title: Illegitimate translation causes unexpected gene expression from on-target out-of-frame alleles created by CRISPR-Cas9.
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.
authors.affiliation: Mutagenesis and Genomics Team, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Makino
authors.affiliation: Mutagenesis and Genomics Team, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Fukumura
authors.affiliation: Mutagenesis and Genomics Team, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Gondo
mesh_terms: D000483:Alleles; D000818:Animals; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D016368:Frameshift Mutation; D015870:Gene Expression; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D018390:Gene Targeting; D005822:Genetic Vectors; D016678:Genome; D057809:HEK293 Cells; D006720:Homozygote; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000235:genetics; D016366:Open Reading Frames; D014176:Protein Biosynthesis / Q000235:genetics*; D016364:Reading Frames; D017422:Sequence Analysis, DNA; D000074290:Zinc Finger Protein Gli3 / Q000235:genetics
chemical_list: C065683:GLI3 protein, human; C465571:Gli3 protein, mouse; D009419:Nerve Tissue Proteins; D000074290:Zinc Finger Protein Gli3