Hui Wang, Jian Ding, Jingyan Zhu, Xiaoshuang Liu, Rongfang Xu, Ruiying Qin, Dongfang Gu, Min Li, Pengcheng Wei, Juan Li
{"title":"开发用于水稻核心启动子编辑的 CRISPR/FrCas9 系统","authors":"Hui Wang, Jian Ding, Jingyan Zhu, Xiaoshuang Liu, Rongfang Xu, Ruiying Qin, Dongfang Gu, Min Li, Pengcheng Wei, Juan Li","doi":"10.1007/s42994-024-00157-5","DOIUrl":null,"url":null,"abstract":"<div><p>Small mutations in the core promoter region of a gene may result in substantial changes in expression strengths. However, targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible Cas9s. In this study, we engineered a unique FrCas9 system derived from <i>Faecalibaculum rodentium</i> for plant genome editing. Our findings indicate that this system is efficient in rice when the TATA sequence is used as a PAM. In addition, FrCas9 demonstrated activity against all 16 possible NNTA PAMs, achieving an efficiency of up to 35.3% in calli and generating homozygous or biallelic mutations in 31.3% of the T<sub>0</sub> transgenic plants. A proof-of-concept experiment to examine editing of the rice <i>WX</i> core promoter confirmed that FrCas9-induced mutations could modify gene expression and amylose content. Multiplex mutations and deletions were produced by bidirectional editing, mediated by FrCas9, using a single palindromic TATA sequence as a PAM. Moreover, we developed FrCas9-derived base editors capable of programmable conversion between A·T and G·C pairs in plants. This study highlights a versatile FrCas9 toolset for plant core promoter editing, offering great potential for the fine-tuning of gene expression and creating of new germplasms.</p></div>","PeriodicalId":53135,"journal":{"name":"aBIOTECH","volume":null,"pages":null},"PeriodicalIF":4.6000,"publicationDate":"2024-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s42994-024-00157-5.pdf","citationCount":"0","resultStr":"{\"title\":\"Developing a CRISPR/FrCas9 system for core promoter editing in rice\",\"authors\":\"Hui Wang, Jian Ding, Jingyan Zhu, Xiaoshuang Liu, Rongfang Xu, Ruiying Qin, Dongfang Gu, Min Li, Pengcheng Wei, Juan Li\",\"doi\":\"10.1007/s42994-024-00157-5\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Small mutations in the core promoter region of a gene may result in substantial changes in expression strengths. However, targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible Cas9s. In this study, we engineered a unique FrCas9 system derived from <i>Faecalibaculum rodentium</i> for plant genome editing. Our findings indicate that this system is efficient in rice when the TATA sequence is used as a PAM. In addition, FrCas9 demonstrated activity against all 16 possible NNTA PAMs, achieving an efficiency of up to 35.3% in calli and generating homozygous or biallelic mutations in 31.3% of the T<sub>0</sub> transgenic plants. A proof-of-concept experiment to examine editing of the rice <i>WX</i> core promoter confirmed that FrCas9-induced mutations could modify gene expression and amylose content. Multiplex mutations and deletions were produced by bidirectional editing, mediated by FrCas9, using a single palindromic TATA sequence as a PAM. Moreover, we developed FrCas9-derived base editors capable of programmable conversion between A·T and G·C pairs in plants. This study highlights a versatile FrCas9 toolset for plant core promoter editing, offering great potential for the fine-tuning of gene expression and creating of new germplasms.</p></div>\",\"PeriodicalId\":53135,\"journal\":{\"name\":\"aBIOTECH\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.6000,\"publicationDate\":\"2024-04-22\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://link.springer.com/content/pdf/10.1007/s42994-024-00157-5.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"aBIOTECH\",\"FirstCategoryId\":\"1091\",\"ListUrlMain\":\"https://link.springer.com/article/10.1007/s42994-024-00157-5\",\"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/s42994-024-00157-5","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}
Developing a CRISPR/FrCas9 system for core promoter editing in rice
Small mutations in the core promoter region of a gene may result in substantial changes in expression strengths. However, targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible Cas9s. In this study, we engineered a unique FrCas9 system derived from Faecalibaculum rodentium for plant genome editing. Our findings indicate that this system is efficient in rice when the TATA sequence is used as a PAM. In addition, FrCas9 demonstrated activity against all 16 possible NNTA PAMs, achieving an efficiency of up to 35.3% in calli and generating homozygous or biallelic mutations in 31.3% of the T0 transgenic plants. A proof-of-concept experiment to examine editing of the rice WX core promoter confirmed that FrCas9-induced mutations could modify gene expression and amylose content. Multiplex mutations and deletions were produced by bidirectional editing, mediated by FrCas9, using a single palindromic TATA sequence as a PAM. Moreover, we developed FrCas9-derived base editors capable of programmable conversion between A·T and G·C pairs in plants. This study highlights a versatile FrCas9 toolset for plant core promoter editing, offering great potential for the fine-tuning of gene expression and creating of new germplasms.