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Background: The genus Borrelia comprises spirochaetal bacteria maintained in natural transmission cycles by tick vectors and vertebrate reservoir hosts. The main groups are represented by a species complex including the causative agents of Lyme borreliosis and relapsing fever group Borrelia. Borrelia miyamotoi belongs to the relapsing fever group of spirochetes and forms distinct populations in North America, Asia, and Europe. As all Borrelia species B. miyamotoi possess an unusual and complex genome consisting of a linear chromosome and a number of linear and circular plasmids. The species is considered an emerging human pathogen and an increasing number of human cases are being described in the Northern hemisphere. The aim of this study was to produce a high quality reference genome that will facilitate future studies into genetic differences between different populations and the genome plasticity of B. miyamotoi. Results: We used multiple available sequencing methods, including Pacific Bioscience single-molecule real-time technology (SMRT) and Oxford Nanopore technology (ONT) supplemented with highly accurate Illumina sequences, to explore the suitability for whole genome assembly of the Russian B. miyamotoi isolate, Izh-4. Plasmids were typed according to their potential plasmid partitioning genes (PF32, 49, 50, 57/62). Comparing and combining results of both long-read (SMRT and ONT) and short-read methods (Illumina), we determined that the genome of the isolate Izh-4 consisted of one linear chromosome, 12 linear and two circular plasmids. Whilst the majority of plasmids had corresponding contigs in the Asian B. miyamotoi isolate FR64b, there were only four that matched plasmids of the North American isolate CT13-2396, indicating differences between B. miyamotoi populations. Several plasmids, e.g. lp41, lp29, lp23, and lp24, were found to carry variable major proteins. Amongst those were variable large proteins (Vlp) subtype Vlp-α, Vlp-γ, Vlp-δ and also Vlp-β. Phylogenetic analysis of common plasmids types showed the uniqueness in Russian/Asian isolates of B. miyamotoi compared to other isolates. Conclusions: We here describe the genome of a Russian B. miyamotoi clinical isolate, providing a solid basis for future comparative genomics of B. miyamotoi isolates. This will be a great impetus for further basic, molecular and epidemiological research on this emerging tick-borne pathogen.

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Chronic hepatitis B is a severe liver disease caused by hepatitis B virus (HBV) infection. Covalently closed circular DNA (cccDNA), a super-spiralized, double-stranded form of the HBV genome, is the major determinant of viral persistence. CRISPR/Cas9 nucleases have been recently shown to introduce double-stranded DNA breaks into HBV cccDNA. The inflicted damage results predominantly in erroneous repair of cccDNA by non-homologous end-joining (NHEJ). NHEJ has been suggested to enhance anti-HBV activity of CRISPR/Cas9 and increase cccDNA mutation. In this study, we assessed anti-HBV activity of CRISPR/Cas9 and cccDNA repair outcomes in an altered NHEJ/HR environment. NU7026, a strong inhibitor of NHEJ, prevented CRISPR/Cas9-mediated degradation of cccDNA and resulted in frequent on-target deletions. We conclude that CRISPR/Cas9 is a highly effective tool to degrade cccDNA and first demonstrate that inhibiting NHEJ impairs cccDNA degradation.