Advancements in CRISPR screens for the development of cancer immunotherapy strategies

CRISPR screen technology enables systematic and scalable interrogation of gene function by using the CRISPR-Cas9 system to perturb gene expression. In the field of cancer immunotherapy, this technology has empowered the discovery of genes, biomarkers, and pathways that regulate tumor development and progression, immune reactivity, and the effectiveness of immunotherapeutic interventions. By conducting large-scale genetic screens, researchers have successfully identified novel targets to impede tumor growth, enhance anti-tumor immune responses, and surmount immunosuppression within the tumor microenvironment (TME). Here, we present an overview of CRISPR screens conducted in tumor cells for the purpose of identifying novel therapeutic targets. We also explore the application of CRISPR screens in immune cells to propel the advancement of cell-based therapies, encompassing T cells, natural killer cells, dendritic cells, and macrophages. Furthermore, we outline the crucial components necessary for the successful implementation of immune-specific CRISPR screens and explore potential directions for future research.


INTRODUCTION
[20][21] CRISPR screen technologies involve the use of guide RNAs (gRNAs) to guide the Cas9 enzyme to precise genomic locations, where it induces double-stranded breaks. 1,2,22These breaks stimulate DNA repair mechanisms, resulting in either random insertions or deletions that disrupt gene function or the replacement of the target gene with an exogenous DNA sequence. 1,2The resulting perturbation in gene expression allows researchers to assess the functional consequences of individual gene knockouts (KOs) or knockins in a high-throughput manner, and this can be accomplished through two primary approaches: pooled CRISPR screens and multiplexed arrayed CRISPR screens. 12ior to the advent of CRISPR-based screens, loss-of-function screens relied primarily on RNAi-based technologies.RNAi uses small interfering RNA and short hairpin RNA to bind to messenger RNA of the target gene, thereby controlling its protein translation. 23Because of the intrinsic difference between gene KO and knockdown, CRISPR screens can produce more consistent genotypes than the hypomorphic mutations generated by RNAi screens.Parallel comparative studies have shown that CRISPR screens identified essential genes with greater accuracy and mitigated off-target effects more effectively than RNAi screens. 24As a pre-transcriptional regulatory tool, CRISPR-based technologies are capable to screen noncoding regions, offering a broader coverage compared with RNAi screens.
The CRISPR screen workflow encompasses several essential steps: gRNA design for targeting specific genes, construction of a gRNA library, delivery of CRISPR components via viral vectors or transfection, phenotypic or functional selection to identify desired genetic alterations, subsequent sequencing and data analysis, and validation through additional experiments. 12,15This comprehensive approach enables various applications, such as the discovery of gene functions, identification of potential drug targets, functional annotation of genomes, and mapping of pathways and disease mechanisms. 25,26,27otably, the CRISPR screen surpasses previous screening methods because of its ability to investigate gene function at a genome-wide scale, facilitating the identification of both known and novel genes involved in specific biological processes or disease phenotypes. 12,162][33][34][35][36] These discoveries have led to the development of innovative immunomodulatory drugs and optimized therapies.][39] This knowledge has guided the design of combination therapies targeting multiple resistance mechanisms to overcome treatment resistance and improve patient outcomes.1][42] This enables tailored treatment strategies based on individual genetic profiles, maximizing efficacy and minimizing side effects.
In summary, the use of CRISPR screen technology has made substantial strides in enhancing our comprehension of the genetic foundations of cancer immunotherapy.Through systematic manipulation of genes and large-scale screening, this technology has facilitated the identification of crucial genes and pathways involved in anti-tumor immune responses and the emergence of treatment resistance.
This review provides a comprehensive overview of the current applications of CRISPR screens in cancer immunotherapy (Figure 1).It emphasizes the use of CRISPR screens in tumor cells for the identification of novel therapeutic targets and the use of CRISPR screens in therapeutic cells to advance cell-based therapies (Figure 2).Furthermore, the review addresses the existing limitations of CRISPR screen technology and explores future research directions in this field (Figures 3 and 4).

CRISPR SCREENS IN TUMOR CELLS TO IDENTIFY NEW THERAPEUTIC TARGETS
The use of CRISPR screens in tumor cell lines has provided a rapid and comprehensive approach to investigate numerous genes, enabling the identification of key regulators within extensively studied signaling networks.These networks encompass crucial biological processes such as antigen presentation, IFN-g signaling, TNF-a signaling, natural killer (NK) cytotoxicity sensitivity, and macrophage recruitment (Table 1).Furthermore, these screens have facilitated the discovery of previously unknown pathways associated with antigen presentation.Here we summarize the novel biological insights garnered from these CRISPR screens and discuss the prospective avenues they have illuminated for future research directions in this field.

