Modulation of alternative cleavage and polyadenylation events by dCas9-mediated CRISPRpas

Methods Enzymol. 2021;655:459-482. doi: 10.1016/bs.mie.2021.04.016. Epub 2021 Jun 4.

Abstract

The CRISPR/Cas9 technology is revolutionizing genomic engineering. The high efficiency and selectivity of the system have inspired the development of various derived tools for gene regulation at different levels, such as transcriptional activation or inhibition, epigenetic modification, splicing, and base editing. Cleavage and polyadenylation (CPA) is an essential 3' end maturation step for almost all eukaryotic mRNAs. CPA is tightly coupled with transcriptional termination, and its activity impacts gene expression. Over half of all human genes display alternative polyadenylation (APA), where multiple cleavage and polyadenylation sites (PASs) lead to mRNA isoforms with variable termini. APA isoforms often have distinct metabolisms, and their relative abundance can change drastically in different cells. Here, we describe a method based on delivering a catalytically dead Cas9 (dCas9) to genomic regions nears the PAS, which alters APA site usage in 3'UTRs or introns. This method, named CRISPRpas, allows investigators to examine functional significance of APA isoforms of individual genes. We also describe using the bioinformatics program APAlyzer to examine APA events of interest with RNA-seq data.

Keywords: 3′UTR; APAlyzer; Alternative polyadenylation; CRISPR; Gene regulation; Intronic polyadenylation; Isoform expression; RNA processing; dCas9.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 3' Untranslated Regions
  • Gene Expression Regulation*
  • Humans
  • Polyadenylation*
  • RNA Splicing
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism

Substances

  • 3' Untranslated Regions
  • RNA, Messenger