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.

Poison Exon Splicing Regulates a Coordinated Network of SR Protein Expression during Differentiation and Tumorigenesis

“…Given the redundant function of RS domains on AS regulation Shin et al, 2005) and our own experiments demonstrating that swapping of RRMs between SRSF3 and TRA2b mirrors the effects of the wild-type protein, we hypothesized that their opposing regulation of TRA2b-PE splicing is dependent on binding location rather than differences in RS domain activity. We developed CRISPR artificial SFs (CASFxs) (Du et al, 2020) for each SR protein (CASFx-SR) by fusing the RS domain, which confers splicing activity, to a dCasRx protein that replaces the RRM and carries the RNA binding activity (Figures 6A and 6B). All CASFx-SR proteins are nuclearly localized with an SV40 nuclear localization signal ( Figures S6D and S6G).…”

“…Total intron length in mutant and wild-type minigenes was kept constant. CASFx plasmids (pMAX-CASFx-SR) CRISPR Artificial Splicing Factors SR proteins were constructed by subcloning RS domains from the pCI-neo-HA-SR protein plasmids in place of RBFOX1 domains in the pMAX-RBFOX1N-dCasRx-C plasmid (Du et al, 2020), using SLIC or restriction enzyme digestion and ligation with primers listed in Table S4C. The dCasRx domain and RS domain are separate by a glycine-rich hinge region.…”

Current Genetic Survey and Potential Gene-Targeting Therapeutics for Neuromuscular Diseases

“…For this purpose, an artificial splicing factor (CASFx) was engineered for CRISPR-induced alternative splicing and Cas analog Cpf1 directed HDR to correcting SMN2 exon 7 [ 156 , 157 ]. CASFx modulates alternative splicing and allows simultaneous exon inclusion and exclusion by differential positioning of the factor [ 156 , 158 ]. When tackling LGMD, researchers expanded the target group of LGMD2C/SGCG [ 92 ] and explored the mutations in LGMD2A/CAPN3 [ 159 ], LGMD2G/TCAP [ 160 ], and LGMD2B/DYSF [ 161 ].…”

CRISPR-Cas13 mediated Knock Down inDrosophilacultured cells

“…Here, we describe the use and generation of a new toolkit to perturb gene expression in Drosophila. The CRISPR-Cas13 system can be used for RNA knockdown with greater accuracy than RNAi 13,19 , but also to image RNA molecules 12,28 , manipulate the epitranscriptome 29,30 , edit transcripts 14 , and alter splicing 19,31 , but the use of this system in Drosophila has been limited by the potential toxicity of the nucleases 22 Our Drosophila cell-line is compatible with large-scale pooled screening, and therefore could be used to perform pooled, variable-dose screening 33 . Last but not least, we achieved tunable expression of PspCas13b by decreasing its translation through the addition of upstream ORFs of different lengths.…”

“…Last but not least, we achieved tunable expression of PspCas13b by decreasing its translation through the addition of upstream ORFs of different lengths. This strategy has successfully been employed in many biological contexts, including for balancing Cas9 activity and toxicity in Cas13 fusions have been used to precisely edit the epi-transcriptome or alter splicing 14,21,[29][30][31] . Utilizing these reagents in Drosophila could reveal the developmental need for epi-transcriptome modification of transcripts in time and space and could be used to determine the developmental function of particular splicing changes.…”