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Rock, Jeremy

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Rock

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Rock, Jeremy

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2015 - 20162

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Author's Publications: search.filters.isAuthorOfPublication.7c9543e5-b209-4e77-a448-72d09bc69415

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    Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform
    (2016) Rock, Jeremy; Hopkins, Forrest; Chavez, Alejandro; Diallo, Marieme; Chase, Michael; Gerrick, Elias; Pritchard, Justin R.; Church, George; Rubin, Eric; Sassetti, Christopher M.; Schnappinger, Dirk; Fortune, Sarah
    Development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered, and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria. The most efficacious of these proteins, the CRISPR1 Cas9 from Streptococcus thermophilus (dCas9Sth1), typically achieves 20–100 fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9Sth1-mediated gene knockdown is robust when targeted far from the transcriptional start site, thereby allowing high-resolution dissection of gene function in the context of bacterial operons. We demonstrate the utility of this system by addressing persistent controversies regarding drug synergies in the mycobacterial folate biosynthesis pathway. We anticipate that the dCas9Sth1 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping, and drug-target profiling in M. tuberculosis.
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    DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader
    (2015) Rock, Jeremy; Lang, Ulla F.; Chase, Michael; Ford, Christopher B.; Gerrick, Elias; Gawande, Richa; Coscolla, Mireia; Gagneux, Sebastien; Fortune, Sarah; Lamers, Meindert H.
    The DNA replication machinery is an important target for antibiotic development for increasingly drug resistant bacteria including Mycobacterium tuberculosis1. While blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In E. coli, the proofreading subunit of the replisome, the ε-exonuclease, is essential for high fidelity DNA replication2; however, we find that it is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase, DnaE1, encodes a novel editing function that proofreads DNA replication, mediated by an intrinsic 3′-5′ exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by greater than 3,000 fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP-domain mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader.