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CRISPR-Cas9 gene editing causes alternative splicing of the targeting mRNA
- Source :
- Biochem Biophys Res Commun
- Publication Year :
- 2020
- Publisher :
- Elsevier BV, 2020.
-
Abstract
- 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.
- Subjects :
- 0301 basic medicine
Biophysics
Biology
Polymorphism, Single Nucleotide
Biochemistry
Article
Cell Line
Dioxygenases
03 medical and health sciences
Exon
0302 clinical medicine
Genome editing
CRISPR-Associated Protein 9
Humans
CRISPR
RNA, Messenger
Guide RNA
Molecular Biology
Gene
Sequence Deletion
Gene Editing
Histone Demethylases
Base Sequence
Alternative splicing
Nuclear Proteins
Exons
Cell Biology
Cell biology
Alternative Splicing
030104 developmental biology
030220 oncology & carcinogenesis
RNA splicing
Human genome
CRISPR-Cas Systems
RNA, Guide, Kinetoplastida
Subjects
Details
- ISSN :
- 0006291X
- Volume :
- 528
- Database :
- OpenAIRE
- Journal :
- Biochemical and Biophysical Research Communications
- Accession number :
- edsair.doi.dedup.....c64f917040110d66a412d636431f1c4a