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Moosburner, Mark

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Moosburner, Mark

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Author's Publications: search.filters.isAuthorOfPublication.64e02d4d-fc64-43e7-80f9-14073c4f4db2

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    Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing
    (2013) Esvelt, Kevin Michael; Mali, Prashant; Braff, Jonathan; Moosburner, Mark; Yaung, Stephanie J.; Church, George
    The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences and identify a highly targetable protein from Neisseria meningitidis. Our results provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins and adapting them to eukaryotic cells.
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    CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering
    (2013) Mali, Prashant; Aach, John; Stranges, P. Benjamin; Esvelt, Kevin Michael; Moosburner, Mark; Kosuri, Sriram; Yang, Luhan; Church, George
    Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes.1–7. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a novel transcriptional activation–based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator–like (TAL) effector proteins8, 9. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effector proteins can potentially tolerate 1–3 and 1–2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.