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

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.

Keywords: FOXO; FOXO3A; Nickase Cas9; Pseudogene.

Fig. 1

The human genome encodes a FOXO3A-related transcript, FOXO3B. A. BLAT alignment of the ZNF286A, FOXO3A and FOXO3B transcripts to the FOXO3B (aka ZNF286B) locus on 17p11.2 (Hg38 assembly) with the mammalian conservation track shown (UCSC Genome Browser). Red hash marks within the aligned sequence indicate sequence mismatches between the aligned transcript sequence and the genome. B. Protein sequence alignment of FOXO3A and predicted FOXO3B protein. The blue bar above the sequence indicates the forkhead domain, the red asterisk the FOXO3B stop codon and the red ‘P’ the FOXO3A Akt phosphorylation site (Thr32 FOXO3A, Thr117 FOXO3B). C. RT-qPCR measurement of FOXO3B expression across a panel of human cell lines and tissues normalized to 18S rRNA and calculated relative to the expression in fetal liver using the ΔΔCt method. Errors bars are SD of three replicates.

Fig. 2

FOXO3A and FOXO3B can be targeted for deletion independently with the spJCRISPR approach. A. Cartoon depiction of spJCRISPR targeting strategy for FOXO3A and FOXO3B loci. Red indicates 5p and 3p UTRs, green intronic sequence and blue protein coding sequence. Red arrows indicate sgRNAs, black arrows primers used for both RT-PCR and genomic PCR, blue arrows primers used for genomic PCR only and green arrows RT-PCR only. B. A cartoon depiction of the multisite Clonase strategy utilizing modified MuLE entry vectors.

Fig. 3

spJCRISPR achieves specific knockdown of FOXO3A and FOXO3B on the mRNA and protein level. A. Genomic PCR using primers flanking the FOXO3A and FOXO3B target sites for the respective spJCRISPR constructs. LHCNM2 myoblasts were infected with the described constructs and selected on puromycin. A week following selection genomic DNA was harvested and PCR performed (35 cycles). Primers specific to sequence in the FOXO3A 3pUTR were used as a loading control. Band densitometry was done by normalizing all bands to their respective NT construct band intensities. Primer pair locations are annotated in Figure 2A. B. RT-qPCR of FOXO3A mRNA using the FOXO3A-specific 3pUTR primers from RNA harvested in parallel with the gDNA. FOXO3A mRNA measurements were normalized to 18S rRNA and calculated relative to cells infected with the NT construct using the ΔΔCt method. Errors bars are SD of four replicates. Primer locations are annotated in Figure 2A. C. RT-PCR of FOXO3B mRNA using FOXO3B-specific primers and 18S rRNA primers as a loading control from the RNA harvested in parallel with the gDNA (35 cycles). Band densitometry was done as in Figure 3A. Primer locations are annotated in Figure 2A. D. Overexpression of FOXO3B in LHCNM2 myoblasts infected with a Dox-inducible FOXO3B expression construct. +Dox cells were treated with 0.1 ug/ml Dox for 48 hours and protein harvested. The western blot was performed with a monoclonal FOXO3A antibody raised against FOXO3A n-terminal residues with the epitope centered on Glu50 (Cell Signaling #2497). α-Tubulin is blotted as a loading control. E. Western blot of endogenous FOXO3A and FOXO3B in LHCNM2 myoblasts from protein harvested in parallel with the RNA and gDNA. α-Tubulin is used as the loading control.

Fig. 4

FOXO3B is phosphorylated by Akt on Thr117 but does not shuttle from the cytoplasm to the nucleus upon Akt inhibition. A. Western blot of LHCNM2 myoblasts overexpressing FOXO3B treated with DMSO (vehicle) and 200 nM insulin or 2 μM MK-2206 and 200 nM insulin for one hour. Phospho-Thr117 of FOXO3B was detected using an antibody raised against Thr24/32 of FOXO1/3 (Cell Signaling #9464). α-Tubulin is used as the loading control. B. Immunofluorescence of inducible FOXO3B overexpression in LHCNM2 myoblasts. +Dox cells were treated with 0.1 ug/ml Dox for 48 hours and then -/+ Dox cells were treated with DMSO and 200 nM insulin or 2 μM MK-2206 and 200 nM insulin for one hour. Blue is DAPI and green is staining with the FOXO3A antibody (Cell Signaling #2497) at a 1:1000 dilution. The white scale bar is 25 μm. C. A LHCNM2 myoblast line containing a Dox-inducible overexpression construct of wild-type FOXO3A. The cells were treated and stained as in Figure 4B. The white scale bar is 25 μm.