Regulators of antigen presentation
Major histocompatibility class I (MHC I) molecules play a crucial role in presenting tumor self-protein peptides on the cell surface, which allows antigen-presenting cells (APCs) to internalize them and activate cytotoxic T cells (CTLs) that specifically target tumor-specific or tumor-associated proteins. 60Subsequently, primed CTLs can recognize tumor cells expressing the abnormal protein via MHC I and mount cytotoxic responses against them. 61However, tumor cells have developed various mechanisms to circumvent antigen presentation pathways or downregulate the expression of MHC I molecule. 60herefore, it is essential to identify regulator proteins that could potentially disrupt or enhance the antigen presentation in tumor cells, accordingly inhibiting or overexpressing genes encoding those proteins to achieve a more robust CTL-mediated anti-tumor response.CRISPR screen, as a functional and efficient gene editing and assessment tool, has been widely used to explore new antigen-presentation regulators in tumor cells.
Several studies have focused on genes directly encoding the MHC I molecule and controlling its localization on the cell surface.Patel et al. 49 illustrated that KO of essential MHC I genes, TAP2 and B2M, stimulated tumor cell evasion from T-cell-mediated killing.Similarly, in an in vivo screening of genes involved in multiple classes including antigen processing and presentation, cell surface localization, and chromatin remodeling, Manguso et al. 43 demonstrated that in addition to Tap1 and Tap2, KO of H2-T23, a non-classical MHC I gene contributing to the inhibitory regulation of T cells, also enhanced the effect of immune cytotoxicity.Moreover, other studies have investigated the function of protein-coding genes associated with antigen-processing and presentation processes, despite not being part of the MHC I molecule itself.For instance, an in vitro loss-of-function screening determined that inhibition of BCL-ABL in the antigen presentation pathway increased the vulnerability of human chronic myelogenous leukemia (CML) cells to NK-cell-mediated lysis. 33Manguso et al. 43 also illustrated that Ptpn2 KO increases the level of MHC I on tumor cell surfaces.
In conclusion, most studies examining the effects of antigen presentation mechanisms on immune cell-mediated killing have primarily focused on genes directly encoding MHC I subunits or those involved in regulatory pathways.Although the functions of several MHC I subunits, both canonical and non-canonical, have been extensively studied (TAP1/2, B2M, and H2-T23), 43,49 there is still a lack of studies investigating genes indirectly related to MHC I expression and localization (BCL-ABL and Ptpn2), 33,43 which presents promising avenues for future screens.
Together, although IFN-g has complicated interaction with other components of tumor regulation pathways, CRISPR screening has revealed potent modulators that influence PD-L1 expression, antigen presentation, responses to oxidative stress, and downstream IFNg-JAK-STAT pathway. 43,46,47,49Future studies could investigate the cross-talk between IFN-g and other pro-tumor or anti-tumor regulatory molecules and signaling pathways.

