Feng-Zhu Wang, Ying Bao, Zhenxiang Li, Xiangyu Xiong, Jian-Feng Li
{"title":"双功能选择系统可实现拟南芥多基因 CRISPR 突变体的正向选择和无 Cas9 后代的负向选择","authors":"Feng-Zhu Wang, Ying Bao, Zhenxiang Li, Xiangyu Xiong, Jian-Feng Li","doi":"10.1007/s42994-023-00132-6","DOIUrl":null,"url":null,"abstract":"<div><p>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 <i>Cas9</i> 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 <span>d</span>-amino acid oxidase (DAO) in detoxifying <span>d</span>-serine and in metabolizing non-toxic <span>d</span>-valine to a cytotoxic product, we develop a DAO-based selection system that simultaneously enables the enrichment of multigene edited alleles and elimination of <i>Cas9</i>-containing progeny in <i>Arabidopsis thaliana</i>. Among five DAOs tested in <i>Escherichia coli</i>, the one encoded by <i>Trigonopsis variabilis</i> (TvDAO) could confer slightly stronger <span>d</span>-serine resistance than other homologs. Transgenic expression of <i>TvDAO</i> in <i>Arabidopsis</i> allowed a clear distinction between transgenic and non-transgenic plants in both <span>d</span>-serine-conditioned positive selection and <span>d</span>-valine-conditioned negative selection. As a proof of concept, we combined CRISPR-induced single-strand annealing repair of a dead <i>TvDAO</i> with <span>d</span>-serine-based positive selection to help identify transgenic plants with multiplex editing, where <span>d</span>-serine-resistant plants exhibited considerably higher co-editing frequencies at three endogenous target genes than those selected by hygromycin. Subsequently, <span>d</span>-valine-based negative selection successfully removed <i>Cas9</i> and <i>TvDAO</i> transgenes from the survival offspring carrying inherited mutations. Collectively, this work provides a novel strategy to ease CRISPR mutant identification and <i>Cas9</i> transgene elimination using a single selection marker, which promises more efficient and simplified multiplex CRISPR editing in plants.</p></div>","PeriodicalId":53135,"journal":{"name":"aBIOTECH","volume":null,"pages":null},"PeriodicalIF":4.6000,"publicationDate":"2024-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s42994-023-00132-6.pdf","citationCount":"0","resultStr":"{\"title\":\"A dual-function selection system enables positive selection of multigene CRISPR mutants and negative selection of Cas9-free progeny in Arabidopsis\",\"authors\":\"Feng-Zhu Wang, Ying Bao, Zhenxiang Li, Xiangyu Xiong, Jian-Feng Li\",\"doi\":\"10.1007/s42994-023-00132-6\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>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 <i>Cas9</i> 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 <span>d</span>-amino acid oxidase (DAO) in detoxifying <span>d</span>-serine and in metabolizing non-toxic <span>d</span>-valine to a cytotoxic product, we develop a DAO-based selection system that simultaneously enables the enrichment of multigene edited alleles and elimination of <i>Cas9</i>-containing progeny in <i>Arabidopsis thaliana</i>. Among five DAOs tested in <i>Escherichia coli</i>, the one encoded by <i>Trigonopsis variabilis</i> (TvDAO) could confer slightly stronger <span>d</span>-serine resistance than other homologs. Transgenic expression of <i>TvDAO</i> in <i>Arabidopsis</i> allowed a clear distinction between transgenic and non-transgenic plants in both <span>d</span>-serine-conditioned positive selection and <span>d</span>-valine-conditioned negative selection. As a proof of concept, we combined CRISPR-induced single-strand annealing repair of a dead <i>TvDAO</i> with <span>d</span>-serine-based positive selection to help identify transgenic plants with multiplex editing, where <span>d</span>-serine-resistant plants exhibited considerably higher co-editing frequencies at three endogenous target genes than those selected by hygromycin. Subsequently, <span>d</span>-valine-based negative selection successfully removed <i>Cas9</i> and <i>TvDAO</i> transgenes from the survival offspring carrying inherited mutations. Collectively, this work provides a novel strategy to ease CRISPR mutant identification and <i>Cas9</i> transgene elimination using a single selection marker, which promises more efficient and simplified multiplex CRISPR editing in plants.</p></div>\",\"PeriodicalId\":53135,\"journal\":{\"name\":\"aBIOTECH\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.6000,\"publicationDate\":\"2024-01-22\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://link.springer.com/content/pdf/10.1007/s42994-023-00132-6.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"aBIOTECH\",\"FirstCategoryId\":\"1091\",\"ListUrlMain\":\"https://link.springer.com/article/10.1007/s42994-023-00132-6\",\"RegionNum\":4,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"aBIOTECH","FirstCategoryId":"1091","ListUrlMain":"https://link.springer.com/article/10.1007/s42994-023-00132-6","RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
CRISPR/Cas9 技术彻底改变了包括植物在内的多种生物体的定向基因敲除技术。然而,从抗除草剂或抗生素的转基因植物中筛选编辑过的等位基因,特别是那些多重编辑的等位基因,以及分离出 Cas9 转基因,是两个费力的过程。目前促进这些过程的解决方案依赖于不同的选择标记。在这里,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方面的相反功能,我们开发了一种基于 DAO 的选择系统,它能同时在拟南芥中富集多基因编辑的等位基因并淘汰含有 Cas9 的后代。在大肠杆菌中测试的五种 DAO 中,拟南芥变种(TvDAO)编码的 DAO 比其他同源物能赋予稍强的 d-丝氨酸抗性。在拟南芥中转基因表达 TvDAO 可以在 d-丝氨酸条件正选择和 d-缬氨酸条件负选择中明确区分转基因植物和非转基因植物。作为概念验证,我们将 CRISPR 诱导的对死亡 TvDAO 的单链退火修复与基于 d-丝氨酸的正向选择相结合,以帮助鉴定具有多重编辑功能的转基因植株。随后,基于 d-缬氨酸的负选择成功地从携带遗传突变的存活后代中移除了 Cas9 和 TvDAO 转基因。总之,这项工作提供了一种新的策略,利用单一选择标记简化了CRISPR突变体的鉴定和Cas9转基因的消除,有望在植物中实现更高效、更简化的多重CRISPR编辑。
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 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.