CRISPR artificial splicing factors

Abstract

Alternative splicing allows expression of mRNA isoforms from a single gene, expanding the diversity of the proteome. Its prevalence in normal biological and disease processes warrant precise tools for modulation. Here we report the engineering of CRISPR Artificial Splicing Factors (CASFx) based on RNA-targeting CRISPR-Cas systems. We show that simultaneous exon inclusion and exclusion can be induced at distinct targets by differential positioning of CASFx. We also create inducible CASFx (iCASFx) using the FKBP-FRB chemical-inducible dimerization domain, allowing small molecule control of alternative splicing. Finally, we demonstrate the activation of SMN2 exon 7 splicing in spinal muscular atrophy (SMA) patient fibroblasts, suggesting a potential application of the CASFx system.

Fig. 1. Exon inclusion induced by CASFx-1 (RBFOX1N-dCasRx-C).

a Schematic of the CASFx-1 and SMN2 minigene. The RNA binding domain of RBFOX1 was substituted by dCasRx to create an RNA-guided CRISPR Artificial Splicing Factor 1 (CASFx-1) RBFOX1N-dCasRx-C. The SMN2 minigene on plasmid pCI-SMN2 contains exons 6 (E6) and 8 (E8) that are constitutively spliced, and exon 7 (E7) that is alternatively spliced, and the intervening introns, driven by the CMV promoter (pCMV). Four designed guide RNA (gRNA) target sites are indicated by numbered boxes 1 through 4 within the intron between E7 and E8. pCI-F and pCI-R indicate primers used for semi-quantitative RT-PCR assays. b Gel image of splicing RT-PCR using primers pCI-F and pCI-R on SMN2 minigene transcripts in cells co-transfected with control GFP plasmid (pmaxGFP), unfused dCasRx, or CASFx-1 (RBFOX1N-dCasRx-C), and the indicated gRNAs, numbers correspond to those in panel a with dash indicating the range of gRNAs used. “C” indicates a control gRNA not matching SMN2 minigene. Upper and lower bands correspond to the E7-included and -excluded transcripts, respectively. Reference splicing bands derived from E7-excluded sample (pCI-SMN2-MS2) and E7-included sample (pCI-SMN2-MS2 + MCP-RBFOX) serve as molecular weight markers (M) for inclusion (Inc) and exclusion (Exc) events. The image shown is representative of two independent experiments. Uncropped gel images are included in the Source Data file.

Fig. 2. Activation and repression of exon by differential positioning of CASFx.

a Schematic of the CRISPR artificial splicing factors, CASFx-1 (RBFOX1N-dCasRx-C), CASFx-2 (RBM38-dCasRx), CASFx-3 (dCasRx-RBM38) and SMN2 minigene, as well as a set of three target sites downstream of E7 (DN: gRNA-1 through 3) and one target site target within E7 (EX). b Upper panel shows the inclusion/exclusion (inc/exc) ratio fold-change assayed by qRT-PCR on SMN2 minigene transcripts in cells co-transfected with dCasRx, CASFx, and the indicated gRNAs, with respect to the GFP control (set to 1). Data are represented as mean ± SD (n = 3). Lower panel shows a gel image of semi-quantitative splicing RT-PCR of the corresponding samples. “C” indicates a control gRNA without matching SMN2 minigene sequence; “DN” indicates a pool of three gRNAs (SMN2-gRNA-1 through 3) targeting downstream of E7; “EX” indicates a gRNA targeting within E7. Uncropped gel images and qRT-PCR values are included in the Source Data file.

Fig. 3. Simultaneous activation and repression of two independent exons by RBFOX1N-dCasRx-C.

