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基因编辑技术已被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/或删除（indel）来特异性删除选定的靶基因。我们最近应用这种技术来破坏矿尘诱导基因（mdig），这是一种先前报道的潜在致癌基因，通过单向导RNA（sgRNA）靶向多种细胞类型中mdig基因的第三外显子，包括人支气管上皮细胞系BEAS-图2B，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 基因之外，我们还意外地注意到生成了几种选择性剪接的 mdig mRNA。在通过逆转录聚合酶链式反应 (RT-PCR) 筛选敲除克隆的过程中，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 周围的九个和二十四个核苷酸缺失。此外，有趣的是，一些 mdig mRNA 显示整个外显子 3 的跳过，或使用新的供体和接受剪接位点在外显子 2 和外显子 8 之间进行选择性剪接，导致外显子 3、4、5、6 和 7 的缺失。因此，在使用 CRISPR-Cas9 策略编辑人类基因时应谨慎，因为目标 mRNA 会出现意外的可变剪接。 CRISPR-Cas9 基因编辑很可能产生新的蛋白质，其中一些可能具有高度致癌性。