Enhanced editing efficiency in Arabidopsis with a LbCas12a variant harboring D156R and E795L mutations  

作　　者：Cuiping Xin Dexin Qiao Junya Wang Wei Sun Zhenghong Cao Yu Lu Yuanyuan Jiang Yiping Chai Xue-Chen Wang Qi-jun Chen 

机构地区：[1]State Key Laboratory of Plant Environmental Resilience,College of Biological Sciences,China Agricultural University,Beijing 100193,People’s Republic of China

出　　处：《aBIOTECH》2024年第2期117-126,共10页生物技术通报（英文版）

基　　金：supported by grants from the National Key Research and Development Program of China(grant no.2023YFD1202905).

摘　　要：Cas12a(Cpf1),a Class 2 Type V CRISPR/Cas nuclease,has several unique attributes for genome editing and may provide a valuable alternative to Cas9.However,a low editing efficiency due to temperature sensitivity and insufficient cleavage activity of the Cas12a nuclease are major obstacles to its broad application.In this report,we generated two variants,ttAsCas12 Ultra and ttLbCas12a Ultra harboring three(E174R,M537R,and F870L)or two(D156R and E795L)mutations,respectively,by combining the mutations from the temperature-tolerant variants ttAsCas12a(E174R)and ttLbCas12a(D156R),and those from the highly active variants AsCas12a Ultra(M537R and F870L)and LbCas12a Ultra(E795L).We compared editing efficiencies of the five resulting Cas12a variants(LbCas12a,ttLbCas12a,ttLbCas12a Ultra,AsCas12a Ultra,and ttAsCas12 Ultra)at six target sites of four genes in Arabidopsis(Arabidopsis thaliana).The variant ttLbCas12a Ultra,harboring the D156R and E795L mutations,exhibited the highest editing efficiency of all variants tested in Arabidopsis and can be used to generate homozygous or biallelic mutants in a single generation in Arabidopsis plants grown at 22 C.In addition,optimization of ttLbCas12a Ultra,by varying nuclear localization signal sequences and codon usage,further greatly improved editing efficiency.Collectively,our results indicate that ttLbCas12a Ultra is a valuable alternative to Cas9 for editing genes or promoters in Arabidopsis.

关 键 词：Genome editing CRISPR/Cas Cas12a ttLbCas12a ultra ARABIDOPSIS 

分 类 号：Q94[生物学—植物学]