Regulators of TNF signaling
TNF exhibited dual functions in the TME, either as anti-tumor regulator or as an immunosuppressive cytokine, which stimulates examination on its complex role with CRISPR screen. 65In an in vitro genome-wide screening, Zhang et al. 50demonstrated that inhibition of RNF31, an E3 ubiquitin ligase, disrupted the cell-ligand-bound TNF receptor complex 1, leading to the loss of A20 and non-canonical IKK complexes and ultimately promoted tumor apoptosis.Besides, to elicit the impact of autophagy on anti-tumor responses, Lawson et al. 47 demonstrated that KO of autophagy-related genes, including Atg12 and Tbk1, enhanced tumor sensitivity to TNF-mediated cytotoxicity.To enhance sensitivity of melanoma cancer to MART-1 T cells, Vredevoogd et al. 58 illustrated that deletion of TNF receptor-associated factor 2 (TRAF2) redirected TNF signaling pathway to promote RIPK1-dependent apoptosis, thereby lowering TNF cytotoxicity threshold in tumor cells.Moreover, Dufva et al. 56 investigated the functions of TNF-related apoptosis-inducing ligand (TRAIL) receptors and found that when TNFRSF1B expression was inhibited, a less differentiated phenotype of acute myeloid leukemia (AML) cells conferred resistance to NK cell cytotoxicity.
Although current studies have primarily focused on improving antitumor responses through perturbations of TNF receptors (i.e., TNF receptor complex 1, TRAF2, and TRAIL receptors), future investigations should also explore the intracellular downstream signaling cascades. 50,56,58gulators of NK cytotoxicity sensitivity NK cells, belonging to the innate lymphoid cell family, play a crucial role in cancer therapy, including autologous and allogeneic NK-cellbased immunotherapy.These cells demonstrate a broad tumor reactivity and are particularly involved in eradicating early tumors and controlling metastasis. 66Tumor cells commonly express stressinducing molecules, such as NK ligands, including MHC class I polypeptide-related sequence A (MICA), MICB, and UL16-binding proteins (ULBPs), which selectively activate NK cells through the NKG2D receptor. 66However, the presence of NK cells is typically reduced in established tumors because of the elevated expression of inhibitory receptors, such as TIGIT, CD96, and TIM3, on tumorinfiltrating NK cells, leading to a substantial impairment of their anti-tumor functionality. 66Moreover, the heterogeneous TME This schematic provides a concise overview of the primary constraints encountered in CRISPR screen technology.These include the propensity for off-target effects resulting from the CRISPR-Cas system (A), incomplete gene knockout efficiency due to the use of suboptimal gRNAs (B), limited coverage of non-coding regions when designing gRNAs (C), the presence of functional redundancy and compensatory mechanisms within certain genes (D), and the technical hurdles associated with library construction (E).Furthermore, the schematic delineates the corresponding remedies proposed for each of these limitations.www.moleculartherapy.orgReview modulates NK cell metabolism within the context of solid tumors, posing a significant constraint on their functionality. 67Prior research has demonstrated that inhibiting TIM3 expression in NK cells enhances their cytotoxicity and IFN-g production, providing a potential strategy to overcome the efficacy limitation of NK-cell-based therapy. 68Consequently, it is crucial to further investigate the molecular mechanisms underlying NK-cell-mediated tumor eradication in order to enhance their killing capacity and clinical application.
Currently, the integration of CRISPR screens in tumor cells has facilitated the examination of modified NK sensitivity, the discovery of novel immune checkpoint targets, and the improvement of cell-based therapies.Zhang et al. 50conducted parallel genome-wide CRISPR-Cas9 KO screens under NK and CD8 + T cell pressure, revealing the involvement of various components, including RNF31, RBCK1, and SHARPIN, in the linear ubiquitination chain assembly complex (LUBAC).This complex holds a pivotal role in TNF signaling, governing cellular survival and death.Inhibition or depletion of RNF31 rendered tumor cells more susceptible to both adaptive and innate immune cells, resulting in heightened apoptosis through reduced NF-kB signaling. 50These findings propose that targeting RNF31 could enhance TNF-mediated killing and empower NK cell anti-tumor activity. 50In a separate study focusing on human glioblastoma stem cells (GSCs), CHMP2A, a chromatin-modifying protein and constituent of the ESCRT-III complex, emerged as a regulator of NK-cell-mediated cytotoxicity.Using a comprehensive "two cell type" whole-genome CRISPR-Cas9 screening system, the KO of CHMP2A triggered augmented migration of NK cells toward tumor cells and increased secretion of chemokines involved in NK cell migration. 51These findings underscore the critical involvement of CHMP2A in mechanisms of immune evasion. 51Previous research also investigated genes that influence the susceptibility of leukemia cells to primary NK cell killing using a CRISPR screen with the CML cell line K562. 33Loss of NCR3LG1, the ligand for natural cytotoxicity receptor NCR3 (NKp30), protected K562 cells from NK-cellmediated killing. 33Additionally, IFNGR2 was found to be responsible for the upregulation of MHC class I molecule on K562 cells following co-incubation with NK cells. 33The results were further corroborated by the observation that decreased expression of IFNGR2 was associated with enhanced overall survival in patients diagnosed with AML and kidney renal clear cell carcinoma (KIRC). 33Moreover, NK-cellsensitive tumor cells were found to exhibit elevated expression of  CDN, cyclic dinucleotide; CHMP2A, charged multivesicular body protein 2A; CML, chronic myelogenous leukemia; DENR, density-regulated protein; GSC, glioblastoma stem cell; FITM2, fat storage-inducing transmembrane protein 2; IFNGR1, interferon-gamma receptor 1; JAK2, Janus kinase 2; KAT2A, lysine acetyltransferase 2A; LGALS2, galectin-2; NCR3LG1, natural killer cell cytotoxicity receptor 3 ligand 1; NUDT21, nudix hydrolase 21; PRMT1, protein arginine methyltransferase 1; PTPN2, protein tyrosine phosphatase, non-receptor type 2; RNF31, ring finger protein 31; SLC19A1, folate-organic phosphate antiporter; TNFRSF1B, TNF receptor superfamily member 1B; TRAF2, TNF receptor-associated factor 2; TNBC, triple-negative breast cancer; XPO1, exportin 1; ZNF143, zinc finger protein 143.
chromatin remodeling complexes, heightened levels of B7-H6 (NCR3LG1), and reduced levels of HLA-E/antigen presentation genes using profiling relative inhibition simultaneously in mixtures (PRISM) phenotypic screens and CRISPR gene-editing studies.The combined use of PRISM and CRISPR provided a "multi-omic" and functional genomic profile of a representative NK-cell-sensitive tumor cell, with implications for NK cell immunotherapies and their correlation with immune checkpoint inhibitor (ICI) resistance. 55urthermore, Dufva et al. 56 conducted genome-wide CRISPR screens to identify genes associated with antigen presentation, interferon signaling, and various factors such as NCR3LG1, apoptotic mediators, TRAIL receptors, CD48, and TNFRSF1B, which influence the resistance of hematologic cancer cells to NK cell cytotoxicity.The results showed that different lineages of hematological malignancies exhibited distinct susceptibility mechanisms to NK cells, and this variability was determined by the lineage-specific expression of susceptibility genes. 56Another study further confirmed the role of CD48 expression in evading NK-cell-mediated immunity in adult T cell leukemia/lymphoma (ATLL) cells.Genome-wide CRISPR screening identified CD48 as a gene whose KO conferred resistance to NK cell cytotoxicity.Reduced CD48 expression was observed in primary ATLL cells and other aggressive peripheral T cell lymphomas, indicating its significance as a biomarker for NK-cell-associated immunotherapies. 69In summary, the successful use of CRISPR screens has greatly contributed to the identification of numerous targets for the improvement of NK cell therapy.Future applications of genome-wide CRISPR screens on effector NK cells hold promise in directly elucidating the molecular mechanisms underlying their anti-tumor efficacy.

