Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Department of Quantitative Health Sciences, Cleveland Clinic Lerner Research Institute, Cleveland, OH, 44195, USA.
Nat Commun. 2019 Sep 6;10(1):4056. doi: 10.1038/s41467-019-12028-5.
The introduction of insertion-deletions (INDELs) by non-homologous end-joining (NHEJ) pathway underlies the mechanistic basis of CRISPR-Cas9-directed genome editing. Selective gene ablation using CRISPR-Cas9 is achieved by installation of a premature termination codon (PTC) from a frameshift-inducing INDEL that elicits nonsense-mediated decay (NMD) of the mutant mRNA. Here, by examining the mRNA and protein products of CRISPR targeted genes in a cell line panel with presumed gene knockouts, we detect the production of foreign mRNAs or proteins in ~50% of the cell lines. We demonstrate that these aberrant protein products stem from the introduction of INDELs that promote internal ribosomal entry, convert pseudo-mRNAs (alternatively spliced mRNAs with a PTC) into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (ESEs). Our results reveal challenges to manipulating gene expression outcomes using INDEL-based mutagenesis and strategies useful in mitigating their impact on intended genome-editing outcomes.
非同源末端连接（NHEJ）途径介导的插入缺失（INDEL）是 CRISPR-Cas9 指导的基因组编辑的机制基础。通过诱导无义介导的降解（NMD）的移码 INDEL 在 CRISPR-Cas9 中安装提前终止密码子（PTC），从而实现对选择性基因缺失。在此，通过检查具有假定基因敲除的细胞系面板中 CRISPR 靶向基因的 mRNA 和蛋白质产物，我们在大约 50%的细胞系中检测到了突变型 mRNA 的产生。我们证明，这些异常蛋白质产物源自促进内部核糖体进入、将假 mRNA（具有 PTC 的可变剪接 mRNA）转化为编码蛋白质的分子，或通过破坏外显子剪接增强子（ESE）诱导外显子跳跃的 INDEL 的引入。我们的结果揭示了使用基于 INDEL 的诱变来操纵基因表达结果的挑战，以及在减轻其对预期基因组编辑结果的影响方面有用的策略。
