Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments.

Nat Protoc
Authors
Keywords
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.

Year of Publication
2018
Journal
Nat Protoc
Volume
13
Issue
5
Pages
946-986
Date Published
2018 05
ISSN
1750-2799
DOI
10.1038/nprot.2018.005
PubMed ID
29651054
PubMed Central ID
PMC6182299
Links
Grant list
F30 DK103359 / DK / NIDDK NIH HHS / United States
R00 HG008399 / HG / NHGRI NIH HHS / United States
U41 HG002371 / HG / NHGRI NIH HHS / United States