Format

Send to

Choose Destination
Nat Protoc. 2018 May;13(5):946-986. doi: 10.1038/nprot.2018.005. Epub 2018 Apr 12.

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

Author information

1
Molecular Pathology Unit and Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
2
Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, California, USA.
3
Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
4
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard University, Boston, Massachusetts, USA.
5
Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
6
Howard Hughes Medical Institute, Boston, Massachusetts, USA.
7
New York Genome Center and Department of Biology, New York University, New York, New York, USA.
8
Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
9
Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
10
Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA.
11
The Broad Institute, Cambridge, Massachusetts, USA.
12
McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
13
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
14
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
15
INSERM U1154, CNRS UMR 7196, Muséum National d'Histoire Naturelle, Paris, France.

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.

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center