CHANGE-seq reveals genetic and epigenetic effects on CRISPR–Cas9 genome-wide activity
- 15 June 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Biotechnology
- Vol. 38 (11), 1317-1327
- https://doi.org/10.1038/s41587-020-0555-7
Abstract
Current methods can illuminate the genome-wide activity of CRISPR–Cas9 nucleases, but are not easily scalable to the throughput needed to fully understand the principles that govern Cas9 specificity. Here we describe ‘circularization for high-throughput analysis of nuclease genome-wide effects by sequencing’ (CHANGE-seq), a scalable, automatable tagmentation-based method for measuring the genome-wide activity of Cas9 in vitro. We applied CHANGE-seq to 110 single guide RNA targets across 13 therapeutically relevant loci in human primary T cells and identified 201,934 off-target sites, enabling the training of a machine learning model to predict off-target activity. Comparing matched genome-wide off-target, chromatin modification and accessibility, and transcriptional data, we found that cellular off-target activity was two to four times more likely to occur near active promoters, enhancers and transcribed regions. Finally, CHANGE-seq analysis of six targets across eight individual genomes revealed that human single-nucleotide variation had significant effects on activity at ~15.2% of off-target sites analyzed. CHANGE-seq is a simplified, sensitive and scalable approach to understanding the specificity of genome editors.Keywords
Funding Information
- Doris Duke Charitable Foundation (2017093, U01EB029373, 2017093)
- U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (U01EB029373, U01EB029373, U01EB029373, U01EB029373, U01EB029373, U01EB029373, U01EB029373)
This publication has 51 references indexed in Scilit:
- CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activityNucleic Acids Research, 2013
- RNA-Guided Human Genome Engineering via Cas9Science, 2013
- Multiplex Genome Engineering Using CRISPR/Cas SystemsScience, 2013
- A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial ImmunityScience, 2012
- ChromHMM: automating chromatin-state discovery and characterizationNature Methods, 2012
- FLASH: fast length adjustment of short reads to improve genome assembliesBioinformatics, 2011
- Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transpositionGenome Biology, 2010
- Fast and accurate short read alignment with Burrows–Wheeler transformBioinformatics, 2009
- Transposon Tn5Annual Review of Genetics, 2008
- Model-based Analysis of ChIP-Seq (MACS)Genome Biology, 2008