruption into the oblivious organ, mesenchymal TNBC cells transform into epithelial cells to relinquish macro metastatic foci. Usually, the ncRNAs have the chief regulation in terms of in-vivo genetic expression, cell proliferation, protein synthesis, transcript targeting, and activation of receptors [119]. However, abnormal behaviour of various classes of ncRNAs has been perceived in TNBC that are discussed in detail [120].  [126]. The miRNA also exhibits anticancer responses without generating reactive confrontation [166]. It tends to express innumerable countenance configurations among TNBC patients and other classes of breast carcinoma [119]. Additionally, miRNAs intimate carcinoma development and screen the prognosis in TNBC patients. Exclusively miRNA has the ability to suppress tumours via regulating EM transmutation in TNBC [141]. For example, wild-type p53 directed transactivation of miRNA of 205 family (miR-205). The miR-205 inhibits EM transmutation, relocation, incursion, and propagation by down-regulating Zinc finger E-box binding homeobox 1 (ZEB1) gene, integrin α5 (ITGA5), E2F1, LAMC1, and ITGA5/Src/Vav2/Rac1 signalling pathway where integrin α5 is the widely occurred heterodimeric transmembrane receptor for extracellular proteome of the cell that plays a pivotal role in cell to cell adhesion and TNBC metastasis [143], while E2F1 govern cell cycle and LAMC1 systematize cell adhesion and metastasis. Similarly, miRNA-199, miRNA-214, and miRNA-3178 inhibit EMT alteration [141], TNBC cell proliferation, invasion, and metastasis by direct downregulation of the NOTCH1 gene [146] that is supposed to be a key driver in terms of tumour cell initiation signalling. The tumour initiation cells (TIC) have a potent ability to reseed carcinoma in distinct places in TNBC patients [137].
Recently, diverse targeting of miRNA has been identified in TNBC poor prognosis and metastasis, for instance, the noncoding single nucleotide polymorphism rs78378222 in the sequence of AATACA, instead of wild-type AATAAA in TP53, generates a targeting site for miRNA-382-5p or miR-382 that in turn provide the site for miRNA-325-3p (miR-325) which is greatly overexpressed in the mammary glands. The unusual behaviour of both miRNA cause upregulation of p53 in breast tumours and sealed neuro-carcinoma proliferation in the brain sites [144]. Also, miRNA-17-5p rheostat TNBC metastasis through suppressing ETV1 oncogene [168], E2F transcription factor 6 (E2F6), and DNA methyltransferase 1 (DNMT1) expression while overexpressing BRCA1 in TNBC, thus proved to be a differential role of miRNA in TNBC suffering [169]. E2F6 and DNMT1 serve as transcriptional precursors and regulators of cell proliferation and its fate [130,169] . Short interfering RNA. Ribonucleic acid interference is a natural phenomenon mainly achieved by siRNA and short hairpin RNA (shRNA), which can ideally complement a specific target gene or miRNA for silencing [5,170]. However, the siRNA can be synthetic and natural, whereas the endogenous double-stranded ribonucleic acid (dsRNA) is typically renowned by a molecular scissor named as dicer, which sliced dsRNA into 20-25 nucleotide pairs in length. These short interfering RNA having two bases overhang at their 3' site [171]. Recently, the underlying mechanism of RNAi has been unveiled where some chromatin-binding RDM15 protein-directed polymerase-V dependent activation accumulation of siRNA at RNAi target site and RNA-directed deoxyribonucleic acid methylation occurred via de novo methylation pathway. The special hydrogen bonding and aromatic cage at the C-terminus of the Tudor domain of RDMI5 precisely identify the monomethyllysine of active histone H3K4me1 peptide and initiate RNAi in living beings [172]. Therefore, the siRNA is a presumptive knockdown gadget to inhibit the genetic expression profile of any gene and explore distinct molecular cascades and genetic/metabolic routes at the posttranslational level [173]. It proposes an innovative therapeutic approach to deal with several disorders or diseases, including TNBC and various other melanomas at the biomolecular level.
Several siRNA depended target therapeutic strategies have been employed to cure TNBC, such as TGF-β expression level modulated with siRNA-based interference that exhibited an efficient decline in metastatic condition since TGF-β involves in cell fate determination, differentiation, apoptosis, and has the ability to interrelate with cell cytoskeleton, integrin's, and epithelial cadherin's based relocation during TNBC metastasis [5]. Similarly, the vascular endothelial growth factor (VEGF) family, such as VEGF-A to VEGF-D and placenta growth factor (PEG) expression, have been successfully meticulated by VEGF siRNA (5'-GCUACUGCCAUCCAAUCGAtt-3') that were highly expressed in TNBC cells, and significantly correlated with worse metastasis, thus enhancing pro-angiogenic impact over endothelial cells, and facade as autocrine for VEGF receptor and survival [174].
TNBC [177]. In this context, the siRNA-based whole-genome screen provides potential elucidation [178]. However, the polar and macromolecular landscape of siRNA deters its cellular admittance to employ its impact [120].

Long noncoding RNAs modalities
The therapeutic targeting of ncRNAs signifies a striking tactic for the therapy of TNBC, as well as many other melanoma and diseases [166]. During the last decade, extensive exertion has been through concerning the clinical application of ncRNA-based therapeutics, employing mostly RNA interference liable techniques, with numerous attaining food drug and administration (FDA) and European medicines agency (EMA) endorsement [120]. However, practical consequences have thus far been indecisive, with some reports noted persuasive impacts while others confirmed limited efficacy or cytotoxicity [123]. Different entities, for instance, lncRNA-based therapeutics are achieving inquisitiveness [153]. The lncRNAs about 200 nucleotides in length are polyadenylated RNAs transcribed by RNA polymerase-II [163], play a part during epigenetic trigger gene expression, steric interruption of secondary structure realization, miRNA silencing, regulation of cell cycle, post-transcriptional inhibition, protein interaction, and signal transduction cascades [179].
Additionally to the transcriptional directives, lncRNAs, have other governing characters [179]. For instance, the long noncoding mammary tumour-associated RNA25 (MaTAR25) downregulated Tensin1 (Tns1) protein in 4T1 cells by interacting with purine-rich element binding protein B (PURB) and heightened tumor cell progression. Inhibition of Tns1 protein exhibits reorganization of actin filaments, weakening of focal adhesion association signalling between extracellular matrix and cytoskeleton, and microvilli [153]. Correspondingly, lncRNA DILA1 can hinder the phosphorylation of cyclin D1 by intermingling Thr286 to stop cyclin D1 deprivation while elevating breast metastasis [185]. However, comprehensive characterization of the action mechanism of most of the lncRNAs for molecular occurrence and precise function in TNBC still needs to be explored.

Ongoing treatments for triple-negative breast cancer
Multiple approaches are applied to treat TNBC, such as conventional (adjuvant and neoadjuvant chemotherapy), surgery, endocrine therapy, and radiation therapy. While, advanced strategies include active and passive targeting by gene manipulation techniques (CRISPR, siRNAs, miRNAs, and lncRNAs), and nanocarriers-based therapies, which prevent the spread of the tumour and eliminate the chances of recurrence of TNBC, are discussed in the sub-sections with detail and illustrated in Fig. 3.

Lumpectomy and mastectomy
Presently, there are no precise strategies for the orthodox treatment of TNBC due to the heterogeneity of this disease depicting varied compulsive structures. However, the initial treatment of TNBC generally depends on the surgery that is divided into two types; one is lumpectomy and the other is mastectomy [186]. The lumpectomy is mainly conducted to preserve the breast, especially when TNBC is at its infancy [187], while mastectomy approaches involve the removal of the entire breast to save the patient's life. It is commonly done when the disease recognizes at the last stage or after chemotherapy. Nonetheless, both surgeries induce 25-60% post-pain syndrome (PPS) in women due to the damage of T4 and T5 peripheral nerves during surgery, ensuing neuroma development along with hypersensitivity [188]. Generally, intercostal T4 and T5 nerve sensory branches exhibited major chronic neuropathic pain aetiology as they supply blood from the chest wall to the breast. Further, during surgery, an acute injury in the sensory part of cutaneous nerves leads to neuroma development and axonal cell membrane successive growth that progresses pacemaker-like commotion causing incessant devastating neuropathic pain syndrome accompanied by poor life quality [189]. It could be obstinate, stimulus sensational agony, and trigger intensified pressure at the position of the neuroma. However, PPS is hardly treated clinically or documented by surgeons. Although, the majority of TNBC patients do not obtain effective treatment even after diagnosis due to a lack of targeted medication. While, some standard medications of breast carcinoma provide ease to relieve pain for a certain period of time, such as non-steroidal anti-inflammatory drugs, corticosteroids, opioid analgesics, interleukin-1β, interleukin-6, interleukin-10, benzodiazepines, gabapentin, antidepressants, pectoral nerve block-I, and serratus anterior plane block [188,190], usually express forged consequences in TNBC.
Some advanced trigger point injections (TPI) are somehow getting interest in suppressing chronic pain. The functional principle of TPI is to inject 2 mL of 50 part 4 mg/mL dexamethasone and 50 part of 0.5% bupivacaine at the site of neuroma that alleviates 91.2% of relatively long-term PPS [189]. Also, perineural infiltration of anaesthesia in combination with dexamethasone or dexamethasone and bupivacaine at the spot of the supposed neuroma showed dormant outcomes [191]. Thus, anaesthetic treatment only affords brief-time relief, nevertheless it supports the diagnosis. However, ongoing strategy induces vilest chronic neuromas by augmenting the pacemaker activity, which consequences in either short-term or rapid PPS relief enforcing effective therapy for TNBC cure and patient ease.

Chemotherapy
Before and after lumpectomy or mastectomy procedure, some undetected deposits in the breast tissues or lymph nodes may lead to relapse in TNBC. However, chemotherapy is one of the most widely practiced conventional first-line short-term therapy to cure postconsequences of TNBC surgery that was introduced in clinics almost half of decades [192]. It has diverse administration modes, such as oral chemotherapy, intramuscular, subcutaneous, intravenous, intrathecal chemotherapy, and types like single agent adjuvant and neoadjuvant chemotherapy (NAC) [193]. The single or multiple agent adjuvant chemotherapy is based on two factors, one being predicted sensitivity concerning a specific methodology and its allied benefits and another being the risk of relapse. Further, implementing any method depends upon the TNBC patient's ailment [194]. The NAC has endorsed surgery formerly to study the impact of medicine in TNBC patients and lessen the magnitude of lumpectomy or mastectomy if patients respond to the chemo-therapeutic dose [193]. Principally, chemotherapeutic agents kill not only tumour cells but also healthy cells. Most accessible treatments may provide anticipated antitumour impact at higher quantities of the alkylating agent, plant alkaloids, antimetabolites, and antitumour antibiotic [195], which is highly hazardous to healthy cells. Further, these cytotoxic chemo-drugs only increased 33% overall survival rate with adverse anemia in TNBC patients [196]. Other after-effects of chemotherapy agents include sickness, fatigue, rash, diarrhea, leukopenia, alopecia, thrombocytopenia, abdominal discomfort, vomiting, peripheral sensory neuropathy, hepatic failure, auto immuno hepatitis, constipation, pneumonia, and fluctuations in weight [15,195,197].
Concisely, among adjuvant, preparative, and neoadjuvant chemotherapies, the NAC is more effective with an advanced ratio of pathologic complete response (pCR), averts tumor relapse in TNBC-bearing patients as compared with non-TNBC patients [193,198]. However, the earlier analysis indicated a higher rate of reversion and distant metastasis during the first 3 years after neoadjuvant chemotherapy [9]. Recently, platinum-based chemotherapies are becoming a new option for solid carcinomas. On the other hand, a high ratio of platinum-based salts exhibits more BRCA-like status in TNBC patients. For example, platinum-based adjuvant chemotherapy (AC) and NAC trials of about 15 studies prove a higher toxic impact that develops worst outcomes after Overview of triple-negative breast cancer traditional and advanced therapeutic applications. Over time, six traditional techniques were widely utilized to overcome breast carcinoma or TNBC (inner ring), while the outer ring indicated their modified mechanics with recent advancements. As discussed in the review, lumpectomy, mastectomy, and chemotherapy have been utilized as first-line treatment, while others are used as advanced treatments to relocate and treat relapse and metastasis in clinical translation.
recurrences. Additionally, it induces severe neuropathy and thrombocytopenia in all grade patients, followed by neutropenia. Also, it has only enhanced disease-free survival (DFS) but not overall survival (OS) response (HR ¼ 0.98, p ¼ 0.87; CI ¼ 0.75-1.27, 95%). Therefore, it was suggested that this chemotherapy could not be a better option as a first-line treatment for TNBC patients [196]. Similarly, in another randomized trial (NCT00861705, CALGB 40603) with stage II and III TNBC patients (n ¼ 443), the used drug such as Carboplatin and Bevacizumab along with Paclitaxel, followed by Cyclophosphamide and Doxorubicin in NAC setting did not improve long-term OS. However, Cyclophosphamide and Paclitaxel are considered standard therapy for TNBC [199]. Thus, the new therapy is an unmet need to increase the life span of TNBC patients.

