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

DOI PDF Web Site 被引用文献14件 参考文献26件 オープンアクセス

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<jats:title>Abstract</jats:title><jats:p>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 <jats:italic>Gli3</jats:italic> 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 <jats:italic>Gli3</jats:italic> 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 <jats:italic>in vitro</jats:italic> prior to the design and construction of any genome-editing vectors. <jats:italic>In vitro</jats:italic> 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.</jats:p>

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  • Scientific Reports

    Scientific Reports 6 (1), 39608-, 2016-12-21

    Springer Science and Business Media LLC

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