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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 |
ISSN | 0021-9525 |
EISSN | 1540-8140 |
卷号 | 214期号:5页码:529-537 |
发表状态 | 已发表 |
DOI | 10.1083/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. |
URL | 查看原文 |
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 |
引用统计 | |
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 |
推荐引用方式 GB/T 7714 | 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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