Liquid biopsy or circulating tumour DNA
The TNBC manifests a higher incidence of relapse and visceral metastasis during the first 36 months after chemotherapy and shortened life span. However, a substantial number of TNBC interventions receive NAC and an unambiguous dichotomy exists in consequence-centered retort to NAC. Almost 1 = 3of TNBC interventions will accomplish 94% pCR with a promising disease-free OS for 3 years. In contrariety, 2 = 3of TNBC interventions exhibit lingering ailment after NAC and are at an extreme hazard of 68% metastasis during 3 years of prediction. Thus, advancements have been subjected to locate the deposition of tumour substantial in the circulatory system of TNBC patients, who are supposed to be disease-free either lumpectomy or mastectomy after NAC. An incipient scheme for non-invading tumour discernment is the examination of circulating tumour DNA (ctDNA) or liquid biopsy and circulating tumour cells (CTCs) to reveal the status of treatment efficacy and tumour prognosis in patients [200]. In a liquid biopsy, the ctDNA is unfettered in the bloodstream to sense CTCs that are usually released in the blood at the site of metastatic lesions or primary tumour cells undergoing necrosis and apoptosis [201]. For most, the fraction of ctDNA matched to the whole cell-free nucleic acid (cfDNA) that would be in a minute quantity of <1%. Lately, thoughtful droplet-based digital polymerase chain reaction (ddPCR), and next-generation sequencing techniques are used to analyse such a minute quantity of ctDNA. However, this blood-based prognostic biomarker in its infancy needs a large prospective study to validate its fruitfulness after NAC in high-risk TNBC intervention population [202].

Immunotherapy
The T-cell is one of the white blood cells that have a central role in cancer immunotherapy. It is a growing field of advanced therapy in which cytotoxic T-cells (CTc) directly act on the microenvironment of tumour that causes minimum after-effects on normal cells. The tumorspecific adaptive immune response controls tumor growth by stabilizing the long-term memory of adaptive T-cells to achieve a possible lasting cure. Mechanistically, activation of CTc initiates anticancer immuno retort by exhibiting expression for immune checkpoint inhibitors such as PD-1 and cytotoxic T-lymphocytes antigen 4 (CTLA-4) [192]. Importantly, PD-1 is a cell surface protein expressed on tumour-infiltrating lymphocytes (TILs) that has been widely studied in TNBC immuno treatments [192,193]. Moreover, The non-self-cells secretion upon genomic mutation actively identified via PD-1 and CTLA-4 leads to tumour cell elimination that ends after overcoming through immune escape process initiated by tumour cells either PD-L1/L2 or downregulating MHC-I/II [18,203]. In recent times, immunotherapy-based drugs, more likely antibodies, proved to be a promising antidote than chemotherapeutic induction alone due to immuno-responsive nature of TNBC [193].
In this context, exogenous cytokines-based vaccines (that enhance the rate of tumour-specific T-cells), adoptive T-cells, adaptive cells, several immune checkpoint inhibitors, chimeric antigen receptors (CAR) T-cells and co-stimulatory receptors have been greatly explored to overcome immune-suppressive response under tumour microenvironment and decrease TNBC burden in women [35,104,106,[203][204][205]. However, there are various limitations in which unpredictability of method outcome is the biggest. For instance, only a few subsets of tumour patients exhibit immune responses, while others relapse after initial retort. Further, different kinds of tumour respond variably, maybe due to the presence of their own intrinsic resistance/tolerance and immunosurveillance mechanism [206]. In addition to the method, it is extremely expensive. For example, advanced immunotherapy costs about 1 million US $/visit, which creates an unbearable burden on average for mediocre earner patients, and psychology also weakens the immune system. Other limitations regarding immunotherapy are tumour heterogeneity, tumour microenvironment (TME), paucity of adequate neoantigens, insufficient production and malfunction of tumour-specialized CD8 T-cells, and epigenetic alteration resulting inefficient activation of tumour-specialized immune retort [204,206,207].
Apart from this, several pre-clinical and clinical trials are assessing to establish chemo-immuno therapy regimes. For example, NAC coupled with immunotherapy evaluated the efficacy and safety of combinatorial treatment and Durvalumab administration in I-III stage PD-L1 positive TNBC patients (n ¼ 59). Additionally, Pembrolizumab (Keytruda, MK-3475), Atezolizumab, or Durvalumab are first-line treatments in PD-L1 positive TNBC patients. The Durvalumab was provided with nab-Paclitaxel followed by dose-dense Cyclophosphamide and Doxorubicin (ddAC) where 44% pCR was reported from Phase-II together with 31% adverse events such as anemia and neutropenia. In addition, Guillain-Barre syndrome (1.9%), hyperglycaemia (3.9%), colitis (3.9%) and death (1.9%) were also observed [208]. Likewise, the COLET Phase-II trial evaluated Cobimetinib in combination with Paclitaxel, or placebo combined with Paclitaxel in cohort-I, Cobimetinib along with Paclitaxel and Atezolizumab in cohort-II, and Cobimetinib together with Atezolizumab and nab-Paclitaxel in cohort-III, did not demonstrate an advantage in relapse free survival (RFS) as first-line therapy for metastatic TNBC (mTNBC) irrespective of MAPK/MEK and PD-L1 [20]. Further studies are ongoing to comprehend the unmet need for chemo-immunotherapy drugs better.

Radiotherapy
Radiation or radiotherapy has exhibited fewer after-effects than chemotherapy in breast tumour vulnerable populations [38]. It is practiced to execute residual tumours residing either in the breast or lymph node to device relapse of breast carcinoma [209]. It is mainly sectioned into external radiation beam that is traditional or whole breast radiotherapy with highly focused x-ray beam for 2-3 min as many as five days in a week [113] with a total dose of about 4500-5000 cGy (cGy) for 5-7 weeks followed by 1 week of booster dose of 1000-2000 cGy [210]. However, treatment doses may vary from patient to patient and the type of radiotherapy. Other kinds of radiotherapy include internal breast radiation or brachytherapy [113]. It is an advanced technique to deliver radioactive liquid compounds via injection at the site of affected tumour tissues, which is supposed to activate the expression of CD4 þ and CD8 þ T cells in the presence of neoantigens triggered by somatic de novo mutation. Specifically, neoantigens-dependent CD8 þ T cells easefully targeted irradiated tumour tissues, while CD4 þ T cells involved in manipulating Th1 cytokines and neoepitopes spread. In advance, genes expression rely upon radiation intensity which upregulated death receptors FAS/CD95, DRS and MHC-II molecules on the plasma membrane of the carcinoma cells [205,211]. On the contrary, it is believed that those patients who have failed to stimulate T cells-based anti-tumour activity, showed shorter life spin during pathogenesis. Further, some antagonist-genes/proteome like myeloid-specific immune checkpoint CD47 protein bind to its SIRPα ligand that is located on the macrophage's surface, thereby phosphorylating the cytoplasmic tail of CD47 ligand to mediate the anti-phagocytic signals to limiting macrophages ability and inducing radio-resistance in the tumour-bearing breast [38].
Although recent advancements that favoured mastectomy combined with adjuvant radiotherapy [205] or immunotherapy along with radiation [212], yield somehow favourable outcomes for internal lymph node melanoma in TNBC. Further, the late hypofractionated accelerated radiotherapy with the dose of 50-52 Gy, which was assumed to be relatively safe for breast carcinoma treatment, resulted in 2.2% symptomatic breast oedema, 1.9% asymptomatic fibrosis, 2.2% contralateral breast, 3.7% arm lymphedema, and 1.6% other melanoma in 290 investigated patients during amifostine abridged primary radiotherapy dermatitis [213]. However, radiation therapy could only recover the prognosis risk in the low-risk TNBC population (P ¼ 0.00056), and revealed non-significant betterment in medium and high-risk patients with short existence duration and mortality within 5 years from diagnosis due to the acquirement of radiotherapy tolerance [214]. Moreover, radiotherapy impairs healthy cells surrounding by tumour cells, induces a rare tumour named angiosarcoma, heaviness and swelling in the breast, numerical/copy number alteration and structural chromosomal aberrations, and infertility [210]. Therefore, applying more advanced technologies, such as CRISPR/Cas gene-editing tool, is necessary to overcome the relapse and metastatic progression in TNBC patients.

Phototherapy
Phototherapy, such as photodynamic therapy (PDT) and photothermal therapy (PTT), is the modernization in the conventional tumor therapeutic techniques that attracted significant attention for TNBC clinical treatments and are being explored extensively in preclinical models that are discussed in the following sections.

Photodynamic therapy (PDT)
PDT is an alternative therapeutic methodology that utilizes a photosensitizer (PS), a suitable excitation light source, and oxygen molecules for treatment [215]. It was begun in the mid-1900s, and Dougherty and his co-workers developed its modernized expression in1975. Much research has demonstrated that PDT has significant potential due to its non-invasive modality and site-specific-targeted treatment [8]. Basically, a specific wavelength is energized PS that shifts its vitality to the molecular oxygen, consequently producing responsive oxygen species (ROS), for example, hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ), hydroxyl free radical (OH) and superoxide ions (O 2 ) can oxidize macromolecules and prompting tumor cell removal. However, conventional PS has a limited therapeutic effect due to this reason the other excitation illumination sources have been explored, such as chemiluminescence (chemiluminescent emitter transfer light energy to the PS), Cerenkov radiation (charged particles produced electromagnetic radiation when passed through dielectric medium, resulting triggering local polarization), and X-rays (transposition of X-rays by energy transducer to optical fluoresces for PDT and radiotherapy) [215]. In recent years, PDT has been proven efficient in various types of breast-related carcinoma. For example, Ag decorated TiO 2 nanorod (200 μg mL 1 ) was used to generate excessive intracellular ROS under 30 min of 5.6 mW cm 2 UV irradiation, resulting in activation of apoptosis and 90% breast cancer cell killing [216]. Further, PDT does not produce any ionizing radiations and systemic toxicity, which can alter the biological medium. Therefore, PDT has very special characteristics when contrasted with conservative diagnostic techniques because of its repeatability deprived of lesser injuriousness, reduced long-term morbidity, minimal invasiveness, and better life quality of the patients [215]. In spite of the extensively growing applications, PDT still has some complications in clinical adoption because of solid tumour hypoxia conditions. The greater consumption of oxygen molecules creates a hypoxia environment that further enhances tumour cell propagation, metastasis, and invasion, thereby rendering the impact of PDT [217]. Some other restrictions on the effectiveness of PDT include deficiency of an ideal PS, selecting a specific wavelength for effective treatment, formulating PS, and monitoring the treatment response [218]. To enhance PDT effect, it is crucial to synthesize excellent PS, improve their solubility in aqueous mediums, and respond to the near-infrared region (NIR) range for deep tissue penetration [219]. Importantly, materials that can absorb light energy in the NIR region exhibited promising potential in PTT and related imagining [220,221].

