RRC ID 48754
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)