气候变化领域集成服务门户(CCPortal): Very fast CRISPR on demand

CCPortal

DOI	10.1126/science.aay8204
	Very fast CRISPR on demand
	Liu Y.; Zou R.S.; He S.; Nihongaki Y.; Li X.; Razavi S.; Wu B.; Ha T.
发表日期	2020
ISSN	0036-8075
起始页码	1265
结束页码	1269
卷号	368 期号:6496
英文摘要	CRISPR-Cas systems provide versatile tools for programmable genome editing. Here, we developed a caged RNA strategy that allows Cas9 to bind DNA but not cleave until light-induced activation. This approach, referred to as very fast CRISPR (vfCRISPR), creates double-strand breaks (DSBs) at the submicrometer and second scales. Synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells respond to Cas9-induced DSBs within minutes and can retain MRE11 after DNA ligation. Phosphorylation of H2AX after DNA damage propagated more than 100 kilobases per minute, reaching up to 30 megabases. Using single-cell fluorescence imaging, we characterized multiple cycles of 53BP1 repair foci formation and dissolution, with the first cycle taking longer than subsequent cycles and its duration modulated by inhibition of repair. Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single-allele resolution. vfCRISPR enables DNA-repair studies at high resolution in space, time, and genomic coordinates. © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
关键词	Cas9 endonuclease Streptococcus pyogenes double strand break repair protein MRE11 H2AX protein, human histone MRE11 protein, human CRISPR Cas system DNA cleavage DNA repair double stranded DNA break fluorescence imaging gene editing genetics HEK293 cell line human light metabolism phosphorylation procedures radiation response single cell analysis CRISPR-Associated Protein 9 CRISPR-Cas Systems DNA Breaks, Double-Stranded DNA Cleavage DNA Repair Gene Editing HEK293 Cells Histones Humans Light MRE11 Homologue Protein Optical Imaging Phosphorylation Single-Cell Analysis
语种	英语
来源机构	Science
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/133439
推荐引用方式 GB/T 7714	Liu Y.,Zou R.S.,He S.,et al. Very fast CRISPR on demand[J]. Science,2020,368(6496).
APA	Liu Y..,Zou R.S..,He S..,Nihongaki Y..,Li X..,...&Ha T..(2020).Very fast CRISPR on demand.,368(6496).
MLA	Liu Y.,et al."Very fast CRISPR on demand".368.6496(2020).