Photothermal therapy (PTT)
PTT is an emerging theranostics therapy that reveals advanced therapeutic precision, screens therapeutic impact, diminishes the harm to non-cancerous cells and standardises the tumor therapy in time. PTT mainly utilized photothermal agents, those are triggered through nearinfrared lasers that can penetrate about 1-2 cm in tissues [222]. After penetration, it generates hyperthermia, which induces thermal ablation or immunogenic death of the cancer cells. However, PTT-based imagining involves laser-emitted light radiation absorbed by the living tissues and transformed into heat. Meanwhile, biological tissues can create supersonic rays by thermal vibration, and acoustic signals induced via illumination produce photoacoustic signal [8]. The photoacoustic signal created by tissues can change optical absorption to visual pictures.
PTT can reduce distant metastasis in breast tumour in a combinatorial trials such as radiotherapy and chemotherapy. In addition, the PTT agents are generally metal-based nanoparticles, carbon nanotubes, and graphene oxide. It stands out due to its minimal invasiveness, great specificity, and particular spatial-temporal perception. Moreover, diverse mechanisms of action are linked with PDT and PTT therapy, such effective treatments can be utilized as single or combinatorial trials to produce a synergistic impact against TNBC and therefore obtain better efficacy in irradiating tumour cells [215]. However, PTT has several disadvantages that preclude its clinical translation. For example, high-temperature PPT induces massive thermal diffusion by inhibiting heat-shock molecular chaperons/proteins, which cause more destruction in normal tissues compared to tumour mass, ultimately declining the PTT antitumour effectiveness. In contrast, low-temperature PPT (43-45 C) suffers due to a decrease in tumour cell killing impact [223]. Other limitations include poor targeting capability of photothermal materials that can be improved by conjugating specialized targeting ligands or proteins with nanomaterials [224]. Nonetheless, more proteins or ligands-loaded nanomaterials may have a lesser dose of therapy drugs, which shows a weakened antitumour effect resulting in recurrence and metastasis or secondary melanoma development [223].

Sonodynamic therapy (SDT)
Sonodynamic therapy is chiefly based on the PDT and has revolutionized as advance cancer modality since it induces systemic immuno responses via ultrasonic radiation and chemical agents. Unlike PDT, SDT has deep penetration in the tumour tissues through sonosensitizers (ultrasound stimulated sensitizer) and create ample cytotoxicity by triggering ROS to destroy breast tumour cells, therefore can be utilized as a cancer vaccine to treat TNBC [219]. Further, investigations have proved that SDT showed intense penetration in living cells compared to PDT. Mechanistically, non-thermal ultra-sound waves penetrate the targeted tissues, activating sonosensitizers that induce ROS-based cytotoxicity within the targeted sites without causing undesirable injury in the untargeted tissues. However, the most significant difficulty is the development of suitable parameters for SDT-based therapeutic efficacy. The unmet need for SD therapy is the development of steady and flexible sonosensitizers with excellent quality and safety. Moreover, to date, no effective criterion has been identified that provides ease in its application in clinical studies [218,219].
Additionally, recent studies have shown some effectiveness of SDT in tumour cell killing, such as a multifunctional system based on manganese-protoporphyrin (FA-MnPs) encapsulated in the folateliposomes. The FA-MnPs nanosonosensitizer not only penetrated about 8 cm into the targeted tissues but also triggered immune responses in the TNBC mice model. Further, FA-MnPs nanosonosensitizer suppressed the growth of the superficial tumour, deep lesion, and re-polarized M2 (immunosuppressive) macrophages into M1 (antitumour) macrophages. Also, it induced T-lymphocytes, natural killer-cells, and dendritic cells to inhibit the growth of the tumour and initiate immunogenic cell death of TNBC cells [219]. Apart from inevitable success, SDT still exhibited limitations in terms of tumour shrinkage and tumor growth retardation that decrease its effectiveness in cancer studies. Additionally, current databases have revealed that no long-term clinical study will evaluate the impact of sonosensitizers and ultrasonic waves on normal human breast, TNBC, and prolonged exposure with SDT after-effects. Also, there is no standard setting for temperature management, sonosensitizers screening, tumour positioning, and ROS generation in TNBC and it is effective for some solid carcinomas [225]. Moreover, the common SDT sonosensitizers are organic lipophilic materials with poor circulation, short-lived, low loading capacity and accumulated asymmetrically in the tumour sites [218]. Thus, advance CRISPR/Cas-based technology is the most suitable and consistent solution for current issues.

CRISPR/Cas (CRISPR)-Based advances in TNBC gene therapy
Currently, genetic engineering-based technologies have been anticipated for the treatment or therapy of TNBC that is able to target any molecule in the pretentious part, as well as knockout and silent gene aberration/alternations [14,226]. However, DNA binding domain-dependent orthodox techniques, specifically zinc finger nuclease (ZFNs) and transcriptional activator-like effector nuclease (TALENs) gained exceptional importance in genetically engineered breast tissue organoids, cellular carcinoma models, and gene therapy investigations [14,227]. However, due to their intricacy and inefficient approaches, an extensive application of this therapy has been constrained [43]. On the other hand, RNAi has reflected a prodigious establishment for gene expression in tumour therapy. Despite its transitory silencing impact, it must be incessantly administrated to achieve a sufficient knockout level [31]. Presently, innovative archaea-derived adaptive immunity mechanisms earn significant amendments that are mechanistically based on double-stranded RNA endonuclease, called CRISPR-associated protein-9 (Cas9) [228]. This system has been explored to treat aggressive tumour pathology, and advanced gene therapy due to its easeful application, straightforwardness, time-saving, and high target efficacy with efficiency and accuracy [39,43,226].
Some other regulatory elements are successfully knocked out by utilizing CRISPR gene technology. For example, BBDIs (Bromodomain inhibitors) targeted inhibition of BET as a candidate therapeutic aid for TNBC relapse, while intrinsic and acquired tolerance against BBDIs frontier clinical potential. However, silencing with CRISPR and JQ1 small-molecule inhibitors revealed BET signalling pathway of activation, SRC, AXL, YAP signalling, systemic regulation and chemo-resistance in TNBC cell lines SUM149, and SUM159 [94]. Similarly, Snail1 is a zing finger protein, abundantly synthesized and enhanced EMT transition via interconnecting 185 genes through cis-regulatory channel and CXCR-LASP1 axis in the SUM-159, T47D, MCF7, BT549, MDA-MB-436, MDA-MB-231, MDA-MB-468, and BT20 cells model. The CRISPR-based dual 10 base pair excision in the first and second exon of transcriptional factor Snail1, uncovered its partial involvement in EMT phenotype in TNBC [248]. Further, CRISPR immensely exploited in the loss-of-function screen of 2240 genes [29], 2500 novel super-enhancer, including TGF-β pseudo receptor dependencies on BAMBI [237], and 104,592 cell's ATAC profiling [249] to detect specific phenotypic susceptibilities in TNBC.

CRISPR/Cas aid in transcriptomic analysis
Previously utilized lncRNA, miRNA, shRNA, and siRNA-dependent interference or silencing is imperfect, and the residual mRNAs may still perform a functional part, thereby preventing identification of the target site that entails mRNA complete deactivation [120,166,250]. This issue can be solved via CRISPR/Cas9 or CRISPR/dCas9 [120]. For instance, synergistic drug matrix, drug target efficacy, and mischaracterization can now be better analysed via CRISPR/siRNA-based silencing, which explains that OTS964, a small antagonist is the potential inhibitor of CDK11 in TNBC, and multiple carcinoma addiction over CDK11 [251]. Similarly, CRISPR/Cas9 directed deletion of the distal promoter region of lncRNA C1orf132 or MIR29B2CHG, residing between CD34 and CD46 protein-coding region, repressed the expression of the miR-29b2 and miR-29c, enhanced chemoresistance and triggered the worst prognosis in TNBC cells. However, upregulation of MIR29B2CHG via CRISPR/Cas9 suppresses progression, EMT, cell migration, and mammary gland growth pathways in TNBC [128] that were substantiated as an idealistic tool for TNBC transcriptome editing, silencing and understanding the complex mechanism of disease regulation. For example, a wider transcriptomic knockout screening via CRISPR/Cas 9 of human endogenous retroviruses indicated that the overexpression of lncRNA TORJAN played an important role in tumour propagation, invasion and poor prognosis in TNBC patients (FUSCC cohort 1-2). Further antisense targeting therapy of TORJAN has significantly decreased the tumour cell proliferation in xenograft in vivo model [162]. This study proves the effectiveness of CRISPR-based gene editing in TNBC model.

CRISPR aids in TNBC biomarker and drug discovery
The CRISPR-dependent genome-wide array screening is a potent strategy to detect and classify biomarkers for early TNBC prognosis and diagnosis. For instance, an estrogen-inducible E3 ligase called RING finger protein 208 (RNF208), overexpression is engineered by CRISPR/ Cas9, induced proteasomal degradation of Vimentin protein resulting suppression in TNBC cell proliferation. CRISPR/Cas9-based activation/ overexpression of RNF208, interact with a serine residue (Ser39) of phosphorylated Vimentin protein and polyubiquitinated its lysine residue (Lys97) of the head domain of Vimentin protein leading to proteasomal degradation, which facilitates suppression of lung metastasis and invasion in TNBC [234]. The negative feedback of RNF208 protein or inhibitors serves as a prognostic biomarker in TNBC drug development. However, drug development for TNBC is a multistep and challenging process in which a series of genetic hurdles such as drug target structure correlation, tissue exposure and disease selectivity, drug target conformation and authentication, clinical dose, drug efficacy [252], prolong testing of about 12 years and huge expense that may exceed $ 1 billion are involved. Therefore, CRISPR/Cas system proved to be suitable for large-scale screening medicine targets. It mainly helped to generate multiple insertion/mutations, knockout, knock-in screening, and activate and suppress desired targets. For example, CRISPR/Cas-based genome-wide screening in patient-derived xenograft model established new combinatorial Verteporfin-Torin1 TNBC therapy. The pharmacological blockage of YAP/TEAD binding by Verteporfin (YAP inhibitor), and mTORC1/2 inhibition by Torin1 (ATP-competitive specialized inhibitor) indicated poor TNBC cell proliferation. Mechanically, 50 nM of Torin1 downregulates mTORC1/2, which provokes macropinocytosis and Verteporfin uptake, while 0.6 μM Verteporfin mediates YAP reduction leads to programmed cell death or apoptosis in TNBC cells. This study revealed the robustness of CRISPR/Cas system where CRISPR blocked dual druggable targets, which produced a better antitumour effect in the TNBC in vivo model. Also, it can be effectively compared with monotherapy approaches for TNBC [59].

