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Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
John G Doench1,5, Nicolo Fusi2,5, Meagan Sullender1,5, Mudra Hegde1,5, Emma W Vaimberg1,5, Katherine F Donovan1, Ian Smith1, Zuzana Tothova1,3, Craig Wilen4, Robert Orchard4, Herbert W Virgin4, Jennifer Listgarten2,5 & David E Root1
CRISPR-Cas9based 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.
npg 201 6 Nature America, Inc. All rights reserved.
The Cas9 protein, an RNA-directed DNA endonuclease, is a powerful tool for manipulating the genome14. The ease of programming Cas9 (CRISPR-associated protein 9) has enabled CRISPR (clustered, regularly interspaced, short palindromic repeats)-based genetic screens5, identifying well-established genes and providing novel insight into gene function for multiple phenotypes68. Initial libraries were designed with little knowledge of sgRNA activity rules, a critical design parameter, as interpreting screening data requires consistency among multiple sgRNAs targeting the same gene to distinguish true hits from false positives. Inactive and nonspecific sgRNAs reduce the effective gene coverage of the library and the accuracy of the hit list.
Many studies indicate that Cas9 off-target activity depends on both sgRNA sequence and experimental conditions913. These studies
have provided qualitative but incomplete understanding of specificity determinants. Finding generalizable patterns is quite challenging, requiring large data sets to adequately sample the vast number of possible imperfect sgRNA-DNA interactions to reveal sequence features for prediction of off-target activity. Here, we present the design and characterization of human and mouse genome-wide sgRNA libraries based on our previously published rules for predicting on-target...