A dual-function selection system enables positive selection of multigene CRISPR mutants and negative selection of Cas9-free progeny in Arabidopsis  

作　　者：Feng-Zhu Wang Ying Bao Zhenxiang Li Xiangyu Xiong Jian-Feng Li 

机构地区：[1]State Key Laboratory of Biocontrol,Guangdong Provincial Key Laboratory of Plant Resources,School of Life Sciences,Sun Yat-sen University,Guangzhou 510275,China

出　　处：《aBIOTECH》2024年第2期140-150,共11页生物技术通报（英文版）

基　　金：supported by the National Key Research and Development Program of China(grant 2019YFA0906202);J.-F.L.,the National Natural Science Foundation of China(grants 31900305 and 32370294);the Natural Science Foundation of Guangdong Province(grant 2020A1515010465)to F.-Z.W.

摘　　要：The CRISPR/Cas9 technology revolutionizes targeted gene knockout in diverse organisms including plants.However,screening edited alleles,particularly those with multiplex editing,from herbicide-or antibiotic-resistant transgenic plants and segregating out the Cas9 transgene represent two laborious processes.Current solutions to facilitate these processes rely on different selection markers.Here,by taking advantage of the opposite functions of a D-amino acid oxidase(DAO)in detoxifying D-serine and in metabolizing non-toxic D-valine to a cytotoxic product,we develop a DAO-based selection system that simultaneously enables the enrichment of multigene edited alleles and elimination of Cas9-containing progeny in Arabidopsis thaliana.Among five DAOs tested in Escherichia coli,the one encoded by Trigonopsis variabilis(TvDAO)could confer slightly stronger D-serine resistance than other homologs.Transgenic expression of TvDAO in Arabidopsis allowed a clear distinction between transgenic and nontransgenic plants in both D-serine-conditioned positive selection and D-valine-conditioned negative selection.As a proof of concept,we combined CRISPR-induced single-strand annealing repair of a dead TvDAO with D-serine-based positive selection to help identify transgenic plants with multiplex editing,where D-serine-resistant plants exhibited considerably higher co-editing frequencies at three endogenous target genes than those selected by hygromycin.Subsequently,D-valine-based negative selection successfully removed Cas9 and TvDAO transgenes from the survival offspring carrying inherited mutations.Collectively,this work provides a novel strategy to ease CRISPR mutant identification and Cas9 transgene elimination using a single selection marker,which promises more efficient and simplified multiplex CRISPR editing in plants.

关 键 词：CRISPR Genome editing D-amino acid oxidase Cas9-free Selection marker 

分 类 号：Q94[生物学—植物学]