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DiCarlo, James

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DiCarlo

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DiCarlo, James

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2006 - 200912010 - 20152

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Author's Publications: search.filters.isAuthorOfPublication.a6d78b95-6632-4f1e-8bb2-5b68136a8f40

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Now showing 1 - 3 of 3
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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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    Object Selectivity of Local Field Potentials and Spikes in the Macaque Inferior Temporal Cortex
    (Elsevier BV, 2006) Kreiman, Gabriel; Hung, Chou P.; Kraskov, Alexander; Quiroga, Rodrigo Quian; Poggio, Tomaso; DiCarlo, James
    Local field potentials (LFPs) arise largely from dendritic activity over large brain regions and thus provide a measure of the input to and local processing within an area. We characterized LFPs and their relationship to spikes (multi and single unit) in monkey inferior temporal cortex (IT). LFP responses in IT to complex objects showed strong selectivity at 44% of the sites and tolerance to retinal position and size. The LFP preferences were poorly predicted by the spike preferences at the same site but were better explained by averaging spikes within approximately 3 mm. A comparison of separate sites suggests that selectivity is similar on a scale of approximately 800 microm for spikes and approximately 5 mm for LFPs. These observations imply that inputs to IT neurons convey selectivity for complex shapes and that such input may have an underlying organization spanning several millimeters.
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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.