本期文章：《自然—生物技术》：Online/在线发表

美国圣犹大儿童研究医院Shengdar Q. Tsai研究小组利用CHANGE-seq技术，揭示了影响CRISPR-Cas9全基因组活性的遗传和表观遗传因素。相关论文于2020年6月15日在线发表在《自然—生物技术》杂志上。

研究人员报道了“通过测序对核酸酶全基因组效应进行高通量分析的循环化”（CHANGE-seq）技术，这是一种可扩展的、基于tagmentation的体外Cas9全基因组活性测量方法。研究人员将CHANGE-seq应用于人类原代T细胞中13个治疗相关基因座上的110个单向导RNA靶标，并鉴定了201,934个脱靶位点，从而能够训练机器学习模型来预测脱靶活性。

通过比较匹配的全基因组脱靶、染色质修饰和可及性以及转录数据，研究人员发现细胞脱靶活性发生在活跃动子、增强子和转录区域附近的可能性增至两倍至四倍。最后，对八个独立基因组中六个目标的CHANGE-seq分析显示，人类单核苷酸变异在约15.2％的脱靶位点上对活性有显著影响。因此，CHANGE-seq是一种简化、敏感且可扩展的方法，可用于了解基因组编辑器的特异性。

据悉，当前的方法可以阐明CRISPR–Cas9核酸酶的全基因组活性，但不容易扩展到充分理解控制Cas9特异性原理所需的通量。

附：英文原文

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

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

Issue&Volume: 2020-06-15

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.

DOI: 10.1038/s41587-020-0555-7

Source: https://www.nature.com/articles/s41587-020-0555-7