气候变化领域集成服务门户(CCPortal): Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis in Drosophila

CCPortal

DOI	10.1073/pnas.2014255118
	Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis in Drosophila
	Koreman G.T.; Xu Y.; Hu Q.; Zhang Z.; Allen S.E.; Wolfner M.F.; Wang B.; Han C.
发表日期	2021
ISSN	00278424
卷号	118 期号:14
英文摘要	CRISPR/Cas9 has emerged as a powerful technology for tissuespecific mutagenesis. However, tissue-specific CRISPR/Cas9 tools currently available in Drosophila remain deficient in three significant ways. First, many existing gRNAs are inefficient, such that further improvements of gRNA expression constructs are needed for more efficient and predictable mutagenesis in both somatic and germline tissues. Second, it has been difficult to label mutant cells in target tissues with current methods. Lastly, application of tissue-specific mutagenesis at present often relies on Gal4-driven Cas9, which hampers the flexibility and effectiveness of the system. Here, we tackle these deficiencies by building upon our previous CRISPRmediated tissue-restricted mutagenesis (CRISPR-TRiM) tools. First, we significantly improved gRNA efficiency in somatic tissues by optimizing multiplexed gRNA design. Similarly, we also designed efficient dual-gRNA vectors for the germline. Second, we developed methods to positively and negatively label mutant cells in tissuespecific mutagenesis by incorporating co-CRISPR reporters into gRNA expression vectors. Lastly, we generated genetic reagents for convenient conversion of existing Gal4 drivers into tissuespecific Cas9 lines based on homology-assisted CRISPR knock-in. In this way, we expand the choices of Cas9 for CRISPR-TRiManalysis to broader tissues and developmental stages. Overall, our upgraded CRISPR/Cas9 tools make tissue-specific mutagenesis more versatile, reliable, and effective in Drosophila. These improvements may be also applied to other model systems. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Co-CRISPR; CRISPR-TRiM; CRISPR/Cas9; Drosophila; GRNA
语种	英语
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/179993
作者单位	Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, United States; Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, United States
推荐引用方式 GB/T 7714	Koreman G.T.,Xu Y.,Hu Q.,et al. Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis in Drosophila[J],2021,118(14).
APA	Koreman G.T..,Xu Y..,Hu Q..,Zhang Z..,Allen S.E..,...&Han C..(2021).Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis in Drosophila.Proceedings of the National Academy of Sciences of the United States of America,118(14).
MLA	Koreman G.T.,et al."Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis in Drosophila".Proceedings of the National Academy of Sciences of the United States of America 118.14(2021).