CRISPR/Cas-based therapeutic in chemotherapy
Apart from drug development, CRISPR augments the effectiveness of chemotherapy as TNBC heterogeneity, including breast cancer stem cells evolution and tumour microenvironments (TME), which are among the protruding aspects accountable for the catastrophe of chemotherapy and chemo-drug resistance [42,253]. For example, MALAT1 lncRNA knockdown enhances Paclitaxel and Doxorubicin sensitivity in TNBC via altering several lncRNA (LINC-PINT, NEAT1, and USP3-AS1), and suppressing STAT1, NUPR1, SREBF1, RELA, interferon regulatory factor 1 (IRF1), ERAP1, aminopeptidase regulator, histone-associated proteins such as H3C12, H1-5, and H2AC4, insulin-like growth factor-binding protein 1 (IGFBP1), granulin precursor (GRN), angiogenin, and oxidative phosphorylation reputable pathway [164]. Similarly, PARP1-generated deficiency in MDA-MB-436 and MDA-MB-231 cell lines prominently decrease the Doxorubicin, Gemcitabine and Docetaxel dose necessities to realize therapeutic effectiveness in even 3D tumour-on-a-chip model [240]. Thus, the intensely expressed transmembrane prostate androgen-induced protein (TMEPAI), induced by TGF-β/Smad signalling, propel TGF-β to perform a tumorigenic role in TNBC patients [254]. More importantly, complete silencing of exon 4 of TMEPAI by sgRNA declined resistance against Paclitaxel, Doxorubicin, but negligible tolerance for Bicalutamide and Cisplatin, indicating partial potency of TMEPAI knockout for chemotherapeutic usage [102]. However, BCL-2 family antagonist BH3 mimetics like S63845 is now widely applied as a potent tool in chemo-immuno combo-therapy [242]. Moreover, after repeated cycles of chemotherapy, drug resistance or chemoresistance is a common response generated by tumour cells, which is the most critical problem. Therefore, most mono and combo-therapies have failed to cure TNBC. However, CRISR/Cas9 gene therapy has offered an advanced therapeutic approach to overwhelm chemoresistance in tumour cells. For example, complete BAG3 silencing via CRISPR/Cas9 downregulated the expression of ZEB1 and SNAI1 that induce EMT in TNBC, reduce TNBC cell migration and penetration in brain tissues as well as overwhelmed therapy resistance [37]. In another study, CRISPR/Cas9-dependent knockdown of serine/threonine and tyrosine protein kinase (DSTYK) induces cell death of chemoresistance cells upon 10 μM Doxorubicin plus 100 nM Docetaxel combinatorial application in vitro and orthotopic mouse model [238]. These findings provide the central role of CRISPR gene technology in developing advanced and quick methods to tackle complex chemoresistance mechanisms in TNBC chemotherapy (Table 3).   LGALS2 Induce tumour macrophages, polarization and proliferation of M2 macrophages by CSF1/CSF1R receptor axis, thereby promoting immunosuppression and TME

CRISPR/Cas-based therapeutic in immunotherapy
The complex and more immuno-based heterogeneous nature of TNBC makes its profiling complicated due to the involvement of various signalling molecules, intra and extra cellular matric, specialized T-cells along with diverse kind of mutations, and genomic instability. In this context, several exogenous inductions of PD-L1 by PI3K/AKT/mTOR, JAK/STAT1/IRF1, NF-κB/JAK/STAT3, ECM, IFN-γ, EGFR, interleukins (ILs), TNF-α, NPM1 or B23, vascular endothelial growth factor receptor-2 (VEGFR2), and PARP1 were used to enhance responses during TNBC relapse [104]. However, the meticulous transcriptional control of PD-L1 in TNBC remains contentious [192]. Furthermore, several ongoing clinical or pre-clinical trials with combinatorial, targeted, ligand-specific, NAC or chemo-immunotherapies are being assessed to classify foreseeable biomarkers that will advance treatment consequences among TNBC interventions. Though the prototype is ever-changing, much consideration is being subjected to improving CRISPR-based treatments for TNBC.
The maximum TNBC patients do not retort to anti-PD-1/PD-L1 immunotherapy [13], which may be due to the immuno exclusion process, and several agents and immune checkpoint markers or inhibitors have been developed to address this issue [13,255]. For instance, CD47 protein jam-packed blocking elevates immune ripostes [38]. Though, immune devastation is one of the imperative factors in TNBC tumorigenesis progression [256], carcinoma cells deter immune effector cells through excretion of extrinsic aspects by disturbing the (TME) [207]. Recently, an innovative effort has been made to understand the immuno exclusion induced by ECM. In this study, the discoidin domain receptor 1 (Ddr1) gene was entirely silenced by All-in-One Lentivector CRISPR/Cas9 system in E0771, M-Wnt and AT-3 mouse mammary tissues, which revealed the potent involvement of untethered-extra cellular domain (ECD) of DDR1 protein. The discoidin domain receptors (DDRs) belong to the non-integrin collagen receptors family that have tyrosine kinase activity, are expressed limitedly in adulthood, and are widely studied in tumorigenesis. The DDR1-ECD interacts with collagen, disturbs fibre alignment, induces immuno exclusion, deter immunity, and uses collagen fibre in tumour defence. Additionally, its membrane-bound intracellular kinase domain assists DDR1-ECD against antitumour T-cells [255]. This CRISPR-based finding gives new insight into why anti-PD-1/PD-L1 immunotherapy is only effective with fewer TNBC patients. Therefore, a new antibody against DDR1 needs to be developed to reduce relapse and metastasis and enhance the overall life span of the TNBC population.
Further, the carcinoma-precise polyclonal memory CD4 þ , and CD8 þ T cells are supposed to be a prime target during immune checkpoint inhibitors documentation [204]. However, the CRISPR/Cas9-interceded gene manipulation has endeavoured to systemic discourse challenges apropos immune mechanism flopping from innumerable prospects [257]. The T cell-dependent immunotherapy is credited to the application of ex vivo exploited T lymphocytes directed to abolish tumours with tumour-associated antigen (TAA) or CAR-T cells, and T cell receptors (TCR)-engineered T lymphocytes as its foremost approaches [207,258]. Interestingly, accumulation of TCRs is directly proportional with better tumour rheostat in patients, as it declines with the aging including former immunological exposures like chronic infection [204]. For CAR-T cell therapy, the T cells possibly originated from an allogeneic (cells derived from another person) or autologous (cells taken from the same person) donor. In contrast, autologous T cells utilization is hypothetically inefficient process and as well as mostly depends on the quantity and quality of autologous T cells harvested from the subjected patient. On the other hand, allogeneic grafted T cells challenged substantial barriers due to the TCR on donor's T lymphocytes, and endogenous MHC class I complex that posse alloreactivity and graft-versus-host disease (GVHD) [207]. Therefore, CRISPR/Cas9 gene-editing technology inserts the CAR gene and confiscates the TCR effectively [257]. For example, EGFR is one of the highly expressed receptors on TNBC cells, whose expression successfully engineered via third-generation CAR-T targeting EGFR in TNBC both in vivo xenograft mouse model and in vitro, revealed limited cytotoxicity, retard tumour cell growth, enhanced IFN-γ, activate PARP, and Fas-associated death domain (FADD), and caspase signalling [259]. Likewise, CRISPR-engineered CAR-T cells with scFv of grafted monoclonal TAB004 antibody coupled to CD3 and CD28 for mutant glycosylated tumour of MUC1 (MUC28z), recognized 95% in TNBC cases, significantly victimized cytotoxicity over an array of human TNBC cells, upon identification of mutant MUC1, MUC28z CAR-T cells elevate synthesis of IFN-γ, granzyme B, Th1 and other cyto-and chemokines [24]. Therefore, a single dose of CRISPR/Cas9 engineered CAR-T cells significantly reduces tumour growth in NOD-Prkdc scid IL2rg null (NSG) mice xenograft model [35].
(NCT04417764), central nervous system tumour (NCT03332030), multiple myeloma (NCT04244656; NCT03399448), relapsed or refractory leukaemia and lymphoma (NCT03398967), relapsed or refractory B-cell melanoma (NCT04035434), T-cell malignancies (NCT03690011), and B acute lymphoblastic leukaemia (NCT04557436). Most of the CRISPR clinical trial is based on PD-1 dependent immunotherapy, while other were conducted to assess the efficacy and safety issues regarding CRISPR [41]. Besides, CRISPR-engineered autologous CAR-T cell's clinical studies are limited to lung cancer or non-small-cell lung cancer [260,261]. Previously, cells were collected, engineered, propagated, and then induced, resulting in low efficacy and frequent death of the individual due to prolonged treatment delay. However, advanced CRISPR-based allogeneic T-cell therapy is more powerful, cost-effective, and rapid than autologous CAR-T cell therapy. Hitherto, the first allogenic CRISPR-engineered CD 19 edited T-cells (CTX 110 ) were injected into the 143 B-cell malignant patients followed by fludarabine and cyclophosphamide standard dose, which initially showed nephrotoxicity in one patient (NCT04035434). In addition, autologous CRISPR-dependent T-cell clinical trials showed fewer off-target and on-target impacts, such as alteration in the non-coding region of genes (1.69%) [262]. Importantly, off-target-induced undesirable mutations are deceased after some time in the patients. Moreover, patients with the higher expression levels of engineered T cell exhibit fewer after-effects like fatigue, rash and fever [262]. This concluded that it might be safe and effective to administer CRISPR-engineered CAR-T cells to the TNBC patients to overcome poor prognosis and increase DFS and OS.

Mechanism of Class-I CRISPRs
Class-I, Type-I complex is identified in Escherichia coli, consists of eleven sub-components of five Cas endonucleases such as Cse-1 1 , Cse-2 2 , Cas5 1 , Cas6e 1 and Cas7 6 that have 405 kDa weight and called CRISPRassociated complex for antiviral defense (CASCADE) where Cas3 and its effectors cleaved CRISPR RNA precursor and retain these products. These cleaved RNAs serve as single small guide RNA, which sufficiently interfere with foreign DNA. Further, Class-I, Type-III CRISPRs widely occur in archaea species such as Cas10 that combine themselves in a cascade-like multi effector complex and then identify and subsequently kill the invaded foreigner RNA [266]. More detail of Class-I CRISPR's action mechanism has been studied elsewhere [43].

Mechanism of Class-II CRISPRs
The most commonly used and explored system in biomedical engineering is Class-II due to its simplicity and ease of targeting efficiency. This system is based on a single effector to detect, bind and target the desired sequence, discussed concisely in sub-sections.

CRISPR/Cas9
CRISPR-Cas9 system generally consists of Cas9 DNA endonuclease enzyme, and single guided-RNA (sgRNA) [264]. The Cas9 protein is sectioned into two lobes, the alpha lobe, and endonuclease activity lobe. This endonuclease lobe is further divided into two domains, named the HNH domain and RuvC domain. The HNH domain cuts the specific targeted sequences of the DNA strand, while RuvC cleaves the non-targeted DNA strand, but it is deactivated due to D10A mutation (Fig. 4) [43]. The sgRNA has great importance in the CRISPR-Cas9-based targeting system efficacy, specificity, and precision [240]. This sgRNA is composed of two diverse RNAs one RNA work as a guider named guide RNA (gRNA) or CRISPR RNA (crRNA), and the other woke for targeting named as trans-activating CRISPR RNA (tracrRNA). The crRNA in the sgRNA can recognize a PAM (proto-spacer adjacent motif) sequence (5'-NGG) that usually consists of about 3-5 nucleotides and then match the remaining 20 nucleotide sequences with the targeted host genome [39]. Further, the starting 10-12 nucleotides sequence at 3' end of the sg RNA that is near to the PAM (proto-spacer adjacent motif) knows as seed sequence that binds at the targeted site in the host for genome editing purpose. Upon complete recognization, tracrRNA pair with crRNA and recruit Cas9 protein to create double-stranded breaks (DSBs) at any PAM-comprising V, variants; ds, double-stranded; ss, single-stranded targeted site within the identify sequences of the targeted host genome. Created DSBs can be either repair by homology-directed repair (HDR) or non-homologous end-joining mechanism (NHEJ). Importantly, NHEJ produce frameshift mutation via spontaneous deletion or insertion, thereby promoting permanent gene silencing as compared to HDR.

CRISPR/Cas12
CRISPR/Cas12 (Cpf1) is another robust system that falls in Type-V of Class-II and was discovered in Francisella and Prevotella in 2015 (Table 4). Unlike CRISPR/Cas9 sgRNA, this system contains only crRNA and Cas12 endonuclease, which generates sticky ends, cleaves hairpin structure of pre-crRNA and produces transitional crRNA that further undergoes processing to become mature crRNAs. Cas12 has more advantages than Cas9 protein due to the small size Cas12 (1.3 kb), guide RNA (42-44 nucleotides) and thymine rich PAM sequences [272] that can address several limitations of CRISPR/Cas9 delivery issues [27,272]. It is a RNA-directed DNA nuclease that offers immunity in bacterium and significantly integrates into the mammalian cell editing system [269,272].

