近日，德国弗莱堡大学Toni Cathomen、Giandomenico Turchiano等研究人员合作，利用CAST-Seq对基因编辑的人类干细胞中的染色体重排进行了定量评估。这一研究成果于2021年2月23日在线发表在《细胞—干细胞》上。

研究人员报道了通过单个靶向接头介导的PCR测序进行染色体畸变分析技术（CAST-Seq），这是一种临床前测定方法，可在人类造血干细胞（HSC）中识别和量化CRISPR-Cas核酸酶或转录激活因子样效应子的核酸酶（TALEN）引起的染色体畸变。根据所使用的设计者核酸酶的不同，CAST-Seq检测到基因编辑的人类CD34⁺HSC的0％–0.5％易位，并且高达20％的目标位点具有总体重排。

此外，CAST-Seq检测到不同类型的染色体畸变，例如同源介导的易位，其由同源重组而不是脱靶活性介导。CAST-Seq是一种灵敏的测定法，除了在脱靶位点上更典型的突变外，还能够鉴定和量化意外的染色体重排。CAST-Seq分析与治疗性基因组编辑密切相关，从而可以在临床应用基因编辑产品之前进行全面的风险评估。 

据介绍，基因组编辑已显示出对临床转化的巨大希望，但同时也揭示了由可编程核酸酶脱靶作用引起的基因毒性风险。

附：英文原文

Title: Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq

Author: Giandomenico Turchiano, Geoffroy Andrieux, Julia Klermund, Georges Blattner, Valentina Pennucci, Melina el Gaz, Gianni Monaco, Sushmita Poddar, Claudio Mussolino, Tatjana I. Cornu, Melanie Boerries, Toni Cathomen

Issue&Volume: 2021-02-23

Abstract: Genome editing has shown great promise for clinical translation but also revealedthe risk of genotoxicity caused by off-target effects of programmable nucleases. Herewe describe chromosomal aberrations analysis by single targeted linker-mediated PCRsequencing (CAST-Seq), a preclinical assay to identify and quantify chromosomal aberrationsderived from on-target and off-target activities of CRISPR-Cas nucleases or transcriptionalactivator-like effector nucleases (TALENs), respectively, in human hematopoietic stemcells (HSCs). Depending on the employed designer nuclease, CAST-Seq detected translocationsin 0%–0.5% of gene-edited human CD34+ HSCs, and up to 20% of on-target loci harboredgross rearrangements. Moreover, CAST-Seq detected distinct types of chromosomal aberrations,such as homology-mediated translocations, that are mediated by homologous recombinationand not off-target activity. CAST-Seq is a sensitive assay able to identify and quantifyunintended chromosomal rearrangements in addition to the more typical mutations atoff-target sites. CAST-Seq analyses may be particularly relevant for therapeutic genomeediting to enable thorough risk assessment before clinical application of gene-editedproducts.

DOI: 10.1016/j.stem.2021.02.002

Source: https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(21)00052-7