Abstract
We report here the engineering of CRISPR Artificial Splicing Factors (CASFx) based on an RNA-targeting CRISPR/Cas system. We showed that simultaneous exon inclusion and exclusion can be induced at distinct targets by differential positioning of CASFx. We also created inducible CASFx (iCASFx) using the FKBP-FRB chemical-inducible dimerization domain, allowing small molecule control of alternative splicing.
Main
Splicing is a process in which segments of a pre-mRNA called introns are removed while segments called exons are joined together to form mature mRNA1. Alternative splicing is a phenomenon in which different exon segments are spliced together to form mature mRNA with different sequences, greatly expanding the protein repertoire by allowing different proteins to be coded by a single gene. The process of alternative splicing is deeply embedded in gene regulatory networks by controlling gene isoform expression of > 90% of human genes2. Given such prevalence, dysregulation of (alternative) splicing has been implicated in many diseases3–5. RNA-seq is a powerful method to “read” transcriptomes for changes in alternative splicing in different cell types, conditions and diseases2,5,6. However, Scalable tools for precisely and reversibly “writing” alterative splicing is lacking.
Fusion of RNA regulatory proteins to heterologous RNA binding domains, such as Pumilio/PUF, MS2 coat protein (MCP), PP7 coat protein (PCP), and λN, have allowed artificial modulation of RNA processes7–12. For example, tethering of serine-rich (RS) domains or Glycine-rich (Gly) domains by engineered PUF domains to exons induce their inclusion or exclusion, respectively9. However, these artificial RNA effectors require either protein engineering or insertion of artificial tags to target RNA, and they depend on short recognition sequences, thus affording only limited targeting flexibility or specificity.
The fields of genetics and epigenetics have greatly benefited by the explosion of technologies based on RNA-guided DNA-targeting CRISPR/Cas systems13. We and others have successfully implemented molecular tools for modifying genetic sequences or epigenetic states of target DNA loci14–22. The exciting prospect of using CRISPR to target RNA was first demonstrated by conversion of the most frequently used DNA-targeting SpCas9 to an RNA nuclease “RCas9” with an addition of a PAMmer, an oligo when bound to target RNA mimics the Protospacer Adjacent Motif (PAM) required for SpCas9 binding16. Targeting of RCas9 to repetitive sequences does not require PAMmer23, however repeat sequences constitute only a small proportion of all RNA cis-regulatory elements. Following the initial report of RCas9, other CRISPR/Cas9 systems were also found to bind to single-stranded RNA in vitro24, 25, but evidence for their in vivo RNA binding in mammalian cells is lacking. RNA-guided RNA nucleases from bacterial CRISPR systems has recently been discovered26–28. Their adaptation to mammalian cells has not only allowed programmable RNA degradation26,28,29 but has also unleashed great potential for engineering novel tools for RNA-guided regulation of endogenous RNAs. These RNA nucleases showed superior specificity compared to RNAi28, and are amenable for engineering to create new functions, e.g., RNA sequence editing27, live cell RNA imaging29, and diagnostics30. In particular, CasRx is the most recently identified type IV-D CRISPR-Cas ribonuclease isolated from Ruminococcus flavefaciens XPD3002 with robust activity in degrading target RNAs matching designed guide RNA (gRNA) sequences28. Furthermore, dCasRx with nuclease domains mutated (R239A/H244A/R858A/H863A) can be programmed to bind splicing elements to inhibit exon splicing, potentially by blocking access of splicing machinery. Induction of exon inclusion, however, has yet to be demonstrated.
