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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.

SUBMITTER: Makino S

PROVIDER: S-EPMC5175197 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Illegitimate translation causes unexpected gene expression from on-target out-of-frame alleles created by CRISPR-Cas9.

Makino Shigeru S Fukumura Ryutaro R Gondo Yoichi Y

Scientific reports 20161221

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 exami ...[more]

PMID: 28000783

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