Inducible CRISPRi-Based Operon Silencing and Selective in Trans Gene Complementation in Borrelia burgdorferi

Bryan T. Murphy, Jacob J. Wiepen, Huan He, Ankita S. Pramanik, Jason M. Peters, Brian Stevenson, Wolfram R. Zückert

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

To accelerate genetic studies on the Lyme disease pathogen Borrelia burgdorferi, we developed an enhanced CRISPR interference (CRISPRi) approach for isopropylb-D-thiogalactopyranoside (IPTG)-inducible repression of specific B. burgdorferi genes. The entire system is encoded on a compact 11-kb shuttle vector plasmid that allows for inducible expression of both the sgRNA module and a nontoxic codon-optimized dCas9 protein. We validated this CRISPRi system by targeting the genes encoding OspA and OspB, abundant surface lipoproteins coexpressed by a single operon, and FlaB, the major subunit forming the periplasmic flagella. As in other systems, single guide RNAs (sgRNAs) complementary to the nontemplate strand were consistently effective in gene repression, with 4- to 994-fold reductions in targeted transcript levels and concomitant reductions of protein levels. Furthermore, we showed that ospAB knockdowns could be selectively complemented in trans for OspA expression via the insertion of CRISPRi-resistant, synonymously or nonsynonymously mutated protospacer adjacent motif (PAM*) ospA alleles into a unique site within the CRISPRi plasmid. Together, this establishes CRISPRi PAM* as a robust new genetic tool to simplify the study of B. burgdorferi genes, bypassing the need for gene disruptions by allelic exchange and avoiding rare codon toxicity from the heterologous expression of dCas9.

Original languageEnglish
JournalJournal of Bacteriology
Volume205
Issue number2
DOIs
StatePublished - Feb 2023

Bibliographical note

Publisher Copyright:
Copyright © 2023 American Society for Microbiology. All Rights Reserved.

Keywords

  • Borrelia burgdorferi
  • CRISPR interference
  • genetic complementation
  • genetic tools
  • spirochetes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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