Enhancement Of CRISPR-mediated Homologous Targeting Efficiency By Small Molecules In Porcine Cells

The double strand break formation and following homology-directed repair(HDR)process mediated by the CRISPR-Cas system at the target site of genome is the gene-editing technology for animals,which has great research and application value.The CRISPR-Cas system is a potentially revolutionary new tool in the field of gene editing that enables efficient and precise editing of genomes.Cas9 effector is currently the most widely used editing system in the Cas family,and Cas12a(Cpf1)is also developed and used for its advantages over Cas9 system.The two effectors can edit DNA with abundant AT and CG content,respectively.Genome-edited pigs have many uses in the field of medical agriculture,but due to the low efficiency of HDR,the work of constructing transgenic modified cell lines and modified animals is too inefficient and time consuming.In order to improve the HDR efficiency in pig genome,this study used porcine fetal fibroblasts(PFF)and porcine kidney(PK-15)cells as research objects.We used ATR inhibitors VE-822 and BAY-1895344 to treat cultured cells,and their effects on HDR efficiency and molecular mechanism were studied by fluorescent reporter vector with different repair methods mediated via CRISPR-Cas9 or Cpf1.The main findings are as follows:(1)We established the a CRISPR-Cpf1/Cas9-based GFP reporter vector,which can quickly detect HDR efficiency and facilitate the screening of small molecule compound for enhancing HDR pathway mediated by CRISPR-Cpf1/Cas9.(2)We transfected PFF and PK-15 cells using CRISPR-Cas9 or CRISPR-Cpf1 system,and tested the integration of EGFP fluorescent reporter gene into porcine ?-actin and GAPDH genomes by HR,HMEJ,MMEJ repair pathways.We screened that ATR inhibitors VE-822 and BAY-1895344 were active in enhancing HDR,with an 1-3 folds increased HDR rates in porcine cells,and no toxic effects were observed.This result indicates that ATR inhibitors VE-822 and BAY-1895344 can be used to promote the efficiency of precise genetic modification in cell lines and establish a precise modified genetic pig model.By comparing the HDR efficiencies induced by the Cas9 and Cpf1 systems,we found that following VE-822 and BAY-1895344 treatment,Cpf1 could enhance HDR more effective than that of Cas9,and promoting effect in PFF cells was more pronounced than that in PK-15 cells,indicating that these ATR inhibitors are affected by many factors in promoting HDR efficiency.(3)Through cell cycle and real-time fluorescence quantitative detection,we found that after treatment of PFF cells with VE-822 and BAY-1895344,the number of cells in G1 phase decreased and the number of cells in S and G2 phases increased;and the expression levels of ATR gene,Rad51 gene and DNA-PK gene were significantly up-regulated.These results imply that enhanced HDR efficiency by ATR inhibition is not due to NHEJ inhibition,but related to the cell cycle arrest,and enhance gene expression in HDR pathways.