CRISPRpas: programmable regulation of alternative polyadenylation by dCas9

Nucleic Acids Res. 2022 Mar 21;50(5):e25. doi: 10.1093/nar/gkab519.

Abstract

Most human protein-coding genes produce alternative polyadenylation (APA) isoforms that differ in 3' UTR size or, when coupled with splicing, have variable coding sequences. APA is an important layer of gene expression program critical for defining cell identity. Here, by using a catalytically dead Cas9 and coupling its target site with polyadenylation site (PAS), we develop a method, named CRISPRpas, to alter APA isoform abundance. CRISPRpas functions by enhancing proximal PAS usage, whose efficiency is influenced by several factors, including targeting strand of DNA, distance between PAS and target sequence and strength of the PAS. For intronic polyadenylation (IPA), splicing features, such as strengths of 5' splice site and 3' splice site, also affect CRISPRpas efficiency. We show modulation of APA of multiple endogenous genes, including IPA of PCF11, a master regulator of APA and gene expression. In sum, CRISPRpas offers a programmable tool for APA regulation that impacts gene expression.

Publication types

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

MeSH terms

  • 3' Untranslated Regions
  • Gene Expression Regulation
  • Genetic Techniques*
  • Humans
  • Introns / genetics
  • Polyadenylation*
  • RNA Isoforms
  • RNA Splice Sites
  • RNA Splicing*
  • RNA, Messenger
  • mRNA Cleavage and Polyadenylation Factors

Substances

  • 3' Untranslated Regions
  • Pcf11 protein, human
  • RNA Isoforms
  • RNA Splice Sites
  • RNA, Messenger
  • mRNA Cleavage and Polyadenylation Factors