Publications by authors named "Zsofia M Szegletes"

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.

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Article Synopsis
  • The diverse CRISPR systems have led to a surge in applications, but researchers often face challenges with complicated cloning processes for custom reagents.
  • The Fragmid toolkit utilizes Golden Gate cloning to streamline the creation of CRISPR cassettes and delivery methods, allowing for quick vector assembly via an online portal.
  • This system enables researchers to evaluate multiple CRISPR technologies simultaneously, accelerating the development and optimization of CRISPR-based solutions and enhancing their usability in various biological studies.
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Cas12a CRISPR technology, unlike Cas9, allows for facile multiplexing of guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here, we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes.

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Background: Glioblastoma is one of the most lethal forms of cancer, with 5-year survival rates of only 6%. Glioblastoma-targeted therapeutics have been challenging to develop due to significant inter- and intra-tumoral heterogeneity. Telomerase reverse transcriptase gene (TERT) promoter mutations are the most common known clonal oncogenic mutations in glioblastoma.

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Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we assess the activity and specificity of WT-Cas9 and 10 SpCas9 variants by benchmarking their PAM preferences, on-target activity, and off-target susceptibility in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A > G and C > T base editors, more than tripling the number of guides and assayable residues.

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