CRISPR/Cas14
CRISPR/Cas14 is solitarily detected in uncultivated symbiotic archaea species and another robust and the simplest CRISPR in CRISPR/ Cas family (Table 4). Cas14 gene has 24 variants grouped in Cas14a, Cas14b and Cas14c. It codes exceptionally small size (400-700 amino acids) RNA-guided endonucleases with a molecular weight about 40-70 kDa, half of all existing Cas proteins. It has a single RuvC catalytic domain, suggesting that it may act as stand-alone effector. It binds and cleaves single-stranded DNA as Cas13. Nonetheless, Cas14-directed cleavage is more specialized than other CRISPR/Cas systems. Moreover, Cas14 does not require PAM sequences to recognize targeted DNA [270], and it can be effectively exploited in TNBC pathogenesis and TME manipulation.

CRISPR/Cas13
The newly discovered Type-VI CRISPR/Cas13 endonuclease can only cleave single-stranded RNA with the help of 28 nucleotides long crRNA at the protospacer flanking site (PFS) [39,273]. Further, this system is similar to Type-V for crRNA processing and maturation but has unique higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains that are exclusively related to RNase not detected in the rest of the CRISPR/Cas system. After specific site recognization and binding, Cas13 activates cleavage activity to the untarget RNAs. Nonetheless, Cas13 collateral cleavage action has not been fully established except it cleaves target sequence in multiple uracil residue sites [273]. Hitherto, this tool has been utilized in some biomedical engineering applications but has much more potential to detect tumour circulating RNA in cancer patients, viral-based pathogenesis [273] in tumour, and manipulation of tumour RNA such as microRNA, lncRNA and mRNA.
Several endonucleases have been designed to enhance efficacy and decrease off-target impacts, such as Cas9 nickase, dead Cas9 (dCas9) and dead Cas12a (dCas12a), whose nickase domain is synthetically pointmutated. Despite prompt breakage, they activate or silence gene expression and maintain the integrity of binding of CRISPR/Cas complex [32,273].

Engineering system for CRISPR-based targeting in TNBC
Recently, CRISPR/Cas system has been synthesized by plasmid, ribonucleoprotein (RNP), and mRNA-based methods to screen, treat, and engineer in vitro and in vivo TNBC genome at their desired location, as shown in Fig. 4.

Plasmid-based genome editing
The plasmid-based CRISPR/Cas9 system, including pX333 [4], pX335 or pX330, pX459 [37,143,153,234], pcDNA-3.1 [13,89,274], pLKO-based plasmid [162,237], and dual vector bearing-plasmid system [83,110] is a modest and lucrative alternative approach for RNP, which signifies tractability in scheme owing to the affluence of DNA sequences integration into plasmids retaining unpretentious molecular cloning systems [94]. Nevertheless, the efficacy of gene engineering is frequently limited by gene expression proficiency and nuclear distribution, which is required to improve the critical gene therapy-dependent multiplexes. This coordination demonstrates advanced steadiness in contrast sgRNA/Cas9 mRNA complex [31]. Plasmid high replication capacity inside the cell makes nuclear ingress easeful via eruption of the nuclear membrane. Nonetheless, the transportation in post-mitotic cells to the nucleus typically befalls through nuclear pores that can be meek by the binding sequences of the transcription factor in the plasmid that intermingle with cellular importins [14]. Generally, in systems cases, comprising a CRISPR tracrRNA, sgRNA-guided Streptococcus pyogenes Cas9 (spCas9), U6promoter, and restriction sites are packed in the same plasmid [275]. Even though this system has great potential, its larger plasmid DNA (pDNA) genome size hinders efficient supply, thereby decreasing biodistribution, efficacy, and excessive on-set deferment in genetic manipulation of targeted gene/genes [34]. Additionally, the high off-target impact is the extra crucial disadvantage of the plasmid-based CRISPR delivery (Table 5) [264].

Cas9-RNA ribonucleoprotein-based genome editing
The Cas9-RNP delivery system into the targeted tumour cells is the hotspot and pragmatic approach [268]. The RNP complex has enough simplified genome engineering stratagem attributable to the fact that translation/transcription consequently promoter assortment, and codon improvement are not prerequisites [43]. This may conceivably increase the proficiency of genome-engineering in hard-to-transfect cells or post-mitotic, in which limitations to translational/transcriptional capability of the cell can lead to worse genome-engineering efficiency when compared to plasmid mRNA/DNA [34]. Together, this scheme also declines the cell type quality since transcriptional directing cannot be engaged [14]. It also signifies a fleeting practical genome-engineering method that can cause lesser off-target impact, immuno stimulation, and cytotoxicity (Table 5) [264]. Nevertheless, some staid challenges inclusively reduce delivery efficacy due to the enlarged size of Cas9 endonucleases protein, which still relics to be cracked [47]. Additional inadequacy of the RNP complex methodology is a considerable endotoxin impurity, which can be created together by bacterial activity and high cost with no mass production [268].

mRNA-based genome editing
The mRNA-based scheme is projected to decrease genome excision deferral wherein mRNA indoctrination sgRNA and Cas9 have been delivered to target tumour cells [43]. Besides, Cas9 endonuclease transitory manifestation in this system might be a two-edged sword, but it can then donate for plummeting the off-target impact, while excessively less expression time resulting in reduced competence [34,277]. The mRNA instability remains one of the main problems for this delivery approach (Table 5) [47]. However, this method has been frequently utilized in embryo engineering and zygote genome editing.

Delivery vehicles for CRISPR/cas-based targeting in TNBC
The CRISPR-mediated genome editing can be achieved via three distinct methods such as (i) physical method for transporting CRISPR/ Cas9 system, (ii) lentiviral/adenoviral-based delivery system, and (iii) nanocarriers-based delivery, which is summarised in Table 6 and demonstrated in Fig. 5.

Physical methods for delivering CRISPR/Cas9
The physical control for spatiotemporal expression of the CRISPR/ Cas9 complex has become popular recently [283]. Its high precision and non-invasive strategies like optical, temperature, magnetic field, pH, ultrasound-responsive elements, including electroporation, and microinjection to deliver CRISPR along with space dimension and time-dependent release [25] are also noticeable. Thus, the high transfection competence can be offered through the physical method, but many serious apprehensions have been articulated together with inaptness for in vivo drives, but rather a poor cell viability and specificity [284]. Conversely, the hydrodynamic mode of delivering CRISPR/Cas can be offered as in vivo experiments [263]. However, distressing physiognomies may cause numerous physical or biological complications, for instance, dysfunction of the heart, high blood pressure, and liver enlargement (hepatomegaly). During the last couple of years, a great revolution has originated in the form of nanoparticle-based CRISPR/Cas9 delivery system [285]. Several of the previously-mentioned obstacles have not been experiential in this technique. Notably, a nonviral-based delivery vehicle proposes many benefits over virus-based delivery, for example, high DNA-wrapping competence, lessen immune-generated stimulation, easeful fabrication, and flexible strategy that are being specialized and targeted the sites in the living tissues or cells with negligible cytotoxicity for healthy cells [192,286]. The strategic inadequacy of these systems is the poor delivery efficacy regarding genes of interest compared with viral delivery systems [285]. Nevertheless, the gene conveyance efficiency and the expression profile of the trans-allele can be augmented ominously through the combination of diverse self-assemble innovative nanocarriers. In the ongoing era, advent of inimitable multifunctional carrier vehicles affords a vigorous auspicious delivery stratagem for TNBC therapy [14].

