Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9

被引：2516

作者：

Doench, John G. ^{[1
]}

Fusi, Nicolo ^{[2
]}

Sullender, Meagan ^{[1
]}

Hegde, Mudra ^{[1
]}

Vaimberg, Emma W. ^{[1
]}

Donovan, Katherine F. ^{[1
]}

Smith, Ian ^{[1
]}

Tothova, Zuzana ^{[1
,3
]}

Wilen, Craig ^{[4
]}

Orchard, Robert ^{[4
]}

Virgin, Herbert W. ^{[4
]}

Listgarten, Jennifer ^{[2
]}

Root, David E. ^{[1
]}

机构：

[1] Broad Inst MIT & Harvard, Cambridge, MA USA

[2] Microsoft Res New England, Cambridge, MA USA

[3] Dana Farber Canc Inst, Div Hematol Malignancies, Boston, MA 02115 USA

[4] Washington Univ, Sch Med, Dept Pathol & Immunol, St Louis, MO USA

来源：

NATURE BIOTECHNOLOGY | 2016年 / 34卷 / 02期

关键词：

GENETIC SCREENS; CELL-LINE; GENOME; CAS9; DNA; RAF; IDENTIFICATION; ENDONUCLEASE; SPECIFICITY; RESISTANCE;

D O I：

10.1038/nbt.3437

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：

CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.

引用

页码：184 / +

页数：12

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