論文 - 詳細
RRC ID | 48754 |
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著者 | Makino S, Fukumura R, Gondo Y. |
タイトル | Illegitimate translation causes unexpected gene expression from on-target out-of-frame alleles created by CRISPR-Cas9. |
ジャーナル | Sci Rep |
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. |
巻・号 | 6 |
ページ | 39608 |
公開日 | 2016-12-21 |
DOI | 10.1038/srep39608 |
PII | srep39608 |
PMID | 28000783 |
PMC | PMC5175197 |
MeSH | Alleles Animals CRISPR-Cas Systems* Clustered Regularly Interspaced Short Palindromic Repeats Frameshift Mutation* Gene Expression Gene Expression Profiling Gene Expression Regulation* Gene Targeting Genetic Vectors Genome HEK293 Cells Homozygote Humans Mice Mice, Transgenic Mutation NIH 3T3 Cells Nerve Tissue Proteins / genetics Open Reading Frames Protein Biosynthesis / genetics* Reading Frames Sequence Analysis, DNA Zinc Finger Protein Gli3 / genetics |
IF | 3.998 |
引用数 | 16 |
WOS 分野 | BIOCHEMISTRY & MOLECULAR BIOLOGY |
リソース情報 | |
遺伝子材料 | pSpCas9-mGli3-exon2_sgRNA (RDB15037) pSpCas9-mGli3-exon3_sgRNA (RDB15038) pCMV-3xFLAG-mGli3_WT (RDB15039) pCMV-3xFLAG-mGli3_WT-HA (RDB15040) pCMV-3xFLAG-mGli3_del97G-HA (RDB15041) pCMV-3xFLAG-mGli3_insGafter97G-HA (RDB15042). |
ヒト・動物細胞 | NIH/3T3(RCB2767) |