Nanocarriers-based delivery for improving CRISPR/Cas targeting in TNBC
The effectual and nontoxic transfer of dynamic DNA, RNA or mRNA, and proteome at the targeted cells or tissues, unsolicited genetic aberrations, inadequate packaging size, and immunogenicity are the principal doubts in translational gene therapeutics and genome editing [263,287]. In this context, nanoparticles (NPs) are getting considerable attention in delivering CRISPR/Cas cargo to the targeted sites due to their dynamic small sizes, excellent loading capacity, easeful penetration through the phospholipid bilayer, and other intracellular barriers that help in amplified pervasion and delivery [288]. In addition, CRISPR/Cas9 stability, biodegradation, and biocompatibility can be meritoriously accomplished by engaging NPs in clinical translation [265,289]. Nanocarriers like cargo-encumbered organic (exosome, dendrimers, micelles, nucleic acid, and liposomes) and inorganic (generally metal and magnetic-based) NPs own unique features that convey the competency to transport the captured CRISPR cargo/complexes into the cell or even within the nucleus's targeted sits [269,283,290]. The shape, size, and surface chemistry of NPs are the crucial factors that regulate nuclear or cellular uptake, biodistribution, and rapid clearance [291,292]. However, various types of NPs are available even for the detection of solid carcinoma, but very few studies have been conducted so far to deliver the NPs-based CRISPR/Cas cargo in TNBC cells that are detailed in the following sections.
6.3.1.2. Lipid-based nanocarriers. The lipid-based CRISPR/Cas delivery vehicles have been conjectured since 2017 [232] to offer improved cargo carriage into the targeting tumour cells, combat antagonistic impact, and aid in the problem of multidrug confrontation [276]. The lipid molecules are devouring amphiphilic characteristics along with the hydrophobic tail and polar head that are allied between the two domains, systematically categorized into ionizable, cationic, and neutral lipids. The ionizable lipids are mostly easily protonated at the least pH value but remain amphipathic or impartial at biological pH. Further, the pH-responsive character of ionizable lipids aids favourable sgRNA delivery in vivo, since neutral lipids rarely interact with the negatively charged membranes of blood cells, thus increasing the biological compatibility of lipid nanoparticles (LNPs) [291,297]. Conversely, cationic lipids possessed a permanent positive charge on the head group [277]. Nonetheless, lipid or lipid-like NPs can be fabricated through diverse materials that provide an extensive diagnostic and therapeutic application for aggressive carcinoma [276] as TNBC therapy and its research. It has excellent targeting capability, biocompatibility, and biodegradability, especially as a nanocarrier, as well as a self-assembly nano/micro emulsified delivery system for CRISPR/Cas [289]. Also, many lipids or lipid-like delivery vehicles, such as nanolipogels (NLGs) and nanoliposomes (NLPs), have been aimed for mRNA, oligonucleotides, and plasmid-derived DNA delivery because of the substantial lessening in immune reaction stimulations, stumpy genotoxicity, the safety of CRISPR/Cas9-sgRNA complex from endonucleases, and rapid renal evacuation. Thus, synthetic lipid NPs believe to be superlative nanocarriers for CRISPR/Cas9-based gene editing into the TNBC cells [233].
The NLPs and NPs are the two foremost vehicles for CRISPR cargo delivery used to regulate effective cargo-loaded circulation and release [283]. This dual structures-based system can be engineered using indistinguishable morphology and surface charges, despite the characteristic transformations existing between their particle elasticity and architecture [192]. NPs retain amorphous or crystalline solid-structured inorganic materials or polymers [298], while NLPs are composed of an aqueous core encapsulated by phospholipid bilayer [291]. However, both NPs and NLPs have size-reliant possessions, prominently distinct from the characteristic of the majority of substances. In addition, somatic signals are well-known for administering cell and NP communications [289]. The particle size of about 100 nm competently escapes the nuclear phagocytosis machinery, thereby extending blood circulation. Also, the surface chemistry of NPs distresses transmission lifetime and biological conveyance [298]. The cationic system displayed considerably non-specific cellular uptake and advanced serum protein captivation compared to the anionic or impartial particle system [287].
Apart from NLPs-NPs, NLGs-NPs hybrid system consisting of alginate hydrogel encapsulated with phospholipid bilayer or liposome offer tuneable characteristics and surface charge proved as an idyllic system to investigate both in vivo and in vitro tumour accumulation and cellular uptake as particle elasticity alter and induce capability to bind cellular surface receptors and squeeze through plasma membrane located channels [299]. Numerous liposome-derived carriers, for example, Myocet®, Doxil®, and AmBisome®, have been commercially manufactured [263]. Since CRISPR/Cas-associated sgRNA is highly anionic, for efficient component delivery, the superficial layer of NLPs or NLGs ought to be cationic to enhance the absorption ratio of charged particles. Despite the encapsulation of Cas9 endonucleases in the LNPs, charge neutralization between Cas9 and negatively charged protein is essential. For that synergy, diverse kinds of agents have been assessed, including polylysine, polyethyleneimine, chitosan, polyethylene terephthalate, powder bed fusion, TAT-peptide, transIT®-LT1, FuGENE® 6, and nuclear localization signal peptide together with gold, magnetic, carbon NPs with variable surface advancement. Among them, Cas9-sgRNA along with polo-like kinase-1 (PLK-1) encapsulated within an innovative cationic polyethylene glycol (PEG) phospholipids NPs delivery system ascertained to be better than the incredulous CRISPR's insufficient transfection efficacy (Fig. 7a). However, in vitro transfection efficiency was about 37.8% in the MCF-7 breast cancer cell lines when compared to the commercial transfection agent as lipofectamine2000, which showed 3.15% transfection (Fig. 7b) [232].
The in vitro tNLG-CRISPR/Cas9 (tNLG-LCN2KO) complex-mediated 80% silencing of LCN2 oncogene in MDA-MB-436 and MDA-MB-231 TNBC cell lines verified the site-specific efficacy and least off-targeting knockout impact (Fig. 8h and i). Moreover, without nanovehicle (Free LCN2KO), CRISPR/Cas9 inhibited LCN2 mRNA expression only 30-50% (Fig. 8i). Further, the tNLG-CRISPR/Cas9-based silencing reduced Lcn2 protein synthesis (Fig. 8j) and transmigration of TNBC cells (Fig. 8k). While MDA-MB-231 tumour-bearing female mice model confirmed 81% inhibition of TNBC progression, 77% tumour volume, and 69% mass in comparison with untreated one (Fig. 8l-o) [233]. Furthermore, tNLG-CRISPR/Cas9 complex exhibited excellent capability to escape efficiently tumour endothelial obstruction (67%) by its high deformability design and selection of alternative pathways such as receptor-based membrane fusion path. Therefore, the CRISPR/Cas9-tNLGs-based system is safe, effective, and precise for TNBC targeted gene therapy. Meanwhile, LNPs have poorer immunogenicity than virus-based vectors; thus, it can assist as a possible substitute for in vivo genomics. In summary, LNPs or lipid-like NPs can be measured as an auspicious delivery vehicle to accelerate the transfection capability of CRISPR/Cas9 gene engineering and successively therapeutic impact in TNBC metastasis. It is prominent that previous research utilized large-size CRISPR pDNA to accomplish gene silencing. Despite the cited benefits, the foremost matter related to plasmid DNA is extraordinary intensities of unintentional gene edits, poor endosomal discharge, and delivery efficacy that relatively limited in vivo application of this approach.
6.3.1.3. Polymer-based nanocarriers. Polymer-based NPs are nanosized materials produced by synthetic or natural biocompatible and decomposable monomers or polymers [287]. They can be synthesized by solvent displacement or diffusion emulsification [301], ionic gelation [302], nanoprecipitation [26], and microfluidics [303]. Based on polymer characteristics and structure, diverse types of multiple, dual, and single CRISPR systems can be engineered inside the NPs core, chemically linked or conjugated, entrapped in the matrix of polymer, or NPs-bound surface polymer that can be delivered and released payload via bulk or superficial erosion, diffusion and inflammation [192]. Polymer-based polymeric NPs, micelles, and dendrimers have been extensively utilized for pDNA, mRNA, oligonucleotides, and nucleic acid delivery and impressively dealt with a distinctive improvement of biological compatibility. It signifies prodigious packaging capability and noteworthy synthetic properties in alleviating desired gene encoding Cas9-sgRNA complex counter to serum-tempted accumulation in TNBC [304]. Moreover, stability, composition, responsivity, superficial charge, kinetics, and loading efficiencies can be accurately restrained [305].
The general forms of polymeric NPs include nanospheres (solid matrix), and nanocapsules (cavities around polymeric shell), which are further divided according to the exterior appearance as in dendrimers, polymersomes, and micelles [287]. The polyamidoamine (PAMAM) or dendrimer is highly branched and high density by possessing primary amine on its periphery, which facilitates a tight junction between nucleic acid and polymer. At the same time, the functional group on its exterior recruits contrast agents or biomolecules over the dendrimer surface [289,306]. However, CRISPR cargo can be encumbered on its interior. Dendrimers have dynamic tuneable 3D architecture like shape, size, mass, and superficial chemistry such as charged polymer polyethylenimine (PEI) [291]. Therefore, numerous dendrimer-centered products are presently running in clinical translation as gene therapy delivery vehicles, contrast and theranostics agents, and topical gels [235,307,308]. Additionally, the charged dendrimer can produce non-dendrimer polymer such as polyelectrolytes having a reiterating electrolyte group, which define their charge variability with pH that improves polyelectrolyte's mucosal transport [309] and bioavailability [310]. Polymersomes are synthetic vesicles usually produced by the amphiphilic block of copolymers that are similar to liposomes and frequent locally receptive but evidenced to have better permanency and payload-retaining efficacy [311], thus making them efficient means of transport for CRISPR/Cas9 delivery in the nucleus at the targeted site. Some applicable polymersomes include hydrophilic blocks of poly (ethylene glycol), and poly (2-methyloxazoline), and hydrophobic blocks of poly (dimethylsiloxane), poly (caprolactone), poly (lactide), and poly (methyl methacrylate). The polymeric micelles are typically self-assemble copolymers of the responsive block, composed of amphiphilic nanospheres core layered by hydrophobic shell through Van der Waals forces, serving to guard CRISPR-sgRNA cargo along with prolonging circulation time, that precludes rapid evacuation under the guidance of reticuloendothelial system (RES) [192]. Moreover, its partial-solid nonpolar core is devised by a biologically demeaned polymer likes polylactic-co-glycolic acid, poly (β-caprolactone), and poly (L-lactide). In addition, micelle polymer measures around 10 nm-100 nm in diameter, perfect for reinforcing various hydra-insoluble gene engineering.
Inclusively, polymer NPs are absolute aspirants for CRISPR-based gene editing due to their biomimetic, biocompatible, biodegradability, hydra soluble, and shelf-stable properties [271]. In addition, the surface modification is very easy for targeted delivery that make them beneficial in TNBC translation. However, the detriments of the polymer include an augmented hazard of particle accumulation and related noxiousness. Very few polymers are approved by FDA for medical usage, although these nanocarriers are presently experiencing challenges in scientific trials for TNBC. For example, PD-L1, a crucial immune checkpoint cell surface receptor direct blockage via efficient and novel CRISPR/Cas9 system delivered through cationic copolymer AMP-modified poly-beta amino ester (aPBAE) (Fig. 9a) with 95% transfection efficacy in 4T1  [232].
carcinoma cells at 80 wt ratio. Also, the cationic copolymer showed higher transfection as compared with conventional vectors even when weight ratio beyond 80, thus proved as versatile gene delivery vector to edit genome (Fig. 9b). The targeting efficacy of cationic copolymer/CRISPR system was as higher because it produced only two bands, one is 200 kDa and other is 400 kDa (Fig. 9c). Further, cyclin-dependent kinase 5 (Cdk5), and p35 proteins expression (Fig. 9d) and PD-L1 relative expression was significantly decreased after aPBAE conjugated Cas9-Cdk5 knockout in the TNBC cells (Fig. 9e). Notably, aPBAE conjugated Cas9-Cdk5 based NPs has provoked strong T cell-facilitated immune reactions in the cancer microenvironment, where the CD8 þ T cells population was considerably improved on the other hand governing T cells population was declin ed, provide the favourable stratagem for the TNBC antitumour clinical treatment and gene therapy (Fig. 9f). Moreover, in vivo antitumour effect of Cdk5 gene manipulation by intravenous injection silent 95% genetic expression of Cdk5, which decreased 75.7% tumour weight, 89.6% tumour volume, protein expression profile, and messenger RNA expression, than anti-PD-L1 antibody and PBS control group (Fig. 9g-p). Further, aPBAE conjugated Cas9-Cdk5-NPs induce the expression of granzyme B and IFN-γ in 4T1 mice, which showed elevation of cytotoxic T-lymphocytes (CTL) retort in TME, leading to effective TNBC immunotherapy that efficiently modulates immunosuppression in TME [26].

Inorganic nanocarriers
Several types of rigid nano-sized inorganic materials, including black phosphorus, carbon (C) nanotubes, calcium carbonate (CaCO 3 ), gold (Au), graphene, silica (SiO 2 ), and iron oxide (Fe 2 O 3 ) NPs are used for nanoformulation due to their strong potential for CRISPR/Cas safe delivery, drug distribution and imaging implication. These inorganic NPs are specifically engineered to have varied types of the nanostructure, surface morphology, sizes, and geometries [312]. Thus far, gold NPs have fascinated significant consideration to emerging pioneering CRISPR/-Cas9 carrier structures for targeting tumours in the murine model with 97% encapsulation efficacy and 79.4% release efficiency [276]. They are the utmost well-researched nanostructures that are exquisitely exploited in innumerable forms, including nanostars, nanospheres, nanocages, nanorods, and nanoshells [313], and widely used as effective breast therapy [265]. In addition, inorganic nanocarriers have inimitable magnetic, physical, optical, and electrical characteristics because of their intrinsic core quality of the material [314].
Evidently, gold nanocarriers own free electrons at their outermost shell that constantly oscillate in a frequency-dependent manner according to the shape, size, and magnitude, benevolent the photothermal character [222]. Further, easeful functionalization concedes extra delivery abilities and surface modification properties [313]. For instance, gold NPs are significantly incorporated in advancement of cell-free DNA-based breast carcinoma diagnosis that was suffered due to effective site-specialized primer design and false-positive signals limitations, prominently overcome via CRISPR/Cas-dependent fluorescent biosensing system and CRISPR/Cas12a@Au nucleic acid-based amplification system (free of fluorescence-based). The fluorescence-free system is superseded over other and mainly based on metal-enhanced fluorescence generated by plasmonic nanoparticles. Upon triggering, CRISPR/Cas12a degrade the cfDNA into single-stranded cfDNA that feasibly monitor through colour-changed chemistry (purple to red) of gold NPs. Also, the CRISPR/Cas12a@Au system can successfully exploit to detect BRCA-1 gene mutations within 30 min [53].
Another inorganic nanocarrier, such as carbon, generating noticeable attention about the expansion of improved carrier delivery systems due to the governable topographies such as wider surface area concerning volume ratio, simple surface alteration to upsurge uptake, tuneable size, immunologically inactive surface, and excellent stability in an internal biological setting [315]. Similarly, iron oxide is a very prevalent examined material for inorganic nanocarrier production, and the majority of them are FDA-permitted nanomedicines or vehicles for gene delivery [269,316,317]. Also, magnetic iron-based gene-editing tools are compounds of maghemite (Fe 2 O 3 ) or magnetite (Fe 3 O 4 ) that retain superparamagnetic characteristics with confined structures or sizes and have shown triumph as gene delivery systems and contrast agents in thermal-based cancer therapy [292]. For example, two tumour-inducing genes such as ferroportin (FPN) and lipocalin2 (LCN2) were knockdown by Cas13a/U6-gRNA with a NF-ĸβ expression controlled "minimal decay promoter" (DMP) (Fig. 10a and b), coated with magnetite nanocarrier that significantly enhanced the ferroptosis in MDA-MB-453 cells line (Fig. 10c). Hence, reducing tumour growth for a longer period in both in vitro and murine-based in vivo model, proving as an idyllic nonviral delivery vehicle for direct CRISPR-based gene engineering [280].
Other common nanocarriers, including mesoporous silica, and calcium phosphate (Ca 3 (PO 4 ) 2 ), both have been utilized efficaciously for safe gene and drug transfer into the target site [318]. Moreover, quantum dots, characteristically prepared by semiconducting nanomaterials such as silicon (Si), are exceptional NPs used principally for in vitro imaging implementation. However, they also have the potential for in vivo therapeutic trials [319]. Currently, silver, cadmium, indium, silicon, and carbon quantum dots are widely applied in cancer genomics due to their higher degree of core cytotoxicity, variable luminescent characteristics, and diverse subcellular fate [320].
In general, struggles to practice nonviral gene editing and transfection strategies have been rigorously restricted by the low delivery efficiency through the plasma membrane of the cell. Correspondingly with inorganic nanocarrier, the efficacy of genome editing is improved when compared with lipid-based formulations and native Cas9 systems. Furthermore, plasmonic or radioactive, photothermal, and magnetic characteristics of inorganic nanocarriers make them ideal for imaging, diagnostics, and photothermal application in cancer genetics. Most inorganic NPs have virtuous stability and biocompatibility and, therefore, block niche applications that need inaccessible properties through organic nanocarriers. Nonetheless, they are inadequate in clinical translation due to the toxicity, and poor solubility disquiets, and specifically heavy metal-based formulations [269,321].

