Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells

Cell Chem Biol. 2022 Feb 17;29(2):321-327.e4. doi: 10.1016/j.chembiol.2021.07.011. Epub 2021 Aug 2.

Abstract

RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to modulate gene expression outcomes. However, protein and CRISPR RNA (crRNA) delivery in human cells can be challenging with rapid crRNA degradation yielding transient knockdown. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes can alter gene expression in primary CD4+ and CD8+ T cells. This system represents a robust and efficient method to modulate transcripts without genetic manipulation.

Keywords: CRISPR type-VI; cas13; chemical modifications; transcriptome regulation.

Publication types

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

MeSH terms

  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Cas Systems / genetics*
  • Cells, Cultured
  • Gene Editing
  • Humans
  • RNA, Guide, CRISPR-Cas Systems / chemical synthesis
  • RNA, Guide, CRISPR-Cas Systems / chemistry
  • RNA, Guide, CRISPR-Cas Systems / genetics*

Substances

  • CRISPR-Associated Proteins
  • RNA, Guide, CRISPR-Cas Systems