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

Autor: Lazzarotto CR; Department of Hematology, St Jude Children's Research Hospital, Memphis, TN, USA., Malinin NL; Department of Hematology, St Jude Children's Research Hospital, Memphis, TN, USA., Li Y; Department of Hematology, St Jude Children's Research Hospital, Memphis, TN, USA., Zhang R; Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA, USA., Yang Y; Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA, USA., Lee G; Department of Hematology, St Jude Children's Research Hospital, Memphis, TN, USA., Cowley E; Roche Sequencing & Life Science, Roche Diagnostics, Indianapolis, IN, USA., He Y; Department of Hematology, St Jude Children's Research Hospital, Memphis, TN, USA.; Department of Human Nutrition, Food and Animal Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI, USA., Lan X; Department of Hematology, St Jude Children's Research Hospital, Memphis, TN, USA., Jividen K; Department of Hematology, St Jude Children's Research Hospital, Memphis, TN, USA., Katta V; Department of Hematology, St Jude Children's Research Hospital, Memphis, TN, USA., Kolmakova NG; National Institute of Standards and Technology, Gaithersburg, MD, USA., Petersen CT; Department of Bone Marrow Transplantation & Cellular Therapy, St Jude Children's Research Hospital, Memphis, TN, USA., Qi Q; Department of Hematology, St Jude Children's Research Hospital, Memphis, TN, USA., Strelcov E; Physical Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA.; Maryland NanoCenter, University of Maryland, College Park, MD, USA., Maragh S; National Institute of Standards and Technology, Gaithersburg, MD, USA., Krenciute G; Department of Bone Marrow Transplantation & Cellular Therapy, St Jude Children's Research Hospital, Memphis, TN, USA., Ma J; Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA, USA., Cheng Y; Department of Hematology, St Jude Children's Research Hospital, Memphis, TN, USA., Tsai SQ; Department of Hematology, St Jude Children's Research Hospital, Memphis, TN, USA. shengdar.tsai@stjude.org.
Jazyk: angličtina
Zdroj: Nature biotechnology [Nat Biotechnol] 2020 Nov; Vol. 38 (11), pp. 1317-1327. Date of Electronic Publication: 2020 Jun 15.
DOI: 10.1038/s41587-020-0555-7
Abstrakt: 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.
Databáze: MEDLINE
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