当前位置:科学网首页 > 小柯机器人 >详情
CRISPR-Cas9切割后原代人类T细胞中频繁出现非整倍体
作者:小柯机器人 发布时间:2022/7/3 18:51:27

以色列特拉维夫大学Adi Barzel等研究人员合作发现,CRISPR-Cas9切割后原代人类T细胞中频繁出现非整倍体。相关论文于2022年6月30日在线发表在《自然—生物技术》杂志上。

利用单细胞RNA测序,研究人员调查了用CRISPR-Cas9和靶向T细胞受体(TCR)链和程序性细胞死亡蛋白1基因的向导RNA转染的原代人类T细胞的基因组编辑结果。转染四天后,研究人员发现多达9%的细胞出现了携带TCRα基因座的14号染色体缺失,多达1.4%的细胞出现了14号染色体增加。携带TCRβ基因座的7号染色体在9.9%的细胞中被截断。

使用荧光原位杂交和数字微滴PCR对畸变进行了验证。非整倍体与增殖减少、诱导P53激活和细胞死亡有关。然而,在转染后11天,0.9%的T细胞仍有14号染色体丢失。因此,非整倍体和染色体截断是CRISPR-Cas9切割的频繁结果,在临床方案中应加以监测并尽量减少。

据悉,异体T细胞治疗的多项临床试验使用定点核酸酶来破坏TCR和其他基因。

附:英文原文

Title: Frequent aneuploidy in primary human T cells after CRISPR–Cas9 cleavage

Author: Nahmad, Alessio David, Reuveni, Eli, Goldschmidt, Ella, Tenne, Tamar, Liberman, Meytal, Horovitz-Fried, Miriam, Khosravi, Rami, Kobo, Hila, Reinstein, Eyal, Madi, Asaf, Ben-David, Uri, Barzel, Adi

Issue&Volume: 2022-06-30

Abstract: Multiple clinical trials of allogeneic T cell therapy use site-specific nucleases to disrupt T cell receptor (TCR) and other genes1,2,3,4,5,6. In this study, using single-cell RNA sequencing, we investigated genome editing outcomes in primary human T cells transfected with CRISPR–Cas9 and guide RNAs targeting genes for TCR chains and programmed cell death protein 1. Four days after transfection, we found a loss of chromosome 14, harboring the TCRα locus, in up to 9% of the cells and a chromosome 14 gain in up to 1.4% of the cells. Chromosome 7, harboring the TCRβ locus, was truncated in 9.9% of the cells. Aberrations were validated using fluorescence in situ hybridization and digital droplet PCR. Aneuploidy was associated with reduced proliferation, induced p53 activation and cell death. However, at 11days after transfection, 0.9% of T cells still had a chromosome 14 loss. Aneuploidy and chromosomal truncations are, thus, frequent outcomes of CRISPR–Cas9 cleavage that should be monitored and minimized in clinical protocols.

DOI: 10.1038/s41587-022-01377-0

Source: https://www.nature.com/articles/s41587-022-01377-0

期刊信息

Nature Biotechnology:《自然—生物技术》,创刊于1996年。隶属于施普林格·自然出版集团,最新IF:31.864
官方网址:https://www.nature.com/nbt/
投稿链接:https://mts-nbt.nature.com/cgi-bin/main.plex