Regulators of macrophage recruitment
Macrophages, a type of phagocytic cells, can be classified into M1 and M2 macrophages, each characterized by unique functions.Although classically activated proinflammatory M1 macrophages mainly contribute to the elimination of pathogens and tumor cells through several ways, such as producing nitric oxide, alternatively activated anti-inflammatory M2 macrophages promote the removal of parasites and homeostasis through the high secretion of polyamines and ornithine. 70However, these versatile cells play an important role in tumor progression and immunosuppression, [70][71][72] especially M2 tumor-associated macrophages (TAMs) which are the major part of tumor myeloid cells. 70By generating various cytokines, such as platelet-derived growth factor (PDGF) and ligands of the epithelial growth factor receptor (EGFR) family, TAM infiltration aggravates tumor development. 73,746][77] Conversely, tumor cells generate cytokines that enhance the differentiation of TAMs, leading to the ultimate reciprocity between TAMs and tumor cells. 78herefore, it is crucial to figure out the mechanisms behind this positive loop and solve the problems accordingly for better tumor treatment outcomes.
On the basis of the nature of macrophages, macrophage-related genes could be potential targets for cancer immunotherapy.Although blocking the recognized immune checkpoints has proved clinically effective, the limited response to treatment highlights the existence of extra immune escape mechanisms.To further deal with this problem, CRISPR screens have been successfully used to identify additional immune checkpoints.For example, for triple-negative breast cancer (TNBC), results of pooled in vivo CRISPR KO screens in syngeneic mouse models have demonstrated that the elimination of the E3 ubiquitin ligase Cop1 in cancer cells could lead to a reduction in the secretion of chemokines associated with macrophages, resulting in decreased infiltration of tumor macrophages. 44As a result, targeting Cop1 improves the anti-tumor immune response and strengthens the response to immune checkpoint blockade. 44Furthermore, in another study of TNBC treatment, two-step customized in vivo CRISPR screens in mouse models with a designed mouse sgRNA library that corresponds to all human disease-related immune (DrIM) genes recognize Lgas2, which promotes the M2-like polarization and macrophage proliferation by activating the colony-stimulating factor 1 (CSF1)/CSF1 receptor (CSF1R) axis, as an important regulation for the progression of TNBC. 45These significant findings suggest the potential of CRISPR screens in the improvement of cancer immunotherapy.

CRISPR screens in T and CAR-T cells
0][91][92] To overcome these limitations and develop more robust CAR-T cell products, it is imperative to elucidate the underlying molecular mechanisms that enhance anti-tumor efficacy while mitigating safety concerns.In this context, the application of CRISPR screens has emerged as a valuable tool, providing comprehensive and detailed insights into the selection of optimal CAR-T cells.CRISPR screens enable the identification of CAR-T cells with enhanced characteristics, such as accelerated proliferation and division rates, potent anti-tumor activity, reduced exhaustion, and improved tumor infiltration capabilities (Table 2).Leveraging the power of CRISPR technology, we can guide the generation of optimized CAR-T cell products tailored for cancer therapy.
hybrid system that combines lentivirus-mediated introduction of traceable sgRNA cassettes with electroporation of Cas9 protein. 35his approach, known as sgRNA lentiviral infection with Cas9 protein electroporation (SLICE), offers several advantages for studying gene function and conducting genome-wide screens in primary cells.First, it enables efficient and specific disruption of target genes, allowing researchers to elucidate their roles in various biological processes.Second, the use of traceable sgRNA cassettes facilitates the tracking and identification of cells in which gene disruption has occurred.This feature is particularly valuable when studying complex cellular processes or conducting pooled screens, as it allows the identification of cells that exhibit specific phenotypic changes resulting from gene disruption.Finally, SLICE can be applied to a wide range of primary cell types, allowing investigations into diverse biological systems and diseases. 35,108variety of selection indicators have been applied to T cells and CAR-T cells.In vitro indicators include the proliferation, 35,95,96,100,103,105,106,108 protein expression level, 93,102,104,107 cytokine production level, 105,94 tumor killing efficacy, 107 differentiation, 95 oxidative stress, 95 and genomic stress.95 Meanwhile, in vivo screens have been carried out to provide useful information regarding the migratory tendency of T and CAR-T cells by measuring their cell number in tumor-infiltrating lymphocytes (TILs), 97 tumors, 98,99 and circulation.101 Some projects have applied multiple screens and considered several phenotypic qualities together.95,105 The combination of multiple selection indicators may lead to more accurate and powerful screens in the future.
Up to now, numerous studies have identified essential genes and proteins to improve the functions and anti-tumor reactivity of T and CAR-T cells: PPARG and BHLHE40 are related to TH2 gene regulation and differentiation 93 ; ARID1A, 94 SNX, 104 and LBTR 103 are related to exhaustion resistance; MAPK14, 95 FLI1, 96 MCAT5 and PDIA3, 102 RASA2, 106 PRODH2, 107 and MED12 or CCNV 105 are related to anti-tumor efficacy; REGNASE-1 is related to BATF function 97 ; DHX37 is related to T cell activation and cytokine production 93 ; PTPN2 is related to CD8 + T-cell-mediated responses to viral infection 100 ; ST3GAL1 is related to the tumor-specific CAR-T cell migration 101 ; USP22 and RNF20 are related to stabilized expression of Foxp3; and DGKA, 35 ACTR6, and RCOR1 108 are related to cell division.
Moreover, it is important to acknowledge the heterogeneity observed among different cancer types.For example, CAR-T cell killing requires the IFN-gR pathway in solid but not liquid tumors. 32By using diverse cancer models as selection approaches, we can specifically design CAR-T cells targeting particular cancers, thereby enhancing both the targeting specificity and therapeutic efficacy of CAR-Tcell-based therapies.9]107 In the future, models of more cancer types are to be explored to gain a complete landscape of cancer immunotherapy.At the same time, it is worth noting that all the screens including cancer models use cell lines to carry out kill assays and other assays.Considering the differences between primary cell culture and cell lines, incorporation of primary tumor cells in future screenings may achieve a more accurate simulation of the interaction between immune cells and tumor cells, thus advancing our understanding of cancer therapy.