Design strategies for effective CRISPR-nano engineering in TNBC
The plasmid-based approach is evident in TNBC pre-clinical applications due to stability, ease of synthesis, low cost and multiple targeting options. However, efficient packaging into the single delivery vehicle and transportation are tremendous tasks [233]. Mechanically, the Cas9 endonuclease and sgRNA-bearing plasmid can target gene/genes by cellular internalization, and upon release, the Cas9 plasmid enters the nucleus for further processing of sgRNA. After transcription, sgRNA and mRNA are delivered to the cytoplasm for translation and then translated RNP (Cas9 protein and sgRNA) redelivered into the nucleus for CRISPR-assisted gene engineering [43]. Importantly, lingering plasmid DNA in the host genome may cause undetectable mutagenesis or off-target impact, leading host immune retort issues.
Systematically, nonviral-mediated plasmid delivery has much more benefits than virus-mediated delivery but suffers due to low transfection efficacy. In this respect, cationic lipid nanocarriers may aid low toxicity and easeful endolysosomal escape [232]. In addition to delivery, it has a complicated process, including transcription, translation and re-translocation in the nucleus, delay or decrease gene engineering precision. Furthermore, using the HDR mechanism by pCas9 plasmid is far from practical, especially in vivo trials [39]. Also, double-stranded Cas9 plasmid can integrate at any site of the host genome and induce constant expression of Cas9 that may elevate off-target impacts. It is suggested to induce silent hindering mutation either in the sg RNA target sequences or PAM sequences that prevent Cas9 plasmid from inducing undesirable mutation repeatedly within the target site. Moreover, before in vivo and ex vivo studies, Sanger sequencing and tracking of indels by decomposition (TIDE or TIDER) can be used to identify design inaccuracies in TNBC in vitro model [231].
The mRNA-based system is greatly easeful, less time-consuming and precise where Cas9 mRNA directly delivers into the cytoplasm after successful endolysosomal escape for functional protein translation and then RNP complex transfer in the nucleus for subsequent gene engineering. This system has a shorter half-life, lesser off-target impact, and faster expression. Recently, approval of the Cas9 mRNA-based vaccine will significantly enhance its importance in clinical application. However, very few studies have utilized this system due to the rapid enzymatic degradation of mRNA. Further, Cas9 mRNA is typically complicated due to heterogeneous action mechanisms. Its mRNA with sgRNA uses the NHEJ mechanism for gene editing, while donor DNA with sgRNA implies HDR mechanism, making its intracellular transportation intricate. Its considerable variations in administration track hamper the synthesis/production for customized delivery. In this case, nanomaterials-mediated delivery is preferential due to nano protection against nucleases-based degradation [39]. However, a more sophisticated method such as systemic administration is better than others, where the least chances of biological hindrance are foreseen. Despite the delivery obstacle, Cas9 mRNA can be better used as a vaccine but can activate autoimmune retort and various recognition receptors in animal cells [278]. This chemical alteration, like pseudouridine or 5-methylcytidine, will be utilized to control immuno retort without distressing the translation of Cas9 mRNA [322].
Unlike Cas9 plasmid and Cas9 mRNA systems, Cas9-RNP is superseded by skipping transcription and translation, resulting in fast genome engineering followed by lower toxicities [40]. Further, protein-based systems are clinically approved and safe. It is directly introduced in the nucleus via vehicle and can be an idealistic choice for TNBC in vivo and ex vivo gene editing. Nonetheless, Cas9-RNPs encapsulation and delivery are the biggest challenges for the geneticist and biomedical engineers due to the large size of RNP. Moreover, negatively dense Cas9 RNPs neither pass through the plasma membrane nor have resistance against enzymatic degradation. Conversely, low charge dense RNP is unstable in forming a complex with delivery vehicles. In this main, modification of Cas9/sgRNA in RNP is mandatory that can be achieved via recombinant protein technology and conjugation biochemistry. Further, modification in spacer length or its sequence is another thought-provoking strategy to decrease off-targets, immune retorts and heighten RNPs delivery efficiency [39]. Recently, lipid-based, polymeric and inorganic nanovehicles have been widely utilized to deal with these issues. Additionally, some small molecules exhibit crRNA stabilizing properties [27,53,269,272] or enlarging nuclear pores to validate CRISPR/Cas9 cargo delivery [53]. Also, NHEJ-based silencing is more reliable than HDR, which shows only 10% delivery efficacy in vitro and non-specific mutation in DNA bases [39]. Thus, these discoveries must be considered while designing the next-generation RNP delivery complex.
Additionally, the successful transfection of Cas protein together with sgRNA at the desired target site is required for fruitful in vivo CRISPRmediated gene editing. For this purpose, the delivery vehicles should have excellent engineering efficacy coupled with low immunogenicity and high capacity of targeted delivery. In this regard, the previously discussed Cas9 plasmid approach is efficient in the mice model, but Fig. 9. The TNBC tumour growth inhibition by aPBAE@Cas9-Cdk5 KO in vitro and 4T1 murine model. (a) Schematic representation of biodegradable cationic polymer, aPBAE@Cas9-Cdk5 knockout-based TNBC therapy. (b) Transfection efficacy of aPBAE/pMax-GFP (green fluoresce proteins) in TNBC cells. (c) The T7E1 cleavage assay was directed to detect aPBAE@Cas9-based silencing of Cdk5 gene in B16F10 murine cells. M, KO and Ctrl stand for DNA leader, knock out, and control, respectively. (d) The CDK5 and co-activator p35 proteins expression profile after distinct treatment by western blot. (e) Relative expression of PD-L1 was measured after the transfection of IFN-g, naked plasmid DNA (pDNA) and aPBAE/Cas9-mediated Cdk5 knockout in the 4T1 cells (n ¼ 4). (f) Immunofluorescences image exhibited aPBAE@Cas9-Cdk5KO-mediated PD-L1 reduction triggered CD4 þ and CD8 þ T-cell infiltration. Scale bar at 100 μm. (g) Treatment timeline of CRISPR/Cas9dependent PD-L1 attenuation by intratumoral administration of aPBAE@Cas9-Cdk5 at 7, 10, 13 and 16 days in 4T1 murine model. (h) Tumour excised from Balb/C female mouse (n ¼ 6) treated with PBS (phosphate-buffered saline), naked pDNA (plasmid DNA), aPBAE/Cas9-Cdk5 (cationic copolymer AMP-modified poly-beta amino ester -CRISPR/Cas9-cyclin-dependent kinase 5), and anti-PD-L1 antibody. (i) Tumour weight reduction in grams after G1 (PBS), G2 (naked pDNA), G3 (aPBAE/ Cas9-Cdk5), and G4 (anti-PD-L1 antibody) treatment (n ¼ 6). (j) Tumour growth reduction after various treatments in mm 3 (n ¼ 6). k | CDK5, P35, PD-L1, and β-Actin proteins expression quantification and, (l) qualification by western blotting that were derived from phosphate buffer saline (PBS) (G1), pDNA (G2), aPBAE@Cas9-Cdk5 (G3) and anti-PD-L1 antibody (G4) treated breast carcinoma bearing mice (n ¼ 3). (m) Heat map of mRNA expression of tested genes compared to GAPDH housekeeping gene was generated by qRT-PCR. (n) Alteration of Cdk5 mRNA expression level, (o) p35 mRNA expression level, and (p) PD-L1 mRNA expression level from PBS (G1), pDNA (G2), aPBAE@Cas9-Cdk5 (G3) and anti-PD-L1 antibody (G4) treated breast carcinoma bearing mice (n ¼ 3). Reproduced by the permission of ELSEVIER [26]. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) delivery, editing specificity and control over the expression of Cas9 are limited. Thus, various virus-based delivery methods have been adopted but suffered due to size, safety and packaging limitations. In the last couple of decades, various nanovehicles have been developed in which cationic lipid-based nanovehicles show superior performance and low safety concerns but have synthesis complications and poor colloidal stability in in vivo systemic administration [219]. Therefore, the prime focus of the ongoing research programs is to improve and develop effective delivery carriers for CRISPR/Cas gene engineering because low engineering precision and toxicity of existing carriers are the foremost constraints in cancer therapeutic. So far, intracellular transportation of Cas9 remains challenging in vivo and as well as in clinical trials.
However, each discussed nanovehicles has its pros and cons due to various biological barriers in vivo. The first obstacle is efficient CRISPR/ Cas complex encapsulation in the nanocarrier. Cas9 plasmid has a relatively large size about 5-10 kDa followed by 1.74 Â 10 4 negative charges, while RNP has a huge size (160 kDa) and negatively charged gRNA, enhancing difficulty and multiple delivery issues [232]. For example, CRISPR-nano complex firstly faces the blood-mediated enzymatic degradation of the CRISPR/Cas component, active clearance either via macrophages or mononuclear phagocyte, irregular accumulation of nanovehicles through the opsonisation process, and lastly filtration by glomerulus. Further, CRISPR-nano complex ought to pass through breast tumour-created barriers before reaching targeted sites such as leaky blood vessels, extracellular matrix, high interstitial pressure, hypoxia, densely acidic condition and tumour microenvironment. Furthermore, transcellular phospholipids bilayer, endosome and nuclear pore pose an additional barrier in the delivery of CRISPR/Cas complex [41]. Before transcription or translation of Cas9 sgRNA, nanovehicles have to overawe all mentioned obstacles.
Recently, animals and preclinical studies have revealed promising transfection capability and ease of targeting of nonviral nanovehicles. Further, the organic and inorganic nanovehicles showed excellent colloidal stability coupled with a series of physical and chemical alteration opportunities. For instance, lipids can be engineered as ionic in lipid-based nanocarriers in which CRISPR is efficiently protected and released under acidic environmental conditions [233]. Further, the dynamic surface modification of lipids-based nanocarriers exhibits the superior capability to escape lysosome and RES clearance and targeted transfection [297]. Likewise, cationic lipid NPs efficiently encapsulate and deliver a large cargo of negatively charged CRISPR/Cas sgRNA system than other like viral, plasmid-based and physical transfection methods [232]. On the other hand, cationic lipid nanocarriers induced in vivo immune retorts and showed adjuvant immuno retort; thus, lipids-based nanovehicles should be wisely exploited in gene editing. As lipid-based nanocarriers, cationic polymers utilize electrostatic interaction to form cargo complexes with CRISPR/Cas sgRNA. They offer an intelligent programmable delivery vehicle system to overwhelm in vivo biological obstructions [307].
Aside from encapsulation, safe and longer circulation in the blood vessels of breast enhance the chances of CRISPR/Cas entry in the tumour cells. A lucrative and even more effective approach is to modify the surface of nanovehicles by diverse kind of FDA approved non-toxic polymer like polyethylene glycol (PEG) [232], chitosan [316], plur-onic™ F 127 (PF127) [224,323] and pluronic™ F 68 (PF68). Furthermore, inorganic nanovehicles aid precise targeted delivery of CRISPR/Cas cargo for in vivo therapy. The next-generation single-chain antibodies [23,233], antigen-binding fragments [23], aptamers (single-stranded oligonucleotides) [235], AS1411 (G rich DNA oligonucleotide) [324], and peptides [55] can be easefully conjugated on the polymer-coated surface of nanovehicles, which will promote targeting in TNBC. After successful internalization of CRISPR-nano complex via endocytosis, the complex face acidic environment and multiple catalytic enzymes of endosome that can inactive CRISPR/Cas components. In this circumstance, pH-responsive or pH-buffering induce nanovehicles can be better option as some cationic polymer exhibit strong proton sponge impact by their tertiary amine groups and resist against acidic environment of endosome thereby activating Cl and H þ antiporter channel. The high amount of Cl and H þ ions and water flow create osmotic pressure that eventually disrupted the membrane of endosome and effectively release CRISPR/Cas-nano complex into the cytosol of tumour cell [41,284]. Nonetheless, ridge cargo design formation enhance higher polyelectrolytes that cause dissociation or accumulation of polymer/CRISPR complex in the host body [39].
Another strategy to reduce engulf of the CRISPR-nano complex by endosome is the direct entry into the cytoplasm via receptor-based endocytosis or caveolae-dependent endocytosis [235]. This direct entry of CRISPR/Cas cargo may have more chance to transport into the Golgi complex or endoplasm reticulum that assisted their immediate transcription in the nucleus [41]. Moreover, tumour microenvironment does play a critical role in CRISPR-nano complex delivery. In this connection, inorganic nanovehicles can be exploited because they provide ample control over time-dependent cargo release upon external or internal stimuli such as light [136,283], photothermal [224,323], pH [311], ultrasound [218], thermal, hypoxia [296], ATP and redox-reagent like glutathione-triggered nanovehicles [296]. For instance, gold and silica-based nanocarriers can provide the advantage of PTT and ultrasound-depended transfection via layer-by-layer assembly method. However, both nanocarriers are susceptible to rendering CRISPR/Cas enzyme or release earlier in the bloodstream [40,222,265]. Emerging CRISPR/Cas delivery systems such as magnetic nanocarriers have appealing characteristics that may enhance delivery efficacy of Cas9 and sgRNA. For example, iron oxide have tuneable size, assessable alteration sites, and MR imaging properties [323]. Comprehensively, new emerging delivery approaches are still in infancy, and required deeper insight of their mechanism of action and related limitation, specifically in terms of biosafety and scale-up production need to be explored in detailed for successful clinical translation of CRISPR in TNBC.

