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

> **NIH NIH R01** · CORNELL UNIVERSITY · 2022 · $318,307

## Abstract

Project Summary
 Recently, new macromolecular systems have been discovered which marries the benefits of both CRISPR
and TNP systems and shows tremendous promise as programmable DNA-insertion tools for genome-editing,
complementing the power of tools such as CRISPR-Cas9. This proposal aims to uncover the molecular
mechanisms governing two as-yet poorly understood phenomena in CRISPR-Transposase (CRISPR-TNP)
systems: target-site immunity and programmed-DNA insertion. The central protein thought to be responsible for
both of these observed behaviors in the multi-component shCAST system is shTnsC. We propose to utilize high-
resolution cryo-EM to determine the structure of DNA-bound shTnsC. In addition, shTnsC is a AAA+ ATPase
whose nucleotide-hydrolysis activity is linked to transposition. We propose to determine the structure of shTnsC
in different nucleotide-bound states in order to reveal the role of ATP-hydrolysis in transposition. Finally, we aim
to uncover the mechanisms governing shTnsC recruitment to the target-site via Cas12k and shTniQ, and how
the association between these factors ultimately initiates shCAST transposition. Our strong preliminary data
indicates that these aims are likely to be successful. In addition, the Kellogg lab is well-supported within the
Cornell community to achieve the goals outlined in this proposal.

## Key facts

- **NIH application ID:** 10344519
- **Project number:** 1R01GM144566-01
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** Elizabeth Kellogg
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $318,307
- **Award type:** 1
- **Project period:** 2022-01-01 → 2026-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10344519

## Citation

> US National Institutes of Health, RePORTER application 10344519, Structural Basis of Programmable DNA-Insertion via Cryo-EM Studies of CRISPR-Associated TnsC (1R01GM144566-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10344519. Licensed CC0.

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