Genetic modification of human leukemic cell lines using CRISPR-Cas9 has become a staple of gene-function studies. Single-cell cloning of modified cells is frequently used to facilitate studies of gene function. Inherent in this approach is an assumption that the genetic drift, amplified in some cell lines by mutations in DNA replication and repair machinery, as well as non-genetic factors will not introduce significant levels of experimental cellular heterogeneity in clones derived from parental populations. In this study, we characterize the variation in cell death of fifty clonal cell lines generated from human Jurkat and MOLT-4 T-cells edited by CRISPR-Cas9. We demonstrate a wide distribution of sensitivity to chemotherapeutics between non-edited clonal human leukemia T-cell lines, and also following CRISPR-Cas9 editing at the NLRP1 locus, or following transfection with non-targeting sgRNA controls. The cell death sensitivity profile of clonal cell lines was consistent across experiments and failed to revert to the non-clonal parental phenotype. Whole genome sequencing of two clonal cell lines edited by CRISPR-Cas9 revealed unique and shared genetic variants, which had minimal read support in the non-clonal parental population and were not suspected CRISPR-Cas9 off-target effects. These variants included genes related to cell death and drug metabolism. The variation in cell death phenotype of clonal populations of human T-cell lines may be a consequence of T-cell line genetic instability, and to a lesser extent clonal heterogeneity in the parental population or CRISPR-Cas9 off-target effects not predicted by current models. This work highlights the importance of genetic variation between clonal T-cell lines in the design, conduct, and analysis of experiments to investigate gene function after single-cell cloning.

使用CRISPR-Cas9对人类白血病细胞系进行遗传修饰已成为基因功能研究的重要内容。修饰细胞的单细胞克隆通常用于促进基因功能的研究。这种方法的固有假设是，在某些细胞系中由于DNA复制和修复机制的突变而导致的遗传漂移以及非遗传因素不会在亲代群体的克隆中引入明显水平的实验性细胞异质性。在这项研究中，我们表征了由CRISPR-Cas9编辑的人类Jurkat和MOLT-4 T细胞产生的五十个克隆细胞系的细胞死亡变化。我们证明了未经编辑的克隆性人类白血病T细胞系之间以及对CRISPR-Cas9编辑后对化疗药物敏感性的广泛分布NLRP1位点，或转染非靶向sgRNA对照后。克隆细胞系的细胞死亡敏感性分布在整个实验中是一致的，并且无法恢复为非克隆亲本表型。由CRISPR-Cas9编辑的两个克隆细胞系的全基因组测序揭示了独特且共享的遗传变异，在非克隆亲本群体中具有最小的阅读支持，并且不怀疑CRISPR-Cas9脱靶效应。这些变体包括与细胞死亡和药物代谢有关的基因。人类T细胞系克隆种群细胞死亡表型的变化可能是T细胞系遗传不稳定性的结果，并且在较小程度上是父母群体的克隆异质性或目前尚无法预测的CRISPR-Cas9脱靶效应楷模。