Student or educator? Sign up with your school email and get 6 months of Pro free.
CoChat
Sign Up
PreprintCited by 14CRISPR and Genetic EngineeringBacterial Genetics and BiotechnologyVector-borne infectious diseases

The DOI matches its Crossref record; 1 of the 1 references it cites were found in a registry or an index.

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

Open access
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

Open this paper in CoChat — free accountOpen PDF

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.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1101/2022.12.05.519228

Cites

1 of 1 references verified0 citation errors · 0 not checkable

Statistics

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 1 references

Research works printed but not linked: 68

  1. 2019, The next generation of CRISPR–Cas technologies and applications, Nature Reviews Molecular Cell Biology, 20, 490, 10.1038/s41580-019-0131-5 · DOI · Paper page
  2. 10.1038/s41579-019-0299-x · DOI · Paper page
  3. 10.1016/j.cell.2013.02.022 · DOI · Paper page
  4. 10.1016/j.cell.2013.06.044 · DOI · Paper page
  5. 10.1016/j.ymeth.2019.05.023 · DOI
  6. 10.1016/j.cell.2016.05.003 · DOI · Paper page
  7. Rock JM , Hopkins FF , Chavez A , Diallo M , Chase MR , Gerrick ER , Pritchard JR , Church GM , Rubin EJ , Sassetti CM , Schnappinger D , Fortune SM. 2017. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nature Microbiology 2. · DOI · Paper page
  8. 2016, Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi), ACS Synthetic Biology, 5, 375, 10.1021/acssynbio.5b00216 · DOI · Paper page
  9. Fernandes LGV , Guaman LP , Vasconcellos SA , Heinemann MB , Picardeau M , Nascimento ALTO. 2019. Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira. Scientific Reports 9. · DOI
  10. Takacs CN , Scott M , Chang Y , Kloos ZA , Irnov I , Rosa PA , Liu J , Jacobs-Wagner C. 2021. A CRISPR Interference Platform for Selective Downregulation of Gene Expression in Borrelia burgdorferi. Applied and Environmental Microbiology 87. · DOI
  11. 10.1016/j.idc.2015.02.010 · DOI · Paper page
  12. 10.1146/annurev-genet-011112-112140 · DOI
  13. 10.1038/37551 · DOI · Paper page
  14. 10.1046/j.1365-2958.2000.01698.x · DOI
  15. 2018, Genetic Manipulation of Borrelia Spp, Curr Top Microbiol Immunol, 415, 113
  16. 10.21775/cimb.042 · DOI
  17. 10.1128/JB.182.9.2445-2452.2000 · DOI
  18. 10.1128/JB.185.22.6723-6727.2003 · DOI
  19. 10.1159/000073406 · DOI
  20. 10.1073/pnas.080068797 · DOI
  21. Takacs CN , Kloos ZA , Scott M , Rosa PA , Jacobs-Wagner C. 2018. Fluorescent Proteins, Promoters, and Selectable Markers for Applications in the Lyme Disease Spirochete Borrelia burgdorferi. Appl Environ Microbiol 84. · DOI
  22. 10.1046/j.1365-2958.2001.02256.x · DOI
  23. 10.1128/AAC.50.2.445-452.2006 · DOI
  24. 10.1111/j.1365-2958.2007.05593.x · DOI
  25. 10.1128/AEM.02454-06 · DOI
  26. 10.1128/AEM.02825-08 · DOI
  27. 10.1128/IAI.63.6.2255-2261.1995 · DOI
  28. 10.1128/JB.187.22.7845-7852.2005 · DOI
  29. 10.1084/jem.183.1.271 · DOI
  30. 1995, Expression of outer surface proteins A and C of Borrelia burgdorferi in Ixodes ricinus, Journal of Clinical Microbiology, 33, 1867, 10.1128/jcm.33.7.1867-1869.1995 · DOI
  31. 10.1128/JCM.38.1.382-388.2000 · DOI
  32. 10.1073/pnas.92.7.2909 · DOI · Paper page
  33. 2012, Domain enhanced lookup time accelerated BLAST, Biology Direct, 7, 12, 10.1186/1745-6150-7-12 · DOI · Paper page
  34. 2019, Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi, Nat Microbiol, 4, 244, 10.1038/s41564-018-0327-z · DOI
