Open AccessIllegitimate translation causes unexpected gene expression from on-target out-of-frame alleles created by CRISPR-Cas9
Author(s) -
Seiya Makino,
Ryutaro Fukumura,
Yoichi Gondo
Publication year - 2016
Publication title -
scientific reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.24
H-Index - 213
ISSN - 2045-2322
DOI - 10.1038/srep39608
Subject(s) - frameshift mutation , crispr , cas9 , genome editing , gene , genetics , biology , translation (biology) , computational biology , allele , mutation , gene expression , expression vector , messenger rna , recombinant dna
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.