Illegitimate translation causes unexpected gene expression from on-target out-of-frame alleles created by CRISPR-Cas9.
Sci Rep
; 6: 39608, 2016 12 21.
Article
in En
| MEDLINE
| ID: mdl-28000783
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Protein Biosynthesis
/
Gene Expression Regulation
/
Frameshift Mutation
/
CRISPR-Cas Systems
Type of study:
Etiology_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
Sci Rep
Year:
2016
Document type:
Article
Affiliation country:
Japan