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Modular vector assembly enables rapid assessment of emerging CRISPR technologies.
McGee, Abby V; Liu, Yanjing V; Griffith, Audrey L; Szegletes, Zsofia M; Wen, Bronte; Kraus, Carolyn; Miller, Nathan W; Steger, Ryan J; Velasco, Berta Escude; Bosch, Justin A; Zirin, Jonathan D; Viswanatha, Raghuvir; Sontheimer, Erik J; Goodale, Amy; Greene, Matthew A; Green, Thomas M; Doench, John G.
Affiliation
  • McGee AV; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Liu YV; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Griffith AL; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Szegletes ZM; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Wen B; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Kraus C; RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Miller NW; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Steger RJ; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Velasco BE; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Bosch JA; Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
  • Zirin JD; Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
  • Viswanatha R; Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
  • Sontheimer EJ; RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Goodale A; Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Greene MA; Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
  • Green TM; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Doench JG; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
bioRxiv ; 2023 Oct 27.
Article in En | MEDLINE | ID: mdl-37961518
ABSTRACT
The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly-described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Language: En Journal: BioRxiv Year: 2023 Document type: Article Affiliation country: Estados Unidos
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Full text: 1 Collection: 01-internacional Database: MEDLINE Language: En Journal: BioRxiv Year: 2023 Document type: Article Affiliation country: Estados Unidos