MicroRNA-responsive release of Cas9/sgRNA from DNA nanoflower for cytosolic protein delivery and enhanced genome editing
Introduction
The development of CRISPR/Cas9 system has revolutionized the field of genome editing for various biology and biomedical applications [1]. Guided by a single-guide RNA (sgRNA), CRISPR-associated protein 9 (Cas9) is able to induce double-strand DNA break at specific genomic sequences [2,3]. For therapeutic application, safe and effective delivery of CRISPR/Cas9 system into the nucleus of specific cells is essential for efficient genome editing and reducing off-target events [[4], [5], [6], [7]].
Viral systems have been used as a first resort to transduce CRISPR systems to cells [8]. However, these systems may cause constant Cas9/sgRNA expression, which favors off-target events [9]. Besides, viral vectors are with high immunogenicity, thus not ideal for delivery of CRISPR systems [10]. Nanoparticles, which can deliver Cas9/sgRNA as ribonucleoprotein complex, have transient functional window, which favors low frequencies of off-target events [11]. Moreover, nanoparticles can be chemically functionalized with ligands to achieve targeting specificity [11,12]. For example, R. Shahbazi et al. have developed a CRISPR nano-formulation based on colloidal gold nanoparticles for non-toxic delivery of entire CRISPR payload into cells [13]. In another case, a self-assembled DNA nanoclew has been developed for efficient delivery of CRISPR/Cas9 [14].
Although gold nanoparticles, polymers, MOFs and DNA nanostructures have been used to deliver Cas9/sgRNA ribonucleoprotein in a very efficient and cell-type-specific manner [8,15,16]. The process of how Cas9/sgRNA been released from the nanoparticle has not been fully studied. Meanwhile, delivering Cas9/sgRNA in its active form into cytosol is essential to fully accomplish the genomic editing potential. Therefore, a stimuli-responsive Cas9/sgRNA release platforms are highly desired for cytosolic protein delivery and enhanced genome editing. For example, some thermo-triggered plasmid release [16] and ATP-triggered Cas9/sgRNA release strategies [17] are recently developed for efficient genome editing, which holds great potential for targeted disease treatment.
MicroRNAs (miRNAs) play essential roles in a variety of physiologic and pathologic processes by regulating related mRNAs expression [18]. Some miRNAs are specifically expressed in confined cell types, which are useful as biomarkers for diagnosis applications [19,20]. For example, it has been widely reported that miR-21 is overexpressed in tumor cells and play a causal role in the onset and maintenance of cancer [21,22]. Therefore, a CRISPR/Cas9 platform which can be turned on by specific endogenous miRNA could be an ideal tool for cell-type-specific genome-regulation [23].
Herein, we developed a DNA nanoflower (DNF) based miR-21 responsive Cas9/sgRNA ribonucleoprotein delivery system. DNA-based functional nanomaterial is an ideal tool for loading of Cas9/sgRNA complex by sequence hybridization [14,24]. Besides, DNA nanomaterials can be designed to encode DNA aptamers for specific tumor cell targeting [25] and miRNA-responsive structure for Cas9/sgRNA release by toehold-mediated strand displacement [26,27]. In this manuscript, we demonstrated that, by designing a miRNA-mediated Cas9/sgRNA release strategy using DNF, the genome editing efficiency can be significantly increased comparing with the non-release control. Moreover, this strategy makes it possible to control the expression of endogenous genes by specific endogenous or exogenous miRNAs in a cell-type-specific manner.
The mechanism was illustrated in Scheme 1. DNA nanoflowers (DNFs) were synthesized by rolling circle amplification, which contained multiple replicates of MUC1 aptamers and miR-21 binding sequences [28]. To construct a sgRNA for toehold-mediated Cas9/sgRNA release, a sequence, which is 7 nt shorter than miR-21, was added to the stem-loop of sgRNA to load Cas9/sgRNA on DNF by sequence hybridization. The DNF/Cas9/sgRNA nanoformulation would go through MUC1-mediated endocytosis, Mg2+-induced endosomal/lysosomal escape and be delivered to the cytoplasm [28,29]. The miR-21 in cytoplasm will replace Cas9/sgRNA complex from DNF by toehold-mediated sequence displacement. The released Cas9/sgRNA complex could bypass karyotheca and be transported into cell nucleus by nuclear-localization-signal (NLS) peptides fused to Cas9. By targeting the coding region of EGFP, the Cas9/sgRNA could drive the formation of indels and prevent EGFP expression.
Section snippets
Materials and apparatus
All oligonucleotides in Table S1 were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Cas9 Nuclease (S. pyogenes, EnGen Cas9 NLS), HiScribeTM T7 Quick High Yield RNA Synthesis Kit, and dNTP Mix were purchased from NEB (New England Biolabs, Ipswich, MA). T4 DNA ligase, T4 polynucleotide kinase, Phi29 DNA polymerase, and RiboLock RNase inhibitor were purchased from Life Technologies (Carlsbad, CA). NanoDrop 2000 UV–vis spectrophotometer was
Characterization of the DNF/Cas9/sgRNA nanoparticles
To construct DNF/Cas9/sgRNA nanoparticle, the DNF nano-vehicle was firstly synthesized by rolling circle amplification (RCA), which encoded multiple repeats of MUC1 aptamers and sequences complementary to miR-21. These complementary sequences would hybridize with miR-21-responsive sgRNA, and be replaced by miR-21 in cells through a toehold-mediated strand displacement reaction. In this work, we used a commercial-available and well-characterized Streptococcus pyogenes Cas9 protein fused with
Conclusion
In summary, this miRNA-responsive DNF system provides a promising strategy for direct cytosolic delivery of Cas9/sgRNA and enhanced genome editing. The inner DNF core acted not only as Cas9/sgRNA carrier with miR-21 responsive sequence, but also encoded MUC1 aptamers for tumor cell targeting and was able to trigger lysosome escape in cells. By comparing miR-21-responsive sgRNA and non-responsive control, we demonstrated that the design of Cas9/sgRNA releasing process is key for improving genome
CRediT authorship contribution statement
Jinjin Shi: Methodology, Investigation, Data curation, Writing - original draft. Xue Yang: Investigation, Methodology, Data curation. Yanan Li: Investigation. Danyu Wang: Investigation. Wei Liu: Investigation, Funding acquisition. Zhenzhong Zhang: Funding acquisition. Junjie Liu: Project administration, Funding acquisition. Kaixiang Zhang: Conceptualization, Project administration, Writing - review & editing.
Declaration of competing interest
All the authors declare no competing financial interests related to the work reported in this paper.
Acknowledgements
The work is supported by grants from the National Natural Science Foundation of China (No.21904119), Innovation Talent Support Program of Henan Province (Nos. 19HASTIT006) and Key scientific research projects (Science and Technology Department of Henan Province, No.192102310147). Thanks to the modern analysis and computing center of Zhengzhou University for technical assistance.
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These two authors contributed equally.