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Nat Biotechnol. 2016 Feb;34(2):184-191. doi: 10.1038/nbt.3437. Epub 2016 Jan 18.

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

Author information

1
Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
2
Microsoft Research New England, Cambridge, Massachusetts, USA.
3
Dana Farber Cancer Institute, Division of Hematologic Malignancies, Boston, Massachusetts, USA.
4
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, Missouri, USA.
#
Contributed equally

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.

PMID:
26780180
PMCID:
PMC4744125
DOI:
10.1038/nbt.3437
[Indexed for MEDLINE]
Free PMC Article

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