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

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.