Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library wit...

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Vydané v:Nature communications Ročník 8; číslo 1; s. 15178 - 8
Hlavní autori: Morgens, David W., Wainberg, Michael, Boyle, Evan A., Ursu, Oana, Araya, Carlos L., Tsui, C. Kimberly, Haney, Michael S., Hess, Gaelen T., Han, Kyuho, Jeng, Edwin E., Li, Amy, Snyder, Michael P., Greenleaf, William J., Kundaje, Anshul, Bassik, Michael C.
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: London Nature Publishing Group UK 05.05.2017
Nature Publishing Group
Nature Portfolio
Predmet:
42/41
42/47
631/1647/2163
631/208/4041/3196
631/61/191/1908
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats - genetics
CRISPR
CRISPR-Cas Systems - genetics
Deoxyribonucleic acid
Design
DNA
DNA damage
DNA Damage - genetics
Gene Targeting - methods
Genes
Genomes
Genomic Library
High-Throughput Screening Assays - methods
Humanities and Social Sciences
Humans
multidisciplinary
Polysaccharides - biosynthesis
Ricin - toxicity
RNA Interference
RNA, Guide, CRISPR-Cas Systems - genetics
Science
Science (multidisciplinary)
Toxicity
Toxins
United States > US
California
ISSN:2041-1723, 2041-1723
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Shrnutí:CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens. CRISPR-Cas9 screens are powerful high-throughput tools but can be confounded by nuclease toxicity. Here the authors design a library of variable length gRNAs with thousands of negative controls, including the targeting of ‘safe’ loci to account for on-target site DNA damage toxicity.
Bibliografia:ObjectType-Article-1
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These authors contributed equally to this work.
ISSN:2041-1723
2041-1723
DOI:10.1038/ncomms15178