1. Illegitimate translation causes unexpected gene expression from on-target out-of-frame alleles created by CRISPR-Cas9
- Author
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Ryutaro Fukumura, Yoichi Gondo, and Shigeru Makino
- Subjects
0301 basic medicine ,Reading Frames ,Genetic Vectors ,Gene Expression ,Mice, Transgenic ,Nerve Tissue Proteins ,Biology ,medicine.disease_cause ,Article ,Frameshift mutation ,Mice ,Open Reading Frames ,03 medical and health sciences ,0302 clinical medicine ,Genome editing ,Zinc Finger Protein Gli3 ,medicine ,Animals ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,Frameshift Mutation ,Gene ,Alleles ,Genetics ,Regulation of gene expression ,Mutation ,Genome ,Multidisciplinary ,Expression vector ,Gene Expression Profiling ,Homozygote ,Gene targeting ,Sequence Analysis, DNA ,Molecular biology ,Gene expression profiling ,HEK293 Cells ,030104 developmental biology ,Gene Expression Regulation ,Protein Biosynthesis ,Gene Targeting ,NIH 3T3 Cells ,CRISPR-Cas Systems ,030217 neurology & neurosurgery - Abstract
CRISPR-Cas9 is efficient enough to knock out both alleles directly by introducing out-of-frame mutations. We succeeded in making biallelic on-target frameshift mutations of the endogenous Gli3 gene; however, the GLI3 protein was expressed in all six of the established cell lines carrying homozygous out-of-frame mutations. We developed a dual-tagged expression vector and proved that illegitimate translation (ITL) was the cause of the unexpected Gli3 expression. Thus, gene expression must be examined even if designed on-target out-of-frame sequences are introduced by genome editing. In addition, it is highly recommended to pre-examine the occurrence of ITL in vitro prior to the design and construction of any genome-editing vectors. In vitro assay systems such as the dual-tagged ITL assay system developed in this study should aid the identification and elucidation of ITL-based human diseases and gene expression.
- Published
- 2016
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