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

JG Doench, N Fusi, M Sullender, M Hegde… - Nature …, 2016 - nature.com
JG Doench, N Fusi, M Sullender, M Hegde, EW Vaimberg, KF Donovan, I Smith, Z Tothova
Nature biotechnology, 2016nature.com
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 …
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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