Screeningand Validationof Small Moleculesto Improvethe Efficiencyof Precise Gene Editing Mediatedby CRISPR/Cas9

CRISPR/Cas9 can cleave DNA double-strands with high specificity,and small molecule compounds can enhance the accuracy of CRISPR/Cas9-mediated gene editing by inhibiting Non-homologous end joining(NHEJ)or promoting Homology directed repair(HDR)during DNA repair.Previous studies have shown that small molecule compounds that improve the efficiency of precise gene editing mediated by CRISPR/Cas9 have species-specific and cell-specific characteristics.In this study,we screened and functionally verified small molecule compounds that can enhance the accuracy of CRISPR/Cas9-mediated gene editing in pigs.1)Eight small molecule compounds were selected for the experiment: AZD7762,SCR7,VE-822,Brefeldin A,RS-1,NU7441,L-189,and L755507.Among them,L-189,SCR7,and NU7441 inhibit Non-homologous end joining during DNA repair,while RS-1,Brefeldin A,L755507,VE-822,and AZD7762 enhance Homology directed repair during DNA repair.In this study,the optimal concentrations of the added small molecules were screened based on their cytotoxicity on cells.The results showed that the optimal concentrations of small molecules for in vitro cultured pig PK15 cells were: 0.5 μM for Brefeldin A;10 μM for SCR7;5 μM for L-189;2 μM for AZD7762;2 μM for VE-822;1 μM for NU7441;5 μM for L755507;and 10 μM for RS-1.2)Based on the CRISPR/Cas9 system and the precise gene editing system mediated by single-stranded DNA templates modified with thiol groups,eight different small molecules were added to screen and verify small molecules that could enhance precise gene editing efficiency in pigs.The results showed that compared with the control group,L755507 increased the precise gene editing efficiency by 1.91 times,SCR7 increased it by 1.89 times,RS-1 increased it by 2.10 times,L-189 increased it by 1.71 times,NU7441 increased it by 2.28 times,Brefeldin A increased it by 1.87 times,AZD762 increased it by 1.43 times,and VE-822 increased it by 1.46 times.3)Based on the CRISPR/Cas9 system and the precise gene editing system mediated by single-stranded DNA templates modified with thiol groups,small molecule combinations that enhance Homology Directed Repair and inhibit Non-Homologous End Joining were screened to further improve the efficiency of precise gene editing.The results showed that compared with the control group,the combination of SCR7+L755507 increased the precise gene editing efficiency by 1.90 times,the combination of SCR7+RS-1 increased it by 1.99 times,the combination of SCR7+Brefeldin A increased it by 1.87 times,the combination of L-189+L755507 increased it by 1.81 times,the combination of L-189+RS-1 increased it by 1.89 times,the combination of L-189+Brefeldin A increased it by 1.79 times,the combination of NU7441+L755507 increased it by 1.98 times,the combination of NU7441+RS-1 increased it by 2.11 times,and the combination of NU7441+Brefeldin A increased it by2.03 times.In this study,small molecules that improve the efficiency of CRISPR-mediated precise gene editing in pigs were screened and verified,providing methodological support for the establishment of an efficient and precise gene editing system in pigs.