Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency (CCI), Medical Center - University of Freiburg, 79106 Freiburg, Germany.
Institute of Medical Bioinformatics and Systems Medicine, Medical Center - University of Freiburg, 79110 Freiburg, Germany; German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Freiburg, 79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, 79110 Freiburg, Germany.
Cell Stem Cell. 2021 Jun 3;28(6):1136-1147.e5. doi: 10.1016/j.stem.2021.02.002. Epub 2021 Feb 23.
Genome editing has shown great promise for clinical translation but also revealed the risk of genotoxicity caused by off-target effects of programmable nucleases. Here we describe chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq), a preclinical assay to identify and quantify chromosomal aberrations derived from on-target and off-target activities of CRISPR-Cas nucleases or transcriptional activator-like effector nucleases (TALENs), respectively, in human hematopoietic stem cells (HSCs). Depending on the employed designer nuclease, CAST-Seq detected translocations in 0%-0.5% of gene-edited human CD34+ HSCs, and up to 20% of on-target loci harbored gross rearrangements. Moreover, CAST-Seq detected distinct types of chromosomal aberrations, such as homology-mediated translocations, that are mediated by homologous recombination and not off-target activity. CAST-Seq is a sensitive assay able to identify and quantify unintended chromosomal rearrangements in addition to the more typical mutations at off-target sites. CAST-Seq analyses may be particularly relevant for therapeutic genome editing to enable thorough risk assessment before clinical application of gene-edited products.
基因组编辑在临床转化方面显示出巨大的前景，但也揭示了可编程核酸酶的脱靶效应引起遗传毒性的风险。在这里，我们描述了通过单靶向连接介导的 PCR 测序 (CAST-Seq) 进行的染色体畸变分析，这是一种临床前检测方法，用于识别和定量源自 CRISPR-Cas 核酸酶或转录激活因子样效应核酸酶 (TALEN) 的靶向和脱靶活性的人造血干细胞 (HSCs) 中的染色体畸变。根据所使用的设计核酸酶，CAST-Seq 在基因编辑的人类 CD34+HSCs 中检测到 0%-0.5%的易位，多达 20%的靶向位点存在大片段重排。此外，CAST-Seq 还检测到了不同类型的染色体畸变，如同源介导的易位，这些易位是由同源重组介导的，而不是脱靶活性。CAST-Seq 是一种灵敏的检测方法，能够识别和定量非靶向位点的典型突变以外的非预期染色体重排。CAST-Seq 分析对于治疗性基因组编辑可能特别重要，以便在临床应用基因编辑产品之前进行全面的风险评估。
