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

作者：Lazzarotto, Cicera R; Malinin, Nikolay L; Li, Yichao; Zhang, Ruochi; Yang, Yang; Lee, GaHyun; Cowley, Eleanor; He, Yanghua; Lan, Xin; Jividen, Kasey; Katta, Varun; Kolmakova, Natalia G; Petersen, Christopher T; Qi, Qian; Strelcov, Evgheni; Maragh, Samantha; Krenciute, Giedre; Ma, Jian; Cheng, Yong; Tsai, Shengdar Q^*

来源：Nature Biotechnology, 2020, 38(11): 1317-1327.

DOI：10.1038/s41587-020-0555-7

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.

• 单位
  National Institute of Standards and Technology