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A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture
Henriette O'Geen1, Isabelle M Henry2, Mital S Bhakta1
1Genome Center and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA.
Nucleic Acids Research
|February 26, 2015
Summary
CRISPR-Cas9 genome engineering is highly specific, with minimal off-target binding. Targeted sequence capture effectively identifies any rare cleavage events, confirming CRISPR
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- CRISPR-Cas9 is a powerful genome engineering tool.
- Concerns exist regarding its target specificity and potential off-target effects.
- Current methods for assessing specificity are low-throughput and limited.
Purpose of the Study:
- To comprehensively evaluate the genome-wide binding specificity of CRISPR-Cas9.
- To investigate the relationship between Cas9 binding and actual cleavage activity.
- To establish a high-throughput method for identifying CRISPR-Cas9 off-target effects.
Main Methods:
- Chromatin immunoprecipitation sequencing (ChIP-seq) to map genome-wide Cas9 binding sites.
- Targeted sequence capture to analyze cleavage activity at numerous genomic loci simultaneously.
- Computational prediction of potential off-target sites.
Main Results:
- Cas9 binding was highly specific to the intended target site.
- Off-target binding occurred at significantly lower intensities.
- Targeted sequence capture revealed indels at the on-target site and one predicted off-target site, with no cleavage at other bound regions.
- Cas9-bound regions were enriched for the NGG motif required for Streptococcus pyogenes Cas9 recognition.
Conclusions:
- CRISPR-Cas9 endonucleases exhibit high specificity in genome targeting.
- Targeted sequence capture is an effective high-throughput method for assessing CRISPR-Cas9 off-target activity genome-wide.