CRISPR screens in NK cells
NK cells have emerged as important players in cancer immunotherapy, offering unique capabilities to recognize and eliminate tumor cells without prior sensitization. 66However, several limitations hinder their therapeutic potential, including difficulties in infiltrating solid tumors, 109 alterations in NK activating receptors and ligands within the tumor environment, 110 and their limited lifespan.To overcome these challenges, CRISPR screen techniques have been used to unravel the intricate interactions between NK cells, tumors, and other immune cells, leading to the development of more effective NK cell products.Current CRISPR screens in NK cells primarily use lentivirus transduction and Cas RNP nucleofection methods (Table 3).For instance, using IL-2-dependent NK cell line NK-92 and a self-designed CRISPR library, Huang et al. 111 identified the activation of FCGR3A and CD226 genes enhanced the cytotoxicity of NK cells.
In a recent study, Peng et al. 112 performed perturbomics mapping of tumor-infiltrating NK cells by in vivo adeno-associated virus (AAV)-CRISPR screens in four distinct mouse models of melanoma, breast cancer, pancreatic cancer, and glioblastoma.The CRISPR screens identified CALHM2, a regulator of calcium homeostasis, and showcased substantial efficacy enhancements both in vitro and in vivo upon perturbing CALHM2 in CAR-engineered NK cells. 112

CRISPR screens in other immune cells
In addition to NK cells, macrophages, dendritic cells (DCs), and monocyte-derived DCs (moDCs) contribute significantly to tumor targeting and immunosurveillance (Table 3).Macrophages phagocytose tumor cells and stimulate immune responses through antigen presentation, while DCs and moDCs capture and present tumor antigens to activate T cells.These immune cells work synergistically to mount effective anti-tumor responses, highlighting the importance of understanding their interactions for comprehensive cancer immunotherapy strategies.CRISPR screen studies using lentivirus transduction have focused primarily on TNF-a secretion pathways in macrophages and moDCs, revealing the roles of METTL3-mediated m6A modification of Irakm mRNA and the KO of TLR2 and PTPN6. 113,115DC studies, using retrovirus transduction, have emphasized antigen cross-presentation, uncovering the impact of Wdfy4 loss on cDC1s in mice. 114However, challenges persist, such as the absence of sgRNA barcodes for pooled screens in DCs.This can be addressed by adapting lentiviral sgRNA delivery and Cas9 electroporation for pooled screening.Additionally, targeting highly homologous genes with CRISPR can be improved through enhanced sgRNA design using computational tools and exploring alternative genome editing techniques.Resolving these limitations will contribute to a better understanding of immune cell interactions and facilitate the development of comprehensive cancer immunotherapy strategies. 114ww.moleculartherapy.org

Review CURRENT LIMITATIONS AND OPTIMIZATIONS OF CRISPR SCREEN
Off-target effects CRISPR screens are prone to unintended genetic modifications resulting from off-target activity of the CRISPR-Cas system, leading to false-positive or false-negative outcomes. 1179][120][121] However, the impact of offtarget activity on gene-targeting growth screens is believed to be minimal for CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa). 122CRISPRi and CRISPRa are transcription regulation model that use nuclease-dead Cas9, a nuclease-deficient variant of the Cas9 protein.dCas9 has the same efficiency as Cas9 in binding to specific genomic regions, but it is incapable of creating a double-stranded break at the binding site.In practice, dCas9, when fused with transcriptional repression or activation domains, is recruited to the transcription start site of the target gene to either repress or activate its transcription.Essentially, CRISPRi and CRISPRa introduce reversible transcriptional control without genetically altering the target sequence by recruiting transcription factors artificially.Furthermore, off-target effects may pose more challenges in non-coding screens compared with gene screens. 123vancements in CRISPR technology aim to enhance specificity and reduce off-target effects through the implementation of improved design strategies and delivery methods.This entails the development of more precise gRNA design algorithms, Cas variants with diminished off-target activity, refined delivery approaches, and exploration of alternative CRISPR enzymes that do not strictly require a  cDC1, classical dendritic cell; ELOVL1, elongation of very long chain fatty acids protein 1; FCGR3A, Fc gamma receptor IIIa; IL-2, interleukin-2; METTL3, methyltransferase 3; moDC, monocyte-derived dendritic cell; NHLRC2, NHL repeat containing protein 2; PTPN6, protein tyrosine phosphatase non-receptor type 6; TLR2, Toll-like receptor 2; TM2D3, TM2 domain containing 3; WDFY4, Wdfy family member 4.

