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bioRxiv · 10.1101/2022.12.05.519228

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

Murphy, B. T.·Wiepen, J. J.·He, H.·Pramanik, A. S.·Peters, J. M.·Stevenson, B.·Zueckert, W. R.
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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. SIGNIFICANCEBorrelia 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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Murphy, B. T., Wiepen, J. J., He, H., Pramanik, A. S., Peters, J. M., Stevenson, B., Zueckert, W. R.. 2022-12-08. Inducible CRISPRi-based operon silencing and selective in trans gene complementation in Borrelia burgdorferi. https://doi.org/10.1101/2022.12.05.519228

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