Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase

Mol Cell. 2018 Oct 18;72(2):380-394.e7. doi: 10.1016/j.molcel.2018.09.002. Epub 2018 Oct 4.

Abstract

RNA splicing is a critical mechanism by which to modify transcriptome, and its dysregulation is the underlying cause of many human diseases. It remains challenging, however, to genetically modulate a splicing event in its native context. Here, we demonstrate that a CRISPR-guided cytidine deaminase (i.e., targeted-AID mediated mutagenesis [TAM]) can efficiently modulate various forms of mRNA splicing. By converting invariant guanines to adenines at either 5' or 3' splice sites (SS), TAM induces exon skipping, activation of alternative SS, switching between mutually exclusive exons, or targeted intron retention. Conversely, TAM promotes downstream exon inclusion by mutating cytidines into thymines at the polypyrimidine tract. Applying this approach, we genetically restored the open reading frame and dystrophin function of a mutant DMD gene in patient-derived induced pluripotent stem cells (iPSCs). Thus, the CRISPR-guided cytidine deaminase provides a versatile genetic platform to modulate RNA splicing and to correct mutations associated with aberrant splicing in human diseases.

Keywords: AID; DMD; base editing; exon skipping; gene therapy; polypyrimidine tract; splice site; splicing; targeted AID-mediated mutagenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Cytidine Deaminase / genetics*
  • Dystrophin / genetics
  • Exons / genetics
  • Gene Regulatory Networks
  • HEK293 Cells
  • Humans
  • Introns / genetics
  • Mice
  • Open Reading Frames / genetics
  • RNA Splice Sites / genetics
  • RNA Splicing / genetics*

Substances

  • Dystrophin
  • RNA Splice Sites
  • Cytidine Deaminase