Structural Basis of Programmable DNA-Insertion via Cryo-EM Studies of CRISPR-Associated TnsC

Cornell University, Ithaca NY

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Abstract

The current major focus of the parent award (R01) is to reveal how CRISPR-associated transposons (CAST) exquisitely regulate their insertion activity, which is dependent on a guide- RNA sequence. Specifically, we are interested in how CAST elements: 1. choose their target- sites and 2. how transposase catalytic activity is properly activated at the target site. We believe that the physical arrangement of the CRISPR and transposase functional modules is the key to describe how disparate functionality in guide-RNA recognition and target-DNA binding are integrated and regulated. To accomplish this, cryo-electron microscopy (Cryo-EM) is used to obtain large imaging datasets of megadalton nucleoprotein assemblies, which must then be analyzed and processed using a computationally intensive workflow. The Kellogg lab maintains its own local server, equipped with 4 A100, which are suitable for analyzing one dataset at a time. However, the large size of the datasets currently imaged necessitate an expansion of current computing resources. To this end, we request an administrative equipment supplement to allow us to expand computing by 3-fold through the purchase of an additional server with 8 A100 GPUs. This will allows us to keep up with current computational bottlenecks experienced throughout the NIH funded research program.