Incomplete KO efficiency
CRISPR screens may exhibit incomplete KO efficiency, which can arise from various factors, including suboptimal gRNA design, limited Cas9 activity, inefficient delivery of CRISPR components, or inherent biological constraints in achieving complete gene disruption. 131,132The consequence of incomplete KO efficiency is the potential masking or underestimation of the functional impact of the targeted gene, leading to possible false-negative outcomes in the screen.
To address the issue of incomplete KO efficiency, researchers use several strategies.Optimizing gRNA design to enhance targeting specificity and efficiency is a common approach. 12,130Using high-fidelity Cas9 variants with improved editing precision and using delivery methods that ensure effective transfer of CRISPR components into the target cells are also essential. 125,133Enhancing the efficiency of CRISPR screening can be achieved by engineering the target cells to express the CRISPR-Cas protein, allowing ectopic delivery of only the gRNAs during the screen. 12The inherent low efficiency of large-scale pooled screens in non-immortalized primary cells (e.g., T cells, NK cells, monocytes), which have limited culture expansion potential, posed a significant hurdle. 35To overcome this limitation, a hybrid system was developed using lentivirus-mediated delivery of traceable sgRNA cassettes, followed by electroporation with Cas9 protein, enabling efficient genetic perturbation in primary immune cells. 35,134Validation experiments play a crucial role in mitigating incomplete KO efficiency.Lentiviral transduction of gRNAs targeting test loci, 135,136 followed by evaluating editing efficiency at the DNA level using targeted DNA sequencing, or at the protein level using western blotting or flow cytometry, can provide valuable insights. 137,138Furthermore, using complementary approaches, such as RNAi or small-molecule inhibitors, serves to validate the functional relevance of candidate genes identified through CRISPR screens. 139ddressing incomplete KO efficiency is of utmost importance to ensure the accuracy and reliability of CRISPR screens, as it directly influences the interpretation of gene functions and their impact on cellular processes or disease phenotypes (Figure 3B).

Limited coverage of non-coding regions
More than 98% of human genes consist of noncoding regions, which have been discovered to play a crucial role in gene expression regulation and are associated with 90% of diseases and trait-associated variants. 140,141Currently, in adoptive immunotherapy, CRISPR screens predominantly focus on protein-encoding genes, while the noncoding regions remain relatively unexplored, which results in an incomplete capture of the genetic factors that influence the proliferation, infiltration, and cytotoxicity of immune cells, including CD8 + T cells and NK cells.There are technical challenges involved in conducting effective CRISPR screens on the noncoding genome.The primary issue per-tains to the construction of sgRNA libraries.Given the vast size of the noncoding genome, no available method exists for genomewide, unbiased, and saturated perturbation.Current techniques either enable saturation of a specific locus through CRISPR perturbations or target pre-identified regions on the basis of specific genomic features. 142Additionally, some non-coding regions are relatively small, such as 5-10 bp transcription factor binding sites, requiring precise mutagenesis at specific locations. 143,144Without precise mutagenesis sites, CRISPR screens can only attribute regulatory functions to extended non-coding regions, while the detailed regulatory mechanisms within each region remain unknown.
Despite these limitations, CRISPR screening, as a high-throughput method, serves as an exceptional tool for the functional characterization of the noncoding genome.Future efforts could be directed toward developing improved CRISPR libraries that facilitate large deletions and genetic interaction studies by using paired sgRNA. 144urthermore, the integration of powerful technologies such as single-cell sequencing, cytometry by time of flight (CyTOF), and cellular barcoding into the screening process holds great potential 144 (Figure 3C).

Functional redundancy and compensatory mechanisms
Genes involved in complex biological processes often exhibit functional redundancy or compensatory mechanisms, in which genes have homologous counterparts that perform identical or analogous functions within the genome, or the expression of alternative proteins in the pathways is stimulated. 145,146Consequently, when conducting CRISPR screens to knock out or activate a single gene, the complete functional impact may not be fully understood, as other genes can compensate for the loss or gain of function. 147,148veral methods have been demonstrated to mitigate the redundancy or compensation effects, thereby improving the reliability and efficiency of CRISPR screening.Validating the screening outcomes after identifying gene candidates is the initial crucial step in preventing potential effects, as it confirms the exclusive influence of perturbing the candidate gene on phenotypes. 149This validation process can be achieved through independent CRISPR disruption, confirmation at the protein level, and subsequent rescue experiments. 150,1513][154] By using these diverse approaches, researchers can enhance the robustness of CRISPR screening, enabling a comprehensive understanding of gene function and its impact on phenotypes (Figure 3D).

Technical challenges in library construction and delivery
Generating high-quality CRISPR libraries and effectively delivering them to target cells present technical challenges that can affect the reproducibility and outcomes of screening experiments.The variability in library construction and delivery methods contributes to www.moleculartherapy.orgReview these challenges.One specific challenge in CRISPR screens is the capture of gRNAs. 12,1557][158] To overcome this, two approaches can be considered.First, modifying gRNAs by adding a polyadenylated tail allows their capture.0][161] Alternatively, gRNAs can be directly captured and amplified, enabling the assessment of their expression and its association with the expression of target genes. 161,162other challenge in CRISPR library construction is ensuring library complexity.Designing a comprehensive and diverse gRNA library requires careful consideration to cover the entire genome or specific gene sets of interest.Precise design and optimization are essential to ensure sufficient representation of target genes.Additionally, addressing library representation bias, where certain gRNAs are overrepresented or underrepresented, is crucial to avoid potential biases in the screening Overcoming these challenges is essential to achieve accurate and comprehensive outcomes in CRISPR screens 163 (Figure 3E).

