Modular vector assembly enables rapid assessment of emerging CRISPR technologies

Abby V McGee; Yanjing V Liu; Audrey L Griffith; Zsofia M Szegletes; Bronte Wen; Carolyn Kraus; Nathan W Miller; Ryan J Steger; Berta Escude Velasco; Justin A Bosch; Jonathan D Zirin; Raghuvir Viswanatha; Erik J Sontheimer; Amy Goodale; Matthew A Greene; Thomas M Green; John G Doench

doi:10.1016/j.xgen.2024.100519

Modular vector assembly enables rapid assessment of emerging CRISPR technologies

Cell Genom. 2024 Mar 13;4(3):100519. doi: 10.1016/j.xgen.2024.100519.

Authors

Abby V McGee¹, Yanjing V Liu¹, Audrey L Griffith¹, Zsofia M Szegletes¹, Bronte Wen¹, Carolyn Kraus², Nathan W Miller¹, Ryan J Steger¹, Berta Escude Velasco¹, Justin A Bosch³, Jonathan D Zirin³, Raghuvir Viswanatha³, Erik J Sontheimer⁴, Amy Goodale¹, Matthew A Greene¹, Thomas M Green¹, John G Doench⁵

Affiliations

¹ Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
² RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
³ Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
⁴ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA; Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA; Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
⁵ Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address: jdoench@broadinstitute.org.

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.

Keywords: AAV; CRISPR; CRISPRi; Cas12a; Cas9; Golden Gate; functional genomics; lentivirus.

MeSH terms

CRISPR-Cas Systems* / genetics
Clustered Regularly Interspaced Short Palindromic Repeats* / genetics
Genetic Vectors / genetics

Abstract

MeSH terms

Grants and funding