The technique of CRISPR-Cas9 gene editing has been widely used to specifically delete the selected target genes through generating double strand breaks (DSBs) and inducing insertion and/or deletion (indel) of the genomic DNAs in the cells. We recently applied this technique to disrupt mineral dust-induced gene (mdig), a potential oncogene as previously reported, by single guide RNA (sgRNA) targeting the third exon of mdig gene in several cell types, including human bronchial epithelial cell line BEAS-2B, lung cancer cell line A549, and human triple negative breast cancer cell line MDA-MB-231 cells. In addition to the successful knockout of mdig gene in these cells, we unexpectedly noted generation of several alternatively spliced mdig mRNAs. Amplification of the mdig mRNAs during the screening of knockout clones by reverse transcription-polymerase chain reaction (RT-PCR) and the subsequent sanger sequencing of DNA revealed deletion and alternative splicing of mdig mRNAs induced by CRISPR-Cas9 gene editing. The most common deletions include nine and twenty-four nucleotides deletion around the DSBs. In addition, interestingly, some mdig mRNAs showed skipping of the entire exon 3, or alternative splicing between exon 2 and exon 8 using the new donor and accept splicing sites, leading to deletion of exons 3, 4, 5, 6, and 7. Accordingly, cautions should be taken when using CRISPR-Cas9 strategy to edit human genes due to the unintended alterative splicing of the target mRNAs. It is very likely that new proteins, some of which may be highly oncogenic, may be generated from CRISPR-Cas9 gene editing.

CRISPR-Cas9基因编辑技术已广泛用于通过产生双链断裂（DSB）并诱导细胞中基因组DNA的插入和/或缺失（indel）来特异性删除选定的靶基因。我们最近应用了该技术，通过针对多种细胞类型（包括人支气管上皮细胞系BEAS-）中mdig基因第三个外显子的单个向导RNA（sgRNA）破坏了矿物尘土诱导的基因（mdig）（先前报道的潜在致癌基因）。如图2B所示，肺癌细胞系A549和人三阴性乳腺癌细胞系MDA-MB-231细胞。除了在这些细胞中成功敲除mdig基因外，我们意外地注意到了几种交替剪接的mdig mRNA的产生。通过逆转录-聚合酶链反应（RT-PCR）筛选敲除克隆时mdig mRNA的扩增以及随后的DNA桑格测序揭示了CRISPR-Cas9基因编辑诱导的mdig mRNA的缺失和可变剪接。最常见的缺失包括DSB周围的9和24个核苷酸缺失。另外，有趣的是，一些mdig mRNA显示出跳过了整个外显子3，或使用新的供体并接受了剪接位点，在外显子2和外显子8之间进行了选择性剪接，导致外显子3、4、5、6和7缺失。因此，由于目标mRNA的意外剪接，在使用CRISPR-Cas9策略编辑人类基因时应谨慎行事。很可能是新蛋白质，其中一些可能是高度致癌的，