HDACi和RS-1提高CRISPR/Cas12i介导的HDR编辑效率OA北大核心CSTPCD

CRISPR/Cas12i is a recently developed CRISPR gene editing system by Chinese scholars,which has independent intellectual property rights and has been proven to have targeting efficiency comparable to the CRISPR/Cas9 system.Homology-directed repair(HDR)is one of the main repair mechanisms for double-stranded DNA breaks(DSBs).Gene editing based on the HDR mechanism can be used to correct any form of mutation in the genome,but it is often limited by the generally lower HDR efficiency in mammalian cells.In this study,the activity of the CRISPR/Cas12i system at different target sites in Homo sapiens embryonic kidney cell line HEK293T,the optimal dosage of single-stranded oligonucleotides(ssODN)donor templates for mediating HDR editing,and the appropriate concentration of small molecule drugs were verified through single-strand annealing(SSA)reporter experiment,dose gradient and concentration gradient experiment.The effects of adding different small molecule drugs on the HDR editing efficiency mediated by the CRISPR/Cas12i system in HEK293T cells and sheep(Ovis aries)fetal fibroblasts were then evaluated using flow cytometry sorting,genomic PCR,Sanger sequencing,and online prediction tools.The results showed that the CRISPR/Cas12i system exhibited high activity at 18 different target sites in HEK293T cells,with efficiencies around 80%,except 2 slightly lower sites.Different dosages and lengths of ssODN had some influence on HDR efficiency,and the appropriate concentrations of small molecule drugs varied slightly in different species and cell types.The addition of histone deacetylase inhibitor(HDACi)and RS-1(C20H16Br2N2O3S)significantly enhanced the HDR editing efficiency mediated by the CRISPR/Cas12i system in both HEK293T cells and sheep fetal fibroblasts.Among them,RS-1 showed the least cytotoxicity and did not significantly reduce the insertion/deletion mutation(InDel)efficiency while improving HDR efficiency.Furthermore,Entinostat increased HDR editing efficiency by approximately 148 fold at the bone morphogenetic protein receptor 1B(BMPR1B)site in sheep fetal fibroblasts.In conclusion,the CRISPR/Cas12i system exhibited high activity and could mediate efficient and precise HDR editing using ssODN as a donor in model cells and primary sheep cells.The appropriate concentrations of HDACi and RS-1 could effectively improve HDR editing efficiency.This study provides reference and guidance for the application and popularization of the CRISPR/Cas12i gene editing system.