Future perspectives and conclusions
The complete elimination of triple-negative breast carcinoma mass pDCUg, Cas13a sgRNA coupled with U6 promoter; pDM, a plasmid with DMP; U6p, U6 promoter; DMP, NF-κB decoy-minimal promoter. Reproduced by the permission of Springer Nature [280]. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) and distinct organ metastasis through clinical trials has not been achieved thus far. Since early diagnosis, treatment, and clinical translation of TNBC are more challenging due to tumour microenvironment, heterogeneity of multiple cellular and oncogenic complexities obfuscate the therapy selection, and monotherapy does not give optimum success. Recently, the CRISPR/Cas cutting-edge gene technology has revolutionized cancer therapeutics; but its delivery is troublesome, off-target impact and genotoxicity due to virus-based transfection threat the biotechnology. The serious concerns regarding CRISPR are error-prone targeting and prolonged aggregation of payload in the genome, and related immunogenicity makes clinical trials in TNBC patients still limited. Undeniably, there are some ethical disquiets regarding genomeengineering technology. First, genome-engineering induces permanent alteration and may cause severe health-related issues in the CRISPRnano-treated patients if any mistakes occur during the engineering process. Secondly, CRISPR/Cas-nano system is not yet fully established for genetic-engineering, and could not be pragmatized in breast and ovarian carcinoma-bearing patients until more innovative systems become obtainable. Also, it creates enormous inequities and social conflicts if abused or overused, especially in modulating ovarian-based cancer. Further, heritable mutations such as BRCA-based genome-edited embryos are not allowed to transfer to the person's uterus. Therefore, before planning CRISPR-nano-mediated engineering in breast/ovarian tumour, one should consider the abovementioned concerns. Additionally, CRISPR-based gene therapy is only utilized for lung cancer and blood cancers such as lymphoma and leukaemia in clinics. Thus, several years have been needed by common society to adapt and accept geneengineering-based alteration.
Apart from ethical concerns, transcription-activator like effector (TALENs) and zing-finger nucleases (ZFNs)-based gene editing are more expensive, generate permanent numerical and structural mutations, time-consuming and even not practical for mass production. whereas, CRISPR/Cas technology is rapid, low-cost, relatively more accurate and efficient than the other gene manipulation technologies. Thus, CRISPR/ Cas gene engineering must be established through a wide range of clinical trials, which will restore the condition of TNBC than presently accessible treatment tactics. Moreover, several biological molecules, targeting agents, organic and inorganic nanocarriers are developed, which enhance the transfection efficacy, aid targeting specificity, and silencing the gene or mRNA expression either directly interrelating with driver genes or through changing several signalling molecules and pathways accountable for TNBC progression.
Nonetheless, innumerable challenges discoursed so farthe therapeutics tactic for TNBC ought to emphasize the effective abolition of carcinoma, related tumour subpopulation, and allied blood vessels via synergistic advanced gene technologies. The treatment objectives have implemented the perception of personalized gene therapy for each patient or subpopulation of tumour cells. The elementary consideration of genomics exploring the stimulus of the cellular signalling cascade in tumour propagation and resistance will expose a novel arena for TNBC therapy. Moreover, tumour microenvironment is the most important arena of oncology research. In this main, CRISPR/Cas pro-codon library aid the analysis of tumour microenvironment by silencing a series of genes in a mouse model and revealing how each gene influences tumor growth, immune composition and histopathology [325]. Its robust knockout and knock-in technology resolved the central dogma of immunosuppression and showed a relation between loss of function genes and tumour microenvironment. Also, an individual's immune responses play a fundamental part in defending from carcinogenesis. Recently, the successful application of CAR T-cell and noncoding RNAs-based immuno-genetic engineering requires an exceptional methodological advancement in molecular genetics, pharmacogenomics, nanotechnology, and chemistry.
In addition, an optimal CRISPR/Cas9-sgRNA-centered genome editing should be comprehensively analysed for MHC-I, II, T-cells, and macrophages population's responses, chemically altered to increase pharmacodynamics, and pharmacokinetics, transfected under contemplation of active biodistribution, and clathrin or endolysosomal escape mechanistic, low off-target and high determination of target specificity, and be treated at an optimum level to get the desired impact. Noteworthy improvements in these fields have been made exclusively for CRISPR/Cas therapeutics; nonetheless, its efficacious transformation depends on additional interdisciplinary advancements to improve on-target impact and payload target delivery. More obviously, the stratagems mentioned above can be incorporated by nanomaterials-based photothermal therapy and imaging to securely locate and classify the biological distribution of the CRISPR@nano complex.
Conversely, high therapeutic cargo envisions within the nuclear spaces enhance the ultimate potential of pioneering technology that would fundamentally be based on the innovation and design of dynamic multi-structured nanomaterials systems. For instance, exotic intrinsic properties holding gold biosensors can be unlocked the ssDNase by CRISPR/Cas12a and examine telomerase activity in breast carcinoma subtypes [27,326]. Also, nucleic acid-based multiplex system [271], cell-penetrating peptides, and carbon nanotubes will potentially deliver CRISPR/Cas-sgRNA cargo efficiently. Importantly, plant-derived exosomes [327], and extracellular vesicles-based nanovehiclea potent platform for intercellular crosstalk and communication, engineered vesicles can be loaded and safely delivered CRISPR/Cas9 complex at the targeting sites [328][329][330]. Similarly, a tuneable magnetic system can be devised by a magnetic field that induces controllable delivery of CRISPR/Cas9 along with advanced imaging properties, mass production and precise transfection in TNBC cells will be more effective. These robust vehicles advance TNBC treatment outcomes that will help enhance the patient's lifespan. However, only a limited number of inorganic nanovehicle are FDA approved for clinical translation due to their apparent toxicities. Moreover, safe, simple, cost-effective, and eco-friendly synthesis of inorganic nanoparticles are unmet needs of material science.
Moreover, organic nanovehicles are immuno-friendly, biocompatible and biodegradable but suffer due to poor therapeutic/drug loading capacity and structural drawbacks. Mostly, their ultra-structures have been destroyed during packaging/loading process of the payload. Thus, advancement in the development of nanovehicles with superior biocompatibility and low immunogenicity, such as biomimetics [285] may be incorporated to have a better option for the delivery of CRISPR/Cas complex at the desired targeting site. Further, the transcriptomes, proteomes, driver genes, and extracellular matrix biomolecules are reported as resilient either in masking other addiction genes expression or inducing TNBC prognosis; however, the previous data lack the connecting links among molecules that have been explored as oncogenes. Moreover, a better understanding of the singling cascades, material interaction with organic molecules, and improvements in material technology will eventually develop a more idyllically targeting system in the future.
Furthermore, good manufacturing practice (GMP), ethical compliance, and low cost are among the most critical concerns in designing the CRISPR/Cas complex for its clinical translation. To tackle this, new strategies are required to develop in which clinically appropriate GMPcompliant material such as clinical grade Cas9 and sgRNA will be utilized [331]. Now, National Institute of Health (NIH) has invested more than billions of dollars to achieve the abovementioned issues. For this, several companies developed cGMP unit to produce desirable nuclease for research and diagnoses. However, these CRISPR/Cas nucleases exhibit leaching of divalent cations during purification, which can cause serious issues in drug production practices. Recently, several off-the-shelf techniques have been developed to scale up the previous CRISPR/Cas gene manipulation method. Whereas, some off-targets related unforeseen toxicities are also noticed, especially in the case of CRISPR-based T cell immunotherapies. Further, the off-target based toxicities will be dealt with by utilizing other host mechanisms than SpCas9 and Streptococcus aureus (SaCas9). Some improvements have been made, such as SpyFi™ Cas9 (Integrated DNA Technologies Inc.) that can replace single amino acid, resulting in prominent decreases in off-target effect compared to wild-type SpCas9, which is the main concern of gene therapy.
In summary, the CRISPR-nano complex brings foremost progress in the analysis and therapy of TNBC. The use of CRISPR/Cas9 in the cell culture study, gene manipulation, establishment of animal models, and patient-derived xenograft (PDX) models for TNBC therapy, rapidly augmented due to the system easiness of designing, targeting, recognizing and engineering multiplexed gene target, and cost-effectiveness. However, microbe-based and physical methods are common CRISPR/ Cas intracellular delivery approaches. Nonetheless, recent inundation research disadvantages have been reported for transfection approaches, resulting in the development of effective and safe payload delivery experiencing serious challenges that are prerequisite to be tackled. In recent times, nanovehicle for CRISPR/Cas9 targeted delivery has been fascinated by remarkable courtesy. The copious advanced nanomaterial transfection systems for example; protein, lipids, nanolipogel, polymer, and inorganic materials, have been aimed. Among them, iron, gold, and cationic nanolipogel-based delivery systems have the most prospective in imminent practical solicitations. The CRISPR/Cas9 gene-editing technology has spotted a rebellion in chemo-immunotherapy. Furthermore, this powerful gene engineering technology can be overwhelmed numerous delivery complications and aid success in the forthcoming clinical translation. Consequently, it is believed that the synergy of CRISPR/Cas9 and nanomaterial-dependent transfection embraces the substantial potential for the prospect of TNBC therapy.
Author contributions JF and MZI conceptualized; JF initial drafted, structured, and written the manuscript; MZI, NSH, RZ, QZ and XDK critically analysed; MZI and NSH edited; MZI and XDK supervised the study; JF, MZI and XDK project funding acquisition.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
Data will be made available on request.