a Schematic of the CASFx-1, various gRNA architectures, as well as the RG6 and SMN2 minigenes. SMN2-DN gRNAs is a pool of three gRNAs (SMN2-gRNA-1 through 3), each expressed by a separate plasmid, targeting the corresponding numbered locations on the SMN2 minigene. RG6-SA targets splice acceptor of RG6 cassette exon (CX). DR-SMN2-2-DR is SMN2 target gRNA 2 flanked by two direct repeats (DR). DR-RG6-SA-DR contains spacer against RG6-CX splice acceptor flanked by two DRs. SMN2-DN-RG6-SA is a polycistronic pre-gRNA with spacers targeting three DN sites on SMN2 downstream intron and RG6-CX splice acceptors intervened by DRs. b Upper panel shows inclusion/exclusion (inc/exc) ratio fold-changes assayed by qRT-PCR on SMN2 minigene transcripts in cells co-transfected with the two minigene plasmids, CASFx-1 (RBFOX1N-dCasRx-C) and the indicated gRNAs. SMN2 and RG6 splicing changes are represented by gray and slash pattern filled bars, respectively. Fold-changes are relative to cells transfected with GFP control (set to 1). Data are represented as mean ± SD (n = 3). Lower panel shows a gel image of semi-quantitative splicing RT-PCR of RG6 and SMN2 minigene transcripts in cells co-transfected with the two minigene plasmids, CASFx-1 and the indicated gRNAs. Uncropped gel images and qRT-PCR values are included in the Source Data file.

Fig. 4. Efficiency and specificity comparison of exon activation induced by CASFx and PUF-ESF.

a Schematic of the three PUF-based engineered splicing factors (PUF-ESFs), two all-in-one CASFx-1 plasmids and the target sites of gRNA 1-3 on the SMN2 minigene. ESF mix represents a pool of three PUF-ESF constructs. b Upper panel shows inclusion/exclusion (inc/exc) ratio fold-changes assayed by qRT-PCR on SMN2 minigene transcripts in transfected cells with respect to GFP control (set to 1). Data are represented as mean ± SD (n = 3). Lower panel shows a gel image of semi-quantitative splicing RT-PCR of SMN2 minigene transcripts in cells transfected with indicated constructs. c Plots showing changes in percentage spliced-in (ΔΨ) and rMATS-computed Benjamini-Hochberg False Discovery Rate (FDR) of cassette exons in cells transfected with indicated constructs compared with GFP control, as determined by RNA-seq (n = 2 biological replicates per sample). Exons with significant changes (|ΔΨ | ≥ 0.1 and FDR ≤ 0.01) are colored in red. ΔΨ of SMN2-minigene was calculated based on the RNA-seq reads matching with minigene-specific sequences and is indicated as blue vertical dotted line. Uncropped gel images and qRT-PCR values are included in the Source Data file.

Fig. 5. Chemically inducible exon activation by iCASFx.

a Schematic of the two-peptide CRISPR artificial splicing factors inducible by rapamycin. The RNA binding module (FKBP- dCasRx or dCasRx-FKBP) and effector module (RBFOX1N-FRB-C) containing the splicing activator domains are expressed separately as two peptides, fused to FKBP or FRB, respectively. FKBP and FRB can be induced to interact by rapamycin, bringing together the RNA binding module and the splicing activator module, and when guided by gRNAs, assemble at the target to activate exon inclusion. b Upper panel shows inclusion/exclusion (inc/exc) ratio fold-changes assayed by qRT-PCR on SMN2 minigene transcripts in cells co-transfected with the indicated constructs, and cultured with (“+”) or without (“−”) rapamycin. Fold-changes are relative to GFP-transfected sample. Data are represented as mean ± SD (n = 3). Lower panel shows a gel image of semi-quantitative splicing RT-PCR of SMN2 minigene transcripts in cells co-transfected with the indicated constructs, and cultured with (“+”) or without (“−”) rapamycin. Uncropped gel images and qRT-PCR values are included in the Source Data file.

Fig. 6. Exon activation in SMA patient cells by CASFx.

a Schematic of the two all-in-one CASFx constructs (CASFx-1: RBFOX1N-dCasRx-C and RBFOX1N-dPspCas13b-C) and six gRNAs targeting the corresponding numbered locations on the endogenous intron. b Upper panel shows inclusion/exclusion ratio fold-changes of the endogenous SMN2 gene in patient cells assayed by qRT-PCR 5 days after nucleofection with the indicated CASFx and gRNA relative to GFP control (set to 1). Data are represented as mean ± SD (n = 3). Lower panel shows a gel image of semi-quantitative splicing RT-PCR using primers En-F and En-R on endogenous SMN2 transcripts in the indicated samples. Uncropped gel images and qRT-PCR values are included in the Source Data file.