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

This study is led by Professor Jian-Feng Li(School of Life Sciences, Sun Yat-sen University, Guangzhou, China). By leveraging the dual functions of a D-amino acid oxidase from Trigonopsis variabilis (TvDAO),the authors develop a dual-function selection system to enable bothpositive selection of multigene CRISPR mutants and negative selection of Cas9-free progeny in Arabidopsis. To utilize a DAO-based surrogate selection marker forCRISPR editing, the authors first evaluatedthe catalytic activity of five homologous DAOs in Escherichia coli, finding that the TvDAOcould confer slightly stronger D-serine resistance than other homologs. Furthermore, through transgenic expression of TvDAO in Arabidopsis and D-amino acid-based screening,the authors validated thatTvDAO could be employed for both D-serine-conditioned positive selection and D-valine-conditioned negative selectionof transgenic Arabidopsis plants. As a proof of concept, the authors 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, the authors demonstrated that D-valine-based negative selectionsuccessfully removed Cas9 and TvDAO transgenes from the survival offspring carrying inheritedmutations.

In summary, this work provides aninnovative 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.

See the article:

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

https://link.springer.com/article/10.1007/s42994-023-00132-6