农业生物技术学报2024，Vol.32Issue(10)：2306-2323,18.DOI:10.3969/j.issn.1674-7968.2024.10.010
HDACi和RS-1提高CRISPR/Cas12i介导的HDR编辑效率
HDACi and RS-1 Enhance CRISPR/Cas12i-mediated HDR Editing Efficiency
摘要
Abstract
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.关键词
CPRISPR/Cas12i/同源重组/RS-1(C20H16Br2N2O3S)/组蛋白去乙酰化酶抑制剂(HDACi)/绵羊Key words
CPRISPR/Cas12i/Homologous recombination/RS-1(C20H16Br2N2O3S)/Histone deacetylase inhibitor(HDACi)/Sheep分类
农业科技引用本文复制引用
陈秋崇,李尚朴,苗洱钰,周冰倩,王旭,孟祥宇,王小龙,徐坤..HDACi和RS-1提高CRISPR/Cas12i介导的HDR编辑效率[J].农业生物技术学报,2024,32(10):2306-2323,18.基金项目
国家自然科学基金(32301251 （）
32272848) （）
现代农业产业体系(CARS-39-03) （CARS-39-03）
陕西省地方助学金项目(2022GD-TSLD-46) （2022GD-TSLD-46）
国家级大学生创新创业训练计划(202210712099) （202210712099）
