CRISPR-Cas9 nuclear dynamics and target recognition in living cells
Ma, Hanhui; Tu, Li-Chun; Naseri, Ardalan; Huisman, Maximiliaan; Zhang, Shaojie; Grunwald, David; Pederson, Thoru
2016
发表期刊JOURNAL OF CELL BIOLOGY
ISSN0021-9525
EISSN1540-8140
卷号214期号:5页码:529-537
发表状态已发表
DOI10.1083/jcb.201604115
摘要

The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9 guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as <2 min in a nucleotide identity- and position-dependent manner. We further show that the duration of target residence correlates with cleavage activity. These results reveal that CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time.

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收录类别SCI
WOS研究方向Cell Biology
WOS类目Cell Biology
WOS记录号WOS:000382597700008
WOS关键词SEQUENCE-SPECIFIC CONTROL ; RNA-GUIDED ENDONUCLEASE ; R-LOOP FORMATION ; IN-VIVO ; GENE-EXPRESSION ; CAS9 NUCLEASES ; DNA ; CLEAVAGE ; BINDING ; PROTEIN
原始文献类型Article
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文献类型期刊论文
条目标识符https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/120134
专题个人在本单位外知识产出
通讯作者Pederson, Thoru
作者单位1.Univ Massachusetts, Sch Med, Dept Biochem & Mol Biol, Worcester, MA 01605 USA
2.Univ Massachusetts, Sch Med, RNA Therapeut Inst, Worcester, MA 01605 USA
3.Univ Cent Florida, Dept Comp Sci, Orlando, FL 32816 USA
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Ma, Hanhui,Tu, Li-Chun,Naseri, Ardalan,et al. CRISPR-Cas9 nuclear dynamics and target recognition in living cells[J]. JOURNAL OF CELL BIOLOGY,2016,214(5):529-537.
APA Ma, Hanhui.,Tu, Li-Chun.,Naseri, Ardalan.,Huisman, Maximiliaan.,Zhang, Shaojie.,...&Pederson, Thoru.(2016).CRISPR-Cas9 nuclear dynamics and target recognition in living cells.JOURNAL OF CELL BIOLOGY,214(5),529-537.
MLA Ma, Hanhui,et al."CRISPR-Cas9 nuclear dynamics and target recognition in living cells".JOURNAL OF CELL BIOLOGY 214.5(2016):529-537.
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