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Chavez, Alejandro

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Chavez

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Alejandro

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Chavez, Alejandro

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2015 - 20185

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Author's Publications: search.filters.isAuthorOfPublication.9f077c49-4cdb-42f5-b988-cf8007186287

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Now showing 1 - 5 of 5
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    Highly-efficient Cas9-mediated transcriptional programming
    (2015) Chavez, Alejandro; Scheiman, Jonathan; Vora, Suhani; Pruitt, Benjamin W.; Tuttle, M; Iyer, Eswar; Lin, Shuailiang; Kiani, Samira; Guzman, Christopher D.; Wiegand, Daniel; Ter-Ovanesyan, Dmitry; Braff, Jonathan L.; Davidsohn, Noah; Housden, Benjamin E; Perrimon, Norbert; Weiss, Ron; Aach, John; Collins, James; Church, George
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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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    Safeguarding CRISPR-Cas9 gene drives in yeast
    (2015) DiCarlo, James; Chavez, Alejandro; Dietz, Sven L.; Esvelt, Kevin M.; Church, George
    RNA-guided gene drives capable of spreading genomic alterations made in laboratory organisms through wild populations in an inheritable way could be used to control populations of organisms that cause environmental and public health problems. However, the possibility of unintended genome editing through the escape of strains from laboratories, coupled with the prospect of unanticipated ecological change, demands caution. We report the efficacy of CRISPR-Cas9 gene drive systems in wild and laboratory strains of the yeast Saccharomyces cerevisiae. Furthermore, we address concerns surrounding accidental genome editing by developing and validating methods of molecular confinement that minimize the risk of unwanted genome editing. We also present a drive system capable of overwriting the changes introduced by an earlier gene drive. These molecular safeguards should enable the development of safe CRISPR gene drives for diverse organisms.
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    Comparative Analysis of Cas9 Activators Across Multiple Species
    (2016) Chavez, Alejandro; Tuttle, Marcelle; Pruitt, Benjamin W; Ewen-Campen, Ben; Chari, Raj; Ter-Ovanesyan, Dmitry; Haque, Sabina J; Cecchi, Ryan J; Kowal, Emma J K; Buchthal, Joanna; Housden, Benjamin E; Perrimon, Norbert; Collins, James; Church, George
    Several groups have generated programmable transcription factors based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems and assess the role of cooperativity in maximizing gene expression.
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    A CRISPR Cas9-based gene drive platform for genetic interaction analysis in Candida albicans
    (2018) Shapiro, Rebecca S.; Chavez, Alejandro; Porter, Caroline B. M.; Hamblin, Meagan; Kaas, Christian S.; DiCarlo, James E.; Zeng, Guisheng; Xu, Xiaoli; Revtovich, Alexey V.; Kirienko, Natalia V.; Wang, Yue; Church, George; Collins, James
    Candida albicans is the leading cause of fungal infections; yet, complex genetic interaction analysis remains cumbersome in this diploid pathogen. Here, we developed a CRISPR-Cas9-based ‘gene drive array’ (GDA) platform to facilitate efficient genetic analysis in C. albicans. In our system, a modified DNA donor molecule acts as a selfish genetic element, replaces the targeted site, and propagates to replace additional wild-type loci. Using mating-competent C. albicans haploids, each carrying a different gene drive disabling a gene of interest, we are able to create diploid strains that are homozygous double-deletion mutants. We generate double-gene deletion libraries to demonstrate this technology, targeting antifungal efflux and biofilm adhesion factors. We screen these libraries to identify virulence regulators and determine how genetic networks shift under diverse conditions. This platform transforms our ability to perform genetic interaction analysis in C. albicans and is readily extended to other fungal pathogens.