A dual-function selection system enables positive selection of multigene CRISPR mutants and negative selection of Cas9-free progeny in Arabidopsis
Author:
Wang Feng-Zhu, Bao Ying, Li Zhenxiang, Xiong Xiangyu, Li Jian-FengORCID
Abstract
AbstractThe 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 non-transgenic 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.
Funder
National Key Research and Development Program of China
Publisher
Springer Science and Business Media LLC
Subject
Plant Science,Genetics,Agronomy and Crop Science,Biochemistry, Genetics and Molecular Biology (miscellaneous),Molecular Biology,Biotechnology
Reference39 articles.
1. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743 2. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823 3. Cullot G, Boutin J, Toutain J, Prat F, Pennamen P, Rooryck C, Teichmann M, Rousseau E, Lamrissi-Garcia I, Guyonnet-Duperat V, Bibeyran A, Lalanne M, Prouzet-Mauléon V, Turcq B, Ged C, Blouin JM, Richard E, Dabernat S, Moreau-Gaudry F, Bedel A (2019) CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations. Nat Commun 10:1136 4. de Jong OG, MurphyDE MI, Willms E, Garcia-Guerra A, Gitz-Francois JJ, Lefferts J, Gupta D, Steenbeek SC, van Rheenen J, Andaloussi SE, Schiffelers RM, Wood MJA, Vader P (2020) A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA. Nat Commun 11:1113 5. Endo M, Iwakami S, Toki S (2021) Precision genome editing in plants via gene targeting and subsequent break-induced single-strand annealing. Plant Biotechnol J 19:563–574
|
|