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

John G. Doench; Nicolo Fusi; Meagan Sullender; Mudra Hegde; Emma W. Vaimberg; Katherine F. Donovan; Ian Smith; Zuzana Tothova; Craig Wilen; Robert Orchard; Herbert W. Virgin; Jennifer Listgarten; David E. Root

doi:10.1038/nbt.3437

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

John G. Doench, Nicolo Fusi, Meagan Sullender, Mudra Hegde, Emma W. Vaimberg, Katherine F. Donovan, Ian Smith, Zuzana Tothova, Craig Wilen, Robert Orchard, Herbert W. Virgin, Jennifer Listgarten, David E. Root

Research output: Contribution to journal › Article › peer-review

2340 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	184-191
Number of pages	8
Journal	Nature biotechnology
Volume	34
Issue number	2
DOIs	https://doi.org/10.1038/nbt.3437
State	Published - Feb 1 2016
Externally published	Yes

ASJC Scopus subject areas

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology
Molecular Medicine
Biomedical Engineering

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10.1038/nbt.3437

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title = "Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9",

abstract = "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.",

author = "Doench, {John G.} and Nicolo Fusi and Meagan Sullender and Mudra Hegde and Vaimberg, {Emma W.} and Donovan, {Katherine F.} and Ian Smith and Zuzana Tothova and Craig Wilen and Robert Orchard and Virgin, {Herbert W.} and Jennifer Listgarten and Root, {David E.}",

note = "Funding Information: We thank M. Tomko, M. Greene, A. Brown, D. Alan and T. Green for software engineering support, and T. Nguyen, N. Tran and X. Yang for library production support (Broad Institute). Z.T. is funded by NIH 5K12CA087723-12, ASCO Young Investigator Award, LLS Special Fellow Award. J.G.D. is a Merkin Institute Fellow and is supported by the Next Generation Fund at the Broad Institute of MIT and Harvard.",

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AU - Doench, John G.

AU - Fusi, Nicolo

AU - Sullender, Meagan

AU - Hegde, Mudra

AU - Vaimberg, Emma W.

AU - Donovan, Katherine F.

AU - Smith, Ian

AU - Tothova, Zuzana

AU - Wilen, Craig

AU - Orchard, Robert

AU - Virgin, Herbert W.

AU - Listgarten, Jennifer

AU - Root, David E.

N1 - Funding Information: We thank M. Tomko, M. Greene, A. Brown, D. Alan and T. Green for software engineering support, and T. Nguyen, N. Tran and X. Yang for library production support (Broad Institute). Z.T. is funded by NIH 5K12CA087723-12, ASCO Young Investigator Award, LLS Special Fellow Award. J.G.D. is a Merkin Institute Fellow and is supported by the Next Generation Fund at the Broad Institute of MIT and Harvard.

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

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