# Characterizing mechanisms of immune protection by the bacterial type II-A CRISPR systems

> **NIH NIH R03** · UNIVERSITY OF OKLAHOMA · 2024 · $70,990

## Abstract

Project Summary/Abstract
Bacteria employ several distinct defense strategies for protection from phage infection.
CRISPR-Cas is one such mechanism that provides an adaptive immune protection against
recurring phage infections based on genetic memory retained from past infections. The genetic
memory is retained through a process called “adaptation” where short pieces of the intruder
DNA (called prespacers) are selected following certain rules, and site-specifically inserted into
the CRISPR locus as a “spacer”. The spacer is transcribed into guide-RNAs that are essential
for sequence-specific targeting and inactivation of foreign genetic material. Adaptation in type II-
A CRISPR systems is unique compared to other CRISPR systems due to the inherent
capabilities of Cas1 and Cas2 proteins to catalyze the site-specific prespacer insertion without
assistance from cellular factors to maintain fidelity during insertion. This property offers unique
promises for type II-A Cas1-Cas2-based biotechnological and biomedical applications. Previous
work from the PI’s group has identified distinct subgroups of type II-A CRISPR systems based
on conserved DNA motifs present at the site of insertion, which is conserved across many
bacterial genera. Accompanying work had biochemically established unique differences in the
mechanisms of prespacer insertion by the different subgroups. The proposed research aims to
derive molecular mechanisms of the differences in DNA requirements and efficiencies of
prespacer insertion between the different type II-A subgroups. To derive structural and
conformational differences between these subgroups, hydrogen deuterium exchange mass
spectrometry (HDX-MS) and molecular dynamics (MD) simulations will be performed, followed
by biochemical validation of the results from HDX-MS and MD simulations. To assess the
contribution of individual protein and nucleic acid complexes in site-specific DNA insertion,
QM/MM (quantum mechanics/molecular mechanics) calculations will be employed. Based on
the free energy contributions identified, amino acids and DNA nucleotides will be mutated to
change integration efficiency with the end goal of engineering new Cas1-Cas2 variants which
has developed specificity to a newly designed DNA sequence. Since Cas1 and Cas2 based
prespacer insertion is vital to the functioning of all CRISPR systems, the lessons learned can be
translated to other CRISPR systems. Applications include programmed DNA insertions,
enhancing phage therapy to fight drug-resistant bacteria, and to develop bacteria that can fight
off phage infections for the fermentation industry.

## Key facts

- **NIH application ID:** 10811272
- **Project number:** 1R03AI175981-01A1
- **Recipient organization:** UNIVERSITY OF OKLAHOMA
- **Principal Investigator:** Rakhi Rajan
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $70,990
- **Award type:** 1
- **Project period:** 2023-12-12 → 2025-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10811272

## Citation

> US National Institutes of Health, RePORTER application 10811272, Characterizing mechanisms of immune protection by the bacterial type II-A CRISPR systems (1R03AI175981-01A1). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10811272. Licensed CC0.

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