Science for Life Laboratory (SciLifeLab), Research Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Methods Mol Biol. 2021;2162:261-281. doi: 10.1007/978-1-0716-0687-2_15.
Clustered regularly interspaced palindromic repeat (CRISPR) systems are revolutionizing many areas of biology and medicine, where they are increasingly utilized as therapeutic tools for correcting disease-causing mutations. From a clinical perspective, unintended off-target (OT) DNA double-strand break (DSB) induction by CRISPR nucleases represents a major concern. Therefore, in recent years considerable effort has been dedicated to developing methods for assessing the OT activity of CRISPR nucleases, which in turn can be used to guide engineering of nucleases with minimal OT activity. Here we describe a detailed protocol for quantifying OT DSBs genome-wide in cultured cells transfected with CRISPR enzymes, based on the breaks labeling in situ and sequencing (BLISS) method that we have previously developed. CRISPR-BLISS is versatile and scalable, and allows assessment of multiple guide RNAs in different cell types and time points following cell transfection or transduction.
成簇规律间隔短回文重复 (CRISPR) 系统正在彻底改变生物学和医学的许多领域，它们越来越多地被用作纠正致病突变的治疗工具。从临床角度来看，CRISPR 核酸酶引起的非预期脱靶 (OT) DNA 双链断裂 (DSB) 是一个主要关注点。因此，近年来，人们致力于开发评估 CRISPR 核酸酶 OT 活性的方法，这些方法反过来又可用于指导具有最小 OT 活性的核酸酶的工程设计。在这里，我们描述了一种基于我们之前开发的断裂原位标记和测序 (BLISS) 方法，在转染了 CRISPR 酶的培养细胞中定量检测全基因组 OT DSB 的详细方案。CRISPR-BLISS 具有多功能性和可扩展性，并允许在细胞转染或转导后不同的细胞类型和时间点评估多个向导 RNA。
