School of Neurobiology, Biochemistry and Biophysics, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Varda and Boaz Dotan Center for Advanced Therapies, Tel Aviv Sourasky Medical Center and Tel Aviv University, Tel Aviv, Israel.
Nat Biotechnol. 2022 Dec;40(12):1807-1813. doi: 10.1038/s41587-022-01377-0. Epub 2022 Jun 30.
Multiple clinical trials of allogeneic T cell therapy use site-specific nucleases to disrupt T cell receptor (TCR) and other genes. 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 11 days 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.
多项异体 T 细胞治疗的临床试验使用位点特异性核酸酶破坏 T 细胞受体 (TCR) 和其他基因。在这项研究中，我们使用单细胞 RNA 测序，研究了用 CRISPR-Cas9 和靶向 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 T 细胞中的基因组编辑结果。转染后 4 天，我们发现多达 9%的细胞中丢失了携带 TCRα 基因座的 14 号染色体，多达 1.4%的细胞中获得了 14 号染色体。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液滴 PCR 验证了这些畸变。非整倍体与增殖减少、p53 激活和细胞死亡有关。然而，在转染后 11 天，仍有 0.9%的 T 细胞存在 14 号染色体缺失。因此，CRISPR-Cas9 切割的非整倍体和染色体截断是常见的结果，在临床方案中应进行监测并尽量减少。