In contrast to exon exclusion that can be sufficiently induced by binding of dCasRx alone28, we decided to create CASFx to induce exon inclusion which likely requires activity of splicing activator proteins31. We chose the Exon 7 of SMN2 (SMN2-E7) as our test exon as it has implications in diseases and its regulation is well-characterized32. We created CRISPR Artificial Splicing Factors (CASFx) by fusing dCasRx with RBFOX1 or RBM38 splicing factors which were successfully applied in aptamer tethering assays to activate exon inclusion when bound downstream of the target exon in splicing minigenes7, 11. We created RBFOX1N-dCasRx-C by replacing segments containing RNA recognition motif (RRM) of splicing factor RBFOX1 (residues 118–189) with dCasRx and tested its activity to induce inclusion of SMN2-E7 in an SMN2 splicing minigene (Fig 1A). Four guide RNAs (gRNAs gSMN2-1 through gSMN2-4) were designed within the intron between SMN2-E7 and E8. When cells were transfected with pCISMN2 (containing the splicing minigene) and control GFP plasmid (pmaxGFP), SMN2 minigene expressed predominantly exclusion isoform (Fig 1B, lane 1). When transfected with RBFOX1N-dCasRx-C and individual SMN2 intronic gRNAs, inclusion isoform level increased (Fig 1B, lanes 11∼14, see upper bands). Introduction of pools of two, three or four gRNAs simultaneously, further increased levels of E7-included transcripts, as well as deceased the levels of E7-excluded transcripts, switching the splicing pattern to predominantly inclusion (Fig 1B, lanes 15∼16). SMN2-E7 activation is dependent on RBFOX1 effector because dCasRx alone did not result in activation (Fig 1B, lanes 2∼9). Activation is also dependent on binding of the RBFOX1N-dCasRx-C on the SMN2 intron as control gRNAs (“C”) did not induce SMN2-E7 inclusion (Fig 2, lanes 2 and 10). Since dCasRx binding within exon was shown to induce exon exclusion in a previous study, we asked whether RBFOX1N-dCasRx-C can also induce exon exclusion when bound within exon. We designed an exonic (“EX”) gRNA at the middle at SMN2-E7 (Fig 2A). We also created two additional CASFx by fusing RBM38 to N- or C-terminus of dCasRx, resulting in RBM38-dCasRx and dCasRx-RBM38, respectively. The SMN2 minigene expressed predominantly the exclusion isoform in HEK293T cells transfected with control pmaxGFP plasmid (Fig 2B, lane 1). When transfected with one of the CASFx and the pool of DN gRNAs, SMN2 showed a switch to predominantly inclusion isoform (Fig 2B, lanes 6,9,12). Again, SMN2-E7 activation was dependent on the RBFOX1 or RBM38 effectors because dCasRx alone did not result in activation (Fig 2B, lane 3). The activation was also dependent on CASFx binding to the SMN2 intron as control guide did not induce exon inclusion (Fig 2B, lanes 5,8,11). Binding of CASFx within exon 7 induced its exclusion independent of effector fusion since dCasRx alone also induced exon exclusion when bound at this position (Fig 2B, lanes 4,7,10,13), consistent with a previous report28. This demonstrates that exon inclusion or exclusion can be induced by the same CASFx (e.g., RBFOX1N-dCasRx-C) by designing gRNA targeting different locations on target transcripts.
Next, we tested whether more than one splicing event can be modulated simultaneously and differently with CASFx (Fig 3A). We targeted RBFOX1N-dCasRx-C to the splice acceptor site of RG6 (RG6-SA) and DN locations at intron downstream of SMN2-E7 and observed simultaneous repression of RG6 cassette exon (RG6-CX) and activation of SMN2-E7 when both sets of gRNAs were transfected together with CASFx (Fig 3B, lane 4). Since CasRx is capable of processing gRNAs encoded in tandem (pre-gRNA) by cleaving 5’ of the direct repeats (DR) 28, we further tested whether the three SMN2-DN spacers and RG6-SA spacer could be encoded in one polycistronic pre-gRNA to achieve simultaneous modulation of the two splicing events. We first tested if the addition of a preprocessed DR to 3’ end of gRNA support CASFx function (Fig 3A, DR-SMN2–2-DR and DR-RG6-SA-DR). As predicted, these gRNAs remained active in inducing SMN2-E7 inclusion and RG6-CX exclusion (Fig 3B, lanes 5,6). More importantly, a polycistronic pre-gRNA (SMN2-DN-RG6-SA) harboring the three SMN2-DN spacers and the RG6-SA spacer induced simultaneous SMN2-E7 inclusion and RG6-CX exclusion when transfected with RBFOX1N-dCasRx-C (Fig 3B, lane 7), confirming the functionality of such polycistronic pre-gRNA architecture in inducing simultaneous and “bidirectional” splicing modulation of two different targets.