SYNERGISTIC INTEGRATION OF CUTTING-EDGE TECHNOLOGIES WITH CRISPR SCREEN
In the realm of CRISPR screen, the advent of base editors and prime editors has heralded a transformative era.Base editors, distinguished by their surgical precision, facilitate an in-depth exploration of gene function by instigating specific point mutations.This capability permits the dissection of nucleotide-level contributions to phenotypic variations. 164Conversely, prime editing empowers researchers to conduct intricate genome-wide screens, offering the precise modification of genes, promoters, and non-coding elements with unmatched efficiency and minimal off-target effects. 27These technologies are catalyzing a paradigm shift in genetic screening, endowing us with unprecedented versatility and accuracy.Moreover, within this section, we delve into a range of advanced techniques, encompassing multi-omics integration, in vivo and tissue-specific CRISPR screens, single-cell technology integration, high-throughput screening platform development, machine learning and data analysis tool integration, and the synergy of next-generation off-the-shelf cell therapies.These innovations collectively redefine the landscape of CRISPRbased screening methods.

Integration of multi-omics approaches
Numerous state-of-the-art technologies have emerged as synergistic counterparts to CRISPR screens, facilitating the identification of novel tumor targets and augmenting the effectiveness of immunotherapies (Figure 4).Recent advancements in technology, particularly the emergence of CRISPR screen, have revolutionized the analysis of biological systems at multiple levels.These levels encompass various aspects such as DNA sequence data (genomics), RNA expression levels (transcriptomics), expression regulation levels (epigenomics), protein interaction levels (proteomics), and metabolite levels (metabolomics). 165Integrating these diverse biological datasets through multi-omics studies allows a comprehensive understanding of complex diseases such as cancer from a holistic perspective.Unlike investigations focused on a single omics level, multi-omics studies leverage information across different biological activities, providing unparalleled insights into disease mechanisms.To further enhance the efficiency and integration of CRISPR screen with other subfields of biological research, future endeavors aim to establish a unified framework connecting various tools and methodologies for data collection. 166 vivo and tissue-specific CRISPR screens Traditionally, CRISPR screens are carried out in an ex vivo environment.The integration of CRISPR editing into in vivo studies of cancer immunology can generate a more faithful model of cancer by tracking the interactions between tumor and immune cells in a tissue microenvironment. 41Loss-of-function and gain-of-function CRISPR screens have been implemented in somatic cells, with CRISPRa and CRISPRi used together. 167These applications provide the foundation for establishing patient-specific models, leading to precise medicine in the future.Notably, recent studies by Bahrami et al. 168,169 and Wirth et al. 168,169 have demonstrated the feasibility and potency of in vivo CRISPR-Cas9 functional screening in models of AML and chemoresistance, unveiling crucial genes such as BCL2, BRIP1, and COPS2 that govern therapeutic outcomes.
Two models exist for in vivo CRISPR screens, indirect in vivo (transplant-based) and direct in vivo (autochthonous) screens.Indirect in vivo screen includes a transplantation of the mutagenized cell pool into a recipient animal, often subcutaneously, while direct in vivo screening requires delivery of CRISPR components to the target organ site through two delivery systems: viral vectors (e.g., AAVs, lentiviruses, and tissue-tropic viruses) and non-viral systems (e.g., nanoparticles, microinjection, electroporation). 167,170Autochthonous screening, while providing a more authentic representation of physiological contexts by targeting specific organs, faces challenges due to lower delivery and infection efficiency.Consequently, current direct in vivo screening is constrained by limitations in the scale of sgRNA library sizes that can be practically used.

Integration of single-cell technologies
The combination of CRISPR screens with single-cell sequencing technologies, commonly known as CRISP-seq or Perturb-seq, offers a powerful approach for assessing gene function at the single-cell level. 154,155,160This method enables simultaneous characterization of gene perturbations and transcriptome profiling. 155With regard to immunotherapy, this technique allows precise identification of specific gene targets and gene signatures associated with anti-tumor or pro-tumor effects in tumor cells or effector cells, revealing fluctuations in cellular states in response to individual gene perturbations and corresponding genetic interactions. 154,155,160Ultimately, this integrative approach provides valuable insights into cellular heterogeneity, facilitates identification of rare cell populations, and elucidates www.moleculartherapy.orgReview cell-specific gene regulatory networks.identify rare cell populations, and elucidate cell-specific gene regulatory networks. 155

Development of high-throughput screening platform
2][173] For instance, gRNA libraries have been refined to target coding and regulatory regions of drosophila, mouse, and human genomes with exceptional precision, while minimizing off-target effects.Multiple techniques including lentivirus transduction and lipid nanoparticle-loaded CRISPR complexes offer diverse options for gRNA delivery approaches. 172The enhanced high-throughput screening capacity, therefore, facilitates the investigation of larger gene sets, drug libraries, and complex genetic interactions, propelling the field of CRISPR-based research.

