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
Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
bioRxiv. 2023 Oct 27:2023.10.25.564061. doi: 10.1101/2023.10.25.564061.
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.
CRISPR系统的多样性，再加上科学创造力，引发了应用的激增；然而，为了在其模型系统中测试新描述的创新，研究人员通常会着手进行繁琐的一次性克隆项目，以生成针对其生物学问题进行优化的定制试剂。在这里，我们利用金门克隆技术创建了Fragmid工具包，这是一组模块化的CRISPR盒式组件和递送技术，以及一个门户网站，从而形成了一个组合平台，能够在数天内实现可扩展的载体组装。我们进一步证明，使用该资源可以以汇集筛选的形式并行评估多种CRISPR技术，从而能够快速优化新技术和细胞模型。这些结果确立了Fragmid作为一种用于快速设计CRISPR载体的强大系统，并且我们预计这种组装方法将广泛应用于CRISPR技术的系统开发、比较和传播。
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