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Nat Biotechnol DOI:10.1038/nbt.3437

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

Publication TypeJournal Article
Year of Publication2016
AuthorsDoench, JG, Fusi, N, Sullender, M, Hegde, M, Vaimberg, EW, Donovan, KF, Smith, I, Tothova, Z, Wilen, C, Orchard, R, Virgin, HW, Listgarten, J, Root, DE
JournalNat Biotechnol
Date Published2016 Feb
KeywordsAnimals, Cell Line, Tumor, CRISPR-Cas Systems, Drug Resistance, Gene Library, Genetic Engineering, Genome, Genomics, Humans, Mice, RNA, Guide

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.


Alternate JournalNat. Biotechnol.
PubMed ID26780180
PubMed Central IDPMC4744125
Grant ListK12 CA087723 / CA / NCI NIH HHS / United States
T32 AI007163 / AI / NIAID NIH HHS / United States
U19 AI109725 / AI / NIAID NIH HHS / United States
5K12CA087723-12 / CA / NCI NIH HHS / United States