Integration of machine learning and data analysis tools
Machine learning algorithms and advanced data analysis techniques are vital for extracting meaningful information from large-scale CRISPR screen datasets.Integrating computational approaches enhances interpretation, prediction, and prioritization of gene function and therapeutic targets.Currently, using machine deep learning (MDL)-based CRISPR techniques, alongside tools like DESeq2, edgeR, and CERES (Table 4), 175,176 enables advanced feature selection, accurate classification and prediction of on-target activity, and interpretable visualization of screening results.These advancements drive progress in understanding gene function and potential therapeutics.
Combination of next-generation off-the-shelf cell therapy CRISPR screen techniques have emerged as a valuable tool for identifying and studying crucial genes involved in cancer immunotherapy. 27This knowledge can be applied to engineer advanced offthe-shelf cell therapies with improved functionality.Strategies such  www.moleculartherapy.org[186][187][188][189][190]

CONCLUSION
2][193][194][195] Despite the significant progress made, there are certain limitations that hinder the achievement of maximum efficacy in cancer immunotherapy.7][198] Consequently, there is an urgent need to develop novel technologies that can effectively overcome these existing limitations and swiftly address the prevailing difficulties encountered in cancer immunotherapy.Such advancements have the potential to transform our understanding of tumor immunology at a profound level and reshape the current landscape of cancer treatment paradigms. 179,199ISPR screen technology has emerged as a pivotal tool in understanding the genetic basis of diseases, including cancer, and has revolutionized the field of cancer immunotherapy.This review offers a comprehensive overview of the applications and impact of CRISPR screen technology within the realm of cancer immunotherapy.

Figure 1 .
Figure 1.The chronological progression of CRISPR screens in cancer immunotherapy research

Figure 2 .
Figure 2. CRISPR screen overview (A) Schematic showing the CRISPR screen workflow.A genome-wide CRISPR library was constructed and introduced into the target cells via transduction.Various selection assays, including in vitro, in vivo, or other methods, were used to manipulate the cellular composition.Subsequently, the identification of specific genes of interest was achieved through the isolation of genomic DNA and barcode sequencing.Further experimental validation was conducted to investigate and characterize the functional role of the identified genes.(B) Current or potential cell types that could be targeted by CRISPR screen.(C) Three transduction approaches used in CRISPR screen.(D) Four major types of CRISPR gene editing strategies.(E) Selection models involved in the CRISPR screen procedure.HSPC, hematopoietic stem and progenitor cell; iPSC, induced pluripotent stem cell; AAV, adeno-associated virus; CRISPRa, CRISPR activation; CRISPRi, CRISPR interference; KO, knockout.

Figure 3 .
Figure 3. Current limitations and optimizations of CRISPR screenThis schematic provides a concise overview of the primary constraints encountered in CRISPR screen technology.These include the propensity for off-target effects resulting from the CRISPR-Cas system (A), incomplete gene knockout efficiency due to the use of suboptimal gRNAs (B), limited coverage of non-coding regions when designing gRNAs (C), the presence of functional redundancy and compensatory mechanisms within certain genes (D), and the technical hurdles associated with library construction (E).Furthermore, the schematic delineates the corresponding remedies proposed for each of these limitations.spCas9, Streptococcus pyogenes Cas9; SaCas9, Staphylococcus aureus Cas9; StCas9, Streptococcus thermophilus Cas9; NmCas9, Neisseria meningitidis Cas9; CjCas9, Campylobacter jejuni Cas9; Cpf1, class 2 type V CRISPRassociated endonuclease; LVLP, large viral-like particles; KO, knockout; eSpCas9, enhanced Streptococcus pyogenes Cas9; HypaCas9, hyper-accurate Streptococcus pyogenes Cas9; HiFi Cas9, high-fidelity Streptococcus pyogenes Cas9; TSS, transcription start site; OE, overexpression; KD, knockdown; AA, amino acid.

Figure 4 .
Figure 4. Synergistic integration of other technologies with CRISPR screen for the development of next-generation cancer immunotherapy /CROP-seq combinations of CRISPR screens with single-cell sequencing technologies supporting large-scale gene perturbation studies, and allowing the evaluation of gene functions at the single-cell level differential gene expression analysis on the basis of the negative binomial distribution DESeq2 an R/Bioconductor software package used for analyses of comparative RNA-seq data by using shrinkage estimators for dispersion and fold change improving the sensitivity and precision of screen results, leading to the emphasis on the strength of differential expression Empirical analysis of digital gene expression (DGE) in R edgeR an R/Bioconductor software package used for evaluating variations in gene expression on the basis of replicated count data enhancing the reliability of inference, and allowing analyses at the most minimal levels of replication CERES CERES a computational approach for examining genedependency levels with CRISPR-Cas9 essentiality screens while considering the copy numberspecific impact reducing false-positive rates of screens gRNA, guide RNA; sgRNA, single-guide RNA.

Table 1 .
CRISPR screens in tumor cells to identify new targets for cancer immunotherapy (Continued on next page) Molecular Therapy: Oncolytics Vol. 31 December 2023 www.moleculartherapy.orgReview

Table 2 .
CRISPR screens in T cells to develop advanced T-cell-based products for cancer immunotherapy

Table 3 .
CRISPR screens in other immune cells to enhance their anti-tumor reactivity

Table 4 .
Overview of technologies mentioned By enabling systematic exploration of gene function, CRISPR screening has brought about a paradigm shift in functional genomics research.Leveraging the precision and adaptability of the CRISPR-Cas9 system, researchers can delve into the intricate complexities of gene networks and gain novel insights into biological processes and disease mechanisms.The continued refinement and application of CRISPR screening methodologies hold immense promise in unraveling new discoveries in genetics and propelling innovative therapeutic interventions to the forefront.