A splice junction-targeted CRISPR approach (spJCRISPR) reveals human FOXO3B to be a protein-coding gene.

TitleA splice junction-targeted CRISPR approach (spJCRISPR) reveals human FOXO3B to be a protein-coding gene.
Publication TypeJournal Article
Year of Publication2018
AuthorsSanto EE, Paik J
JournalGene
Volume673
Pagination95-101
Date Published2018 Oct 05
ISSN1879-0038
KeywordsAlternative Splicing, Cloning, Molecular, CRISPR-Cas Systems, Cytosol, Deoxyribonuclease I, Embryonic Stem Cells, Forkhead Box Protein O3, Gene Editing, Gene Expression Regulation, Genetic Techniques, HEK293 Cells, Humans, Mutation, Myoblasts, Phosphorylation, Proto-Oncogene Proteins c-akt, RNA, Guide, RNA, Small Interfering
Abstract

The rapid development of CRISPR technology is revolutionizing molecular approaches to the dissection of complex biological phenomena. Here we describe an alternative generally applicable implementation of the CRISPR-Cas9 system that allows for selective knockdown of extremely homologous genes. This strategy employs the lentiviral delivery of paired sgRNAs and nickase Cas9 (Cas9D10A) to achieve targeted deletion of splice junctions. This general strategy offers several advantages over standard single-guide exon-targeting CRISPR-Cas9 such as greatly reduced off-target effects, more restricted genomic editing, routine disruption of target gene mRNA expression and the ability to differentiate between closely related genes. Here we demonstrate the utility of this strategy by achieving selective knockdown of the highly homologous human genes FOXO3A and suspected pseudogene FOXO3B. We find the spJCRISPR strategy to efficiently and selectively disrupt FOXO3A and FOXO3B mRNA and protein expression; thus revealing that the human FOXO3B locus encodes a bona fide human gene. Unlike FOXO3A, we find the FOXO3B protein to be cytosolically localized in both the presence and absence of active Akt. The ability to selectively target and efficiently disrupt the expression of the closely-related FOXO3A and FOXO3B genes demonstrates the efficacy of the spJCRISPR approach.

DOI10.1016/j.gene.2018.06.048
Alternate JournalGene
PubMed ID29925039
PubMed Central IDPMC6064391
Grant ListR01 AG048284 / AG / NIA NIH HHS / United States
R01 CA219957 / CA / NCI NIH HHS / United States
R56 AG048284 / AG / NIA NIH HHS / United States
Related Faculty: 
Ji-Hye Paik, Ph.D.

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