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DOI | 10.1126/science.aah7084 |
Kinetics of dCas9 target search in Escherichia coli | |
Jones, Daniel Lawson; Leroy, Prune; Unoson, Cecilia; Fange, David; Curic, Vladimir; Lawson, Michael J.; Elf, Johan | |
2017-09-29 | |
发表期刊 | SCIENCE
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ISSN | 0036-8075 |
EISSN | 1095-9203 |
出版年 | 2017 |
卷号 | 357期号:6358页码:1420-1423 |
文章类型 | Article |
语种 | 英语 |
国家 | Sweden |
英文摘要 | How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations. |
领域 | 地球科学 ; 气候变化 ; 资源环境 |
收录类别 | SCI-E |
WOS记录号 | WOS:000411880800052 |
WOS关键词 | CRISPR RNA ; LIVING CELLS ; HOMOLOGOUS RECOMBINATION ; LAC REPRESSOR ; INTERROGATION ; ENDONUCLEASE ; SEQUENCE ; DYNAMICS ; IMMUNITY |
WOS类目 | Multidisciplinary Sciences |
WOS研究方向 | Science & Technology - Other Topics |
URL | 查看原文 |
引用统计 | |
文献类型 | 期刊论文 |
条目标识符 | http://119.78.100.173/C666/handle/2XK7JSWQ/196979 |
专题 | 地球科学 资源环境科学 气候变化 |
作者单位 | Uppsala Univ, Dept Cell & Mol Biol, Sci Life Lab, Uppsala, Sweden |
推荐引用方式 GB/T 7714 | Jones, Daniel Lawson,Leroy, Prune,Unoson, Cecilia,et al. Kinetics of dCas9 target search in Escherichia coli[J]. SCIENCE,2017,357(6358):1420-1423. |
APA | Jones, Daniel Lawson.,Leroy, Prune.,Unoson, Cecilia.,Fange, David.,Curic, Vladimir.,...&Elf, Johan.(2017).Kinetics of dCas9 target search in Escherichia coli.SCIENCE,357(6358),1420-1423. |
MLA | Jones, Daniel Lawson,et al."Kinetics of dCas9 target search in Escherichia coli".SCIENCE 357.6358(2017):1420-1423. |
条目包含的文件 | 条目无相关文件。 |
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