Structural rearrangements generate cell-specific, gene-independent CRISPR-Cas9 loss of fitness effects

CRISPR-Cas9 genome editing is widely used to study gene function, from basic biology to biomedical research. Structural rearrangements are a ubiquitous feature of cancer cells and their impact on the functional consequences of CRISPR-Cas9 gene-editing has not yet been assessed.

Utilizing CRISPR-Cas9 knockout screens for 250 cancer cell lines, we demonstrate that targeting structurally rearranged regions, in particular tandem or interspersed amplifications, is highly detrimental to cellular fitness in a gene-independent manner. In contrast, amplifications caused by whole chromosomal duplication have little to no impact on fitness. This effect is cell line specific and dependent on the ploidy status. We devise a copy-number ratio metric that substantially improves the detection of gene-independent cell fitness effects in CRISPR-Cas9 screens. Furthermore, we develop a computational tool, called Crispy, to account for these effects on a single sample basis and provide corrected gene fitness effects.

Our analysis demonstrates the importance of structural rearrangements in mediating the effect of CRISPR-Cas9-induced DNA damage, with implications for the use of CRISPR-Cas9 gene-editing in cancer cells.

CRISPR-Cas9基因组编辑（CRISPR-Cas9 genome editing）被广泛应用于基因功能研究，涵盖基础生物学至生物医学研究的全范畴。结构重排（structural rearrangements）是癌细胞普遍存在的特征，但其对CRISPR-Cas9基因编辑所产生的功能后果的影响尚未得到评估。
本研究依托针对250株癌细胞系的CRISPR-Cas9敲除筛选（CRISPR-Cas9 knockout screens），证实靶向结构重排区域——尤其是串联扩增或散在扩增区域——会以基因非依赖的方式对细胞适应性产生极强的损害作用。与之形成对比的是，全染色体复制所导致的扩增几乎不会对细胞适应性产生影响。该效应具有癌细胞系特异性，且依赖于细胞的倍性状态。本研究设计了一种拷贝数比值指标，可显著提升CRISPR-Cas9敲除筛选中基因非依赖型细胞适应性效应的检测效能。此外，本研究开发了一款名为Crispy的计算工具，可在单样本层面上校正上述效应，并输出校正后的基因适应性效应值。
本研究的分析结果证实，结构重排在介导CRISPR-Cas9诱导的DNA损伤效应中发挥着关键作用，这一发现对CRISPR-Cas9基因编辑在癌细胞中的应用具有重要参考价值。