CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering

被引：1331

作者：

Mali, Prashant ^{[1
]}

Aach, John ^{[1
]}

Stranges, P. Benjamin ^{[1
]}

Esvelt, Kevin M. ^{[2
]}

Moosburner, Mark ^{[1
]}

Kosuri, Sriram ^{[2
]}

Yang, Luhan ^{[3
]}

Church, George M. ^{[1
,2
]}

机构：

[1] Harvard Univ, Sch Med, Dept Genet, Boston, MA 02114 USA

[2] Harvard Univ, Wyss Inst Biol Inspired Engn, Cambridge, MA 02138 USA

[3] Harvard Univ, Sch Med, Biol & Biomed Sci Program, Boston, MA USA

来源：

NATURE BIOTECHNOLOGY | 2013年 / 31卷 / 09期

基金：

美国国家卫生研究院;

关键词：

ZINC-FINGER NUCLEASES; GENE-EXPRESSION; TAL EFFECTORS; RNA; CRISPR; SYSTEMS; IMMUNITY; CELLS; ENDONUCLEASE; CLEAVAGE;

D O I：

10.1038/nbt.2675

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes(1-7). Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA: Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors(8,9). Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA: Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA: Cas9 tool and highlight the critical need to engineer improved specificity.

引用

页码：833 / +

页数：8

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