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

Nature Biotechnology
John G DoenchDavid E Root

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.

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Methods Mentioned

BETA
flow cytometry
protein knockout
GUIDE-Seq
GUIDE-seq.
gene knockout
PCR
Transfection

Software Mentioned

Seq
Cas
GUIDE
OFFinder
lentiGuide
MAGeCK
Addgene
TIDE
CCtop
GENCODE

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