Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments
Name
nihms961575.pdf
Description
Accepted version
Size
1.89 MB
Format
Adobe PDF
Checksum (MD5)
e049ef79058bd5c768f4df28d0fc8f86
Author(s)
Zhang, Feng
Pinello, Luca
Date Issued
April 2018
Journal
Nature Protocols
Publisher
Springer Nature
Citation
Canver, Matthew C. et al. "Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments." Nature Protocols 13 (April 2018): 946-86 ©2018 Author(s)
Version
Author's final manuscript
Abstract
CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts, as well as computational data analysis that can be performed in 1-2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.
MIT Department
Broad Institute of MIT and Harvard
Terms of Use
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1038/NPROT.2018.005