Finally, we tested whether we could implement chemical control for CASFx. We created two-peptide inducible CRISPR Artificial Splicing Factors (iCASFx) by separating the RNA binding module (FKBP-dCasRx, or dCasRx-FKBP) and exon activation module (RBFOX1N-FRB-C, RBM38-FRB, or FRB-RBM38) into two peptides that can be induced to interact via the FKBPFRB domains33 in the presence of rapamycin (Fig 4A). Induction of SMN2-E7 inclusion was observed in cells cultured with rapamycin and transfected with iCASFx plus SMN2-DN-gRNA plasmids (Fig 4B, lanes 2,4,6,8,10,12) but not in cells cultured without rapamycin (Fig 4B, lanes 1,3,5,7,9,11).
In this study, we reported three RNA-guided splicing activators based on CRISPR/dCasRx. These CRISPR Artfiicial Splicing Factors (CASFx) can induce exon inclusion when targeted to bind at the downstream intron, and can induce exon exclusion when guided to bind within the target exon. We showed that simultaneous exon inclusion/exclusion can be achieved by a pool of gRNAs or a polycistronic pre-gRNA encoding spacers matching two target RNAs. Finally, we engineered inducible CRISPR Artificial Splicing Factors (iCASFx) that are inducible by small molecule rapamycin, potentially allowing spatiotemporal and tunable control of alternative splicing.
Material & Methods
Cloning
HEK293T cDNA was used as a source for PCR-amplification of coding sequences of splicing factors or other RNA binding proteins. Alternatively, gBlocks encoding human codon optimized versions of their coding sequences were ordered from IDT to serve as PCR template. The pXR002: EF1a-dCasRx-2A-EGFP 28 plasmid (Addgene #109050) served as PCR template for dCasRx coding sequence. The coding sequences of the CRISPR Artificial Splicing Factors (CASFx) were then cloned into pmax expression vector (Lonza) by a combination of fusion PCR, restriction-ligation cloning and Sequence- and Ligation-Independent Cloning (SLIC)34. gRNA expression cloning plasmids were generated by similar procedures using IDT oligonucleotides encoding CasRx gRNA direct repeat and PCR reaction using a ccdbCam selection cassette (Invitrogen) and a U6-containing plasmid as templates. Two BbsI restriction sites flanking the ccdbCam selection cassette serves as the restriction cloning sites for insertion of target-specific spacers. Target-specific spacer sequences were then cloned into the gRNA expression plasmids by annealed oligonucleotide ligation. Plasmid listing is included in the supplementary information. Plasmids and Genbank files will be available on Addgene. More supplementary data will also be on http://CasFx.org
Cell culture and transfection
HEK293T cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) with 10% fetal bovine serum (FBS)(Lonza), 4% Glutamax (Gibco), 1% Sodium Pyruvate (Gibco) and penicillin-streptomycin (Gibco). Incubator conditions were 37 °C and 5% CO2. For activation experiments, cells were seeded into 12-well plates at 100,000 cells per well the day before being transfected with 600ng (the “quota”) of plasmid DNA with 2.25uL Attractene tranfection reagent (Qiagen). 18 ng of each reporter minigene plasmid was transfected. The remaining quota was then divided equally among the effector and gRNA plasmids. In cases where there were two or more gRNA plasmids, the quota allocated for gRNA plasmids is further subdivided equally. For two-pepide effectors (i.e., the FKBP-FRB systems), the effector plasmid quota was divided equally between the plasmids encoding the individual peptides. Media was changed 24hr after transfection. 100nM (final concentration) of rapamycin was added during media change if applicable. Cells were harvested 48hr after transfection for RT-PCR analysis.
RT-PCR
Cells were harvested for RNA extraction using RNeasy Plus Mini Kit (Qiagen). Equal amount of RNAs from one transfection experiment (either 700ng or 1000ng) were reverse-transcribed using High Capacity RNA-to-cDNA Kit (ThermoFisher). PCR was then performed using 2uL (out of 10uL) of cDNA using Phusion® High-Fidelity DNA Polymerase (NEB) using minigene plasmid-specific primers for 25 cycles. PCR products were then analyzed on a 3% agarose gel.
Footnotes
Manuscript version: 1.0. Get current version and plasmids at http://CasFx.org