CHANGE-seq reveals genetic and epigenetic effects on CRISPR–Cas9 genome-wide activity

Published

June 15, 2020

Author(s)

Samantha Maragh, Natalia G. Kolmakova, Evgheni Strelcov

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.

Citation

Nature Biotechnology

Pub Type

Journals

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DOI Link

Keywords

genome editing, off-target, CRISPR, CRISPR-Cas9, CHANGE-seq, CIRCLE-seq, genome-wide, nuclease activity

Citation

Maragh, S. , Kolmakova, N. and Strelcov, E. (2020), CHANGE-seq reveals genetic and epigenetic effects on CRISPR–Cas9 genome-wide activity, Nature Biotechnology, [online], https://doi.org/10.1038/s41587-020-0555-7 (Accessed April 26, 2024)