CRISPR artificial splicing factors

Nat Commun. 2020 Jun 12;11(1):2973. doi: 10.1038/s41467-020-16806-4.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing*
  • Base Sequence
  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • Cell Line, Tumor
  • Exons / genetics*
  • Fibroblasts / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Muscular Atrophy, Spinal / genetics
  • Muscular Atrophy, Spinal / metabolism
  • Muscular Atrophy, Spinal / pathology
  • RNA / genetics*
  • RNA / metabolism
  • RNA Isoforms / genetics
  • RNA Isoforms / metabolism
  • RNA Splicing Factors / genetics*
  • RNA Splicing Factors / metabolism
  • Survival of Motor Neuron 2 Protein / genetics
  • Survival of Motor Neuron 2 Protein / metabolism

Substances

  • RNA Isoforms
  • RNA Splicing Factors
  • SMN2 protein, human
  • Survival of Motor Neuron 2 Protein
  • RNA