Modular vector assembly enables rapid assessment of emerging CRISPR technologies

• Published in: Cell genomics Vol. 4; no. 3; p. 100519
• Main Authors: McGee, Abby V., Liu, Yanjing V., Griffith, Audrey L., Szegletes, Zsofia M., Wen, Bronte, Kraus, Carolyn, Miller, Nathan W., Steger, Ryan J., Escude Velasco, Berta, Bosch, Justin A., Zirin, Jonathan D., Viswanatha, Raghuvir, Sontheimer, Erik J., Goodale, Amy, Greene, Matthew A., Green, Thomas M., Doench, John G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13-03-2024; Elsevier
• Subjects: AAV; Cas12a; Cas9; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR; CRISPR-Cas Systems - genetics; CRISPRi; functional genomics; Genetic Vectors - genetics; Golden Gate; lentivirus; Technology; CRISPR; CRISPRi; AAV; functional genomics; Golden Gate; Cas9; Cas12a; lentivirus
• ISSN: 2666-979X; 2666-979X
• DOI: 10.1016/j.xgen.2024.100519

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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•Creation of a Golden Gate-based toolkit for combinatorial CRISPR vector assembly•Development of positive control guides to enable benchmarking of CRISPR technologies•Simultaneous assessment of multiple CRISPR technologies in a pooled, barcoded setting
McGee et al. develop Fragmid, a modular toolkit and online portal for the creation of CRISPR vectors compatible with many delivery approaches, enzymes, and mechanisms. They also develop a set of positive control guides to enable benchmarking of CRISPR technologies. They then use this toolkit to create and test vectors for a variety of implementations, including the simultaneous assessment of multiple CRISPR technologies in a pooled approach.