Guide RNA Categorization Enables Target Site Choice in Tn7-CRISPR-Cas Transposons

Cell. 2020 Dec 23;183(7):1757-1771.e18. doi: 10.1016/j.cell.2020.11.005. Epub 2020 Dec 2.

Abstract

CRISPR-Cas defense systems have been coopted multiple times in nature for guide RNA-directed transposition by Tn7-like elements. Prototypic Tn7 uses dedicated proteins for two targeting pathways: one targeting a neutral and conserved attachment site in the chromosome and a second directing transposition into mobile plasmids facilitating cell-to-cell transfer. We show that Tn7-CRISPR-Cas elements evolved a system of guide RNA categorization to accomplish the same two-pathway lifestyle. Multiple mechanisms allow functionally distinct guide RNAs for transposition: a conventional system capable of acquiring guide RNAs to new plasmid and phage targets and a second providing long-term memory for access to chromosomal sites upon entry into a new host. Guide RNAs are privatized to be recognized only by the transposon-adapted system via sequence specialization, mismatch tolerance, and selective regulation to avoid toxic self-targeting by endogenous CRISPR-Cas defense systems. This information reveals promising avenues to engineer guide RNAs for enhanced CRISPR-Cas functionality for genome modification.

Keywords: CRISPR regulation; RNA-directed transposition; guide RNA categorization; self-targeting spacers.

Publication types

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

MeSH terms

  • Bacterial Proteins / metabolism
  • Base Sequence
  • CRISPR-Cas Systems / genetics*
  • DNA Transposable Elements / genetics*
  • Gammaproteobacteria / metabolism
  • Phylogeny
  • Promoter Regions, Genetic / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • Transcription Factors / metabolism
  • Zygote / metabolism

Substances

  • Bacterial Proteins
  • DNA Transposable Elements
  • RNA, Guide, CRISPR-Cas Systems
  • Transcription Factors