Abstract
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.
Highlights
CRISPR-Cas[9] 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
Assays for genome-wide double-strand DNA breaks[26,27,28,32] have indicated these strategies successfully limit off-target cutting. These experiments have so far been limited to measurement of the off-target cutting of a handful of guides, leaving open the question of how these off-targets may interfere with the output of high-throughput screens, and if the varied strategies for off-target reduction can be effective in this domain
In order to determine the effect of singleguide RNAs (sgRNAs) length, the library was designed to contain guides ranging from 17 to 20 bp in length (Supplementary Fig. 1d)
Summary
CRISPR-Cas[9] 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. Studies using Cas[9] have included non-targeting singleguide RNAs (sgRNAs)[22,23,34,35,36], which are overexpressed and loaded into Cas[9], presumably controlling for the potentially disruptive binding of Cas[9] to PAM sites throughout the genome[30,37] These non-targeting sgRNAs may fail to replicate the most dramatic, non-specific effect of Cas[9] gene knockouts: the formation of double-strand breaks in genomic DNA22,23. Assays for genome-wide double-strand DNA breaks[26,27,28,32] have indicated these strategies successfully limit off-target cutting These experiments have so far been limited to measurement of the off-target cutting of a handful of guides, leaving open the question of how these off-targets may interfere with the output of high-throughput screens, and if the varied strategies for off-target reduction can be effective in this domain. Though reduced overall activity of truncated sgRNAs could be responsible for this reduction in off-target activity, low-throughput tests suggest that this is not the case in either human cell lines[40] or yeast[44]
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