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

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

CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), Cas9 can be reprogrammed to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary...

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Main Authors: Doench, John G., Fusi, Nicolo, Sullender, Meagan, Hegde, Mudra, Vaimberg, Emma W., Donovan, Katherine F., Smith, Ian, Tothova, Zuzana, Wilen, Craig, Orchard, Robert, Virgin, Herbert W., Listgarten, Jennifer, Root, David E.
Format: Online
Language:English
Published: 2016
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744125/
id pubmed-4744125
recordtype oai_dc
spelling pubmed-47441252016-07-18 Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 Doench, John G. Fusi, Nicolo Sullender, Meagan Hegde, Mudra Vaimberg, Emma W. Donovan, Katherine F. Smith, Ian Tothova, Zuzana Wilen, Craig Orchard, Robert Virgin, Herbert W. Listgarten, Jennifer Root, David E. Article CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), Cas9 can be reprogrammed 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. 2016-01-18 2016-02 /pmc/articles/PMC4744125/ /pubmed/26780180 http://dx.doi.org/10.1038/nbt.3437 Text en Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Doench, John G.
Fusi, Nicolo
Sullender, Meagan
Hegde, Mudra
Vaimberg, Emma W.
Donovan, Katherine F.
Smith, Ian
Tothova, Zuzana
Wilen, Craig
Orchard, Robert
Virgin, Herbert W.
Listgarten, Jennifer
Root, David E.
spellingShingle Doench, John G.
Fusi, Nicolo
Sullender, Meagan
Hegde, Mudra
Vaimberg, Emma W.
Donovan, Katherine F.
Smith, Ian
Tothova, Zuzana
Wilen, Craig
Orchard, Robert
Virgin, Herbert W.
Listgarten, Jennifer
Root, David E.
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
author_facet Doench, John G.
Fusi, Nicolo
Sullender, Meagan
Hegde, Mudra
Vaimberg, Emma W.
Donovan, Katherine F.
Smith, Ian
Tothova, Zuzana
Wilen, Craig
Orchard, Robert
Virgin, Herbert W.
Listgarten, Jennifer
Root, David E.
author_sort Doench, John G.
title Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
title_short Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
title_full Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
title_fullStr Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
title_full_unstemmed Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
title_sort optimized sgrna design to maximize activity and minimize off-target effects of crispr-cas9
description CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), Cas9 can be reprogrammed 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.
publishDate 2016
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744125/
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