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Norville, Julie

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Norville

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Norville, Julie

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2013 - 20152

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Author's Publications: search.filters.isAuthorOfPublication.f6ea77d4-9b7d-4feb-af4f-fbcc3ce84a5e

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    Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
    (Oxford University Press, 2013) DiCarlo, James; Norville, Julie; Mali, Prashant; Rios, Xavier; Aach, John; Church, George
    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.
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    Biocontainment of Genetically Modified Organisms by Synthetic Protein Design
    (Nature Research, 2015-02-05) Mandell, Daniel; Lajoie, Marc J.; Mee, Michael T.; Takeuchi, Ryo; Kuznetsov, Gleb; Norville, Julie; Gregg, Christopher J.; Stoddard, Barry L.; Church, George
    Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient either because they impose evolutionary pressure on the organism to eject the safeguard, because they can be circumvented by environmentally available compounds, or because they can be overcome by horizontal gene transfer (HGT). Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code to confer metabolic dependence on nonstandard amino acids for survival. The resulting GMOs cannot metabolically circumvent their biocontainment mechanisms using environmentally available compounds, and they exhibit unprecedented resistance to evolutionary escape via mutagenesis and HGT. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by reliance on synthetic metabolites.