PDF(Pubmed)
Abstract：
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 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.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s42994-023-00132-6.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s42994-023-00132-6.
摘要：
CRISPR/Cas9技术彻底改变了包括植物在内的多种生物中的靶向基因敲除。然而,筛选编辑的等位基因，特别是那些有多重编辑的人，从除草剂或抗生素抗性转基因植物和分离出Cas9转基因代表了两个费力的过程。促进这些过程的当前解决方案依赖于不同的选择标记。这里,通过利用d-氨基酸氧化酶（DAO）在解毒d-丝氨酸和将无毒d-缬氨酸代谢为细胞毒性产物中的相反功能，我们开发了一种基于DAO的选择系统，该系统可以同时富集拟南芥中多基因编辑的等位基因并消除含Cas9的后代。在大肠杆菌中测试的五个DAO中，由Trigonopsisvariabilis（TvDAO）编码的一种可以赋予比其他同系物稍强的d-丝氨酸抗性。TvDAO在拟南芥中的转基因表达允许在d-丝氨酸条件的阳性选择和d-缬氨酸条件的阴性选择中明确区分转基因和非转基因植物。作为概念的证明，我们将CRISPR诱导的死亡TvDAO的单链退火修复与基于d-丝氨酸的阳性选择相结合，以帮助鉴定具有多重编辑的转基因植物，其中d-丝氨酸抗性植物在三个内源靶基因上表现出比潮霉素选择的更高的共编辑频率。随后，基于d-缬氨酸的阴性选择成功地从携带遗传突变的存活后代中去除Cas9和TvDAO转基因。总的来说，这项工作提供了一种新策略，以缓解CRISPR突变体的鉴定和Cas9转基因消除使用单一选择标记，它承诺在植物中更有效和简化的多重CRISPR编辑。
■在线版本包含补充材料，可在10.1007/s42994-023-00132-6获得。
■在线版本包含补充材料，可在10.1007/s42994-023-00132-6获得。
