Development Of A Self-Restricting CRISPR-Cas9 System To Reduce Off-Target Effects

Objective: Development of the CRISPR-Cas9 gene editing system has given rise to a new era of gene editing with wide applications in biology,medicine,agriculture,and other fields.However,the overexpression of Cas9 nuclease causes off-target effects and may trigger an immune response in vivo.In order to reduce the overexpression of Cas9 protein and reach an expression mode of less is good,we constructed a self-restricting CRISPR system(SR-CRISPR)to achieve the goal of significantly reducing off-target effects without changing the efficiency of target gene editing.Methods: In this study,we selected programmed cell death protein 1(PD-1)as the target and modified the existing gene editing plasmid pX458-s3 plasmid(contains the gRNA sequence that recognizes the PD-1 gene)in the laboratory.The target gene consensus sequence corresponding to the gRNA was inserted into the Kpn I and Age I sites directly preceding and following the Cas9 promoter,respectively,to construct the selfrestricting CRISPR system(SR-CRISPR plasmid).Because the gRNA also targets the Cas9 gene promoter,transcription is terminated and overexpression is prevented.When transfected into HEK293 T cells this plasmid is designed to induce controlled expression of the Cas9 protein at a level that still allows the formation of a protein complex with the gRNA targeted to PD-1.Thus,this should achieve the goal of reducing off-target effects,while at the same time allowing the Cas9-gRNA complex to cut the PD-1 gene at the target site to form the indel mutation.To verify that the SR-CRISPR system inhibits overexpression of Cas9 protein,we transfected the unmodified px458-s3 plasmid(conventional CRISPR)and the modified SR-CRISPR plasmid(SR-CRISPR system)into HEK293 T cells.The following experiments were performed.1.The confocal microscope was used to observe the expression of EGFP,and the difference in intracellular fluorescence density between the two was analyzed.2.Total RNA and protein of cells were extracted at different time points(12h,18 h,24h,48 h,60h)after transfection,and Cas9 gene expression was detected by Real time PCR and Western blot.3.Primers were designed for the cleavage site of the promoter,and Real Time PCR and high-throughput sequencing were performed to analyze the cleavage efficiency and Indel of the promoter.4.The genome was extracted 60 hours after transfection.Primers were designed on both sides of the target gene and the cutting site that may be off-target.PCR was used to build the library.High-throughput sequencing was used to analyze editing efficiency(off-target efficiency)for target genes and off-target sites.Results: 60 hours after transfection of cells,the Cas9 protein expression in the SR-CRISPR group was only 10% of the wild-type system,and the promoter cut efficiency was about 70%.The SR-CRISPR system's editing efficiency for target genes and homologous recombination efficiency were respectively about 50% and 30%,no significant difference in editing efficiency with the traditional CRISPR group;In this paper,an off-target site was confirmed experimentally.Compared with the conventional CRISPR system,the Indel frequency of the SR-CRISPR system for this off-target site was reduced by 76.7%.In addition,The type of Indel was also significantly reduced(from 13 to 2 in the traditional CRISPR group).Conclusion: The SR-CRISPR system can reduce the overexpression of Cas9 protein in the nucleus.Further,we verified that with this system not only is the editing efficiency of target gene unchanged,but off-target effects are also significantly reduced.