  35. 10.1073/pnas.1810022115 · DOI
  36. 10.1093/nar/gkm219 · DOI
  37. 2020, Designing sgRNAs for CRISPR-BEST base editing applications with CRISPy-web 2.0, Synthetic and Systems Biotechnology, 5, 99, 10.1016/j.synbio.2020.05.005 · DOI
  38. 10.1038/nbt.3026 · DOI · Paper page
  39. 10.1371/journal.pone.0098186 · DOI · Paper page
  40. 10.1038/srep21451 · DOI
  41. 10.1126/science.1246981 · DOI · Paper page
  42. 10.1128/IAI.66.11.5119-5124.1998 · DOI
  43. 10.1099/13500872-141-6-1321 · DOI
  44. 10.1128/IAI.61.5.2207-2210.1993 · DOI
  45. 10.1038/nbt.2647 · DOI · Paper page
  46. 10.1016/j.micpath.2004.02.007 · DOI
  47. 10.1371/journal.pntd.0002514 · DOI
  48. 10.1007/978-1-60327-032-8_6 · DOI
  49. 10.1128/JB.05037-11 · DOI
  50. 10.1371/journal.ppat.1004056 · DOI
  51. 1996, Comparison of infectivities of six tick-derived isolates of Borrelia burgdorferi for rodents and ticks, Journal of Clinical Microbiology, 34, 71, 10.1128/jcm.34.1.71-75.1996 · DOI
  52. 2020, Mismatch-CRISPRi Reveals the Co-varying Expression-Fitness Relationships of Essential Genes in Escherichia coli and Bacillus subtilis, Cell Syst, 11, 523, 10.1016/j.cels.2020.09.009 · DOI
  53. 2022, CRISPRi chemical genetics and comparative genomics identify genes mediating drug potency in Mycobacterium tuberculosis, Nat Microbiol, 7, 766, 10.1038/s41564-022-01130-y · DOI
  54. 10.1371/journal.pone.0164165 · DOI
  55. 10.1073/pnas.1222538110 · DOI
  56. 10.1128/JB.186.9.2745-2756.2004 · DOI
  57. 1984, Isolation and cultivation of Lyme disease spirochetes, Yale J Biol Med, 57, 521
  58. Zückert WR. 2007. Laboratory Maintenance of Borrelia burgdorferi. Current Protocols in Microbiology 4. · DOI
  59. 10.1385/0-89603-310-4:253 · DOI
  60. 10.1084/jem.20031960 · DOI
  61. 10.1073/pnas.97.25.13865 · DOI
  62. 10.1093/nar/28.1.292 · DOI · Paper page
  63. Sambrook J. 2001. Molecular cloning : a laboratory manual. Third edition. Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory Press, [2001] ©2001.
  64. 1983, Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody, Infect Immun, 41, 795, 10.1128/iai.41.2.795-804.1983 · DOI
  65. 1984, Variation in a major surface protein of Lyme disease spirochetes, Infect Immun, 45, 94, 10.1128/iai.45.1.94-100.1984 · DOI
  66. 10.1111/j.1469-0691.2006.01509.x · DOI
  67. 10.1016/j.mimet.2014.09.001 · DOI
  68. 2006, A Reverse Transcriptase–Polymerase Chain Reaction Assay of Borrelia burgdorferi 16S rRNA for Highly Sensitive Quantification of Pathogen Load in a Vector, Vector-Borne and Zoonotic Diseases, 6, 103, 10.1089/vbz.2006.6.103 · DOI

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

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review

How to cite

Cite CoChat for the checks on this page, and the paper for its own findings.

This page
CoChat. (2026). Citation check: Inducible CRISPRi-based operon silencing and selective in trans gene complementation in Borrelia burgdorferi. https://app.cochat.ai/papers/10.1101/2022.12.05.519228
The paper
Murphy, B. T., Wiepen, J. J., He, H., Pramanik, A. S., Peters, J. M., Stevenson, B., & Zückert, W. R. (2022). Inducible CRISPRi-based operon silencing and selective in trans gene complementation in Borrelia burgdorferi. Journal of Bacteriology. https://doi.org/10.1101/2022.12.05.519228

Data sources

Bibliographic data: Semantic Scholar, OpenAlex, Crossref.

Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).

Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.