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PreprintCited by 14CRISPR and Genetic EngineeringBacterial Genetics and BiotechnologyVector-borne infectious diseases

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Inducible CRISPRi-based operon silencing and selective in trans gene complementation in Borrelia burgdorferi

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Murphy, Bryan T., Wiepen, Jacob J., He, Huan, Pramanik, Ankita S., Peters, Jason M., Stevenson, Brian, Zückert, Wolfram R.·2022·Journal of Bacteriology
DOI 10.1101/2022.12.05.519228PMC PMC9945571

Preprint — published as 10.1128/jb.00468-22

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Abstract

To accelerate genetic studies on the Lyme disease pathogen Borrelia burgdorferi , we developed an enhanced CRISPR interference (CRISPRi) approach for 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 non-toxic codon-optimized dCas9 protein. We validated this CRISPRi system by targeting the genes encoding for OspA and OspB, abundant surface lipoproteins co-expressed by a single operon, and FlaB, the major subunit forming the periplasmic flagella. As in other systems, sgRNAs complementary to the non-template strand were consistently effective in gene repression, with 4- to 994-fold reductions in targeted transcript levels and concomitant reductions of in proteins levels. Furthermore, we showed that ospAB knockdowns could be selectively complemented in trans for OspA expression via the insertion of synonymous or non-synonymous CRISPRi-resistant PAM mutant (PAM*) ospA alleles into a unique site within the 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 heterologous expression of dCas9. SIGNIFICANCE: Borrelia burgdorferi , the causative agent of Lyme disease, is a tick-borne pathogen of global importance. Here, we expand the genetic toolbox for studying B. burgdorferi physiology and pathogenesis by establishing a single-plasmid-based CRISPRi system with optional in trans complementation for the functional study of essential and non-essential proteins.

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Metadata

Authors
Murphy, B. T., Wiepen, J. J., He, H., Pramanik, A. S., Peters, J. M., Stevenson, B., & Zückert, W. R.
Affiliations
  • University of Kansas (US)
  • University of Wisconsin–Madison (US)
  • University of Kentucky (US)
Corresponding author
Zückert, Wolfram R.
ORCID
Venue
Journal of Bacteriology
Publisher
openRxiv
Published
2022-12-08
Volume
205
Issue
2
Pages
e0046822-e0046822
ISSN
0021-9193 (ISSN-L)
DOI
10.1101/2022.12.05.519228
PMC
PMC9945571
OpenAlex
W4318620387
Cited by
14
References
1
Citations by year
2026: 1 · 2025: 5 · 2024: 4 · 2023: 3 · 2022: 1
FWCI
1.15
Type
journal article
Language
en
Open access
Locations
  • Journal of Bacteriology (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • University of Arkansas for Medical Sciences
  • National Institutes of Health
  • National Institute of Allergy and Infectious Diseases (R21AI144624, R01AI144126, R21AI147139)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Immunology and Microbiology › Parasitology › Vector-borne infectious diseases
Keywords
Borrelia burgdorferi, CRISPR interference, transcriptional silencing, Lyme disease, pathogenesis
MeSH terms
Antigens, Surface, Bacterial Outer Membrane Proteins, Bacterial Vaccines, Codon, Operon, Borrelia burgdorferi, Clustered Regularly Interspaced Short Palindromic Repeats

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