# Generation of DNA memory by bacterial CRISPR-Cas9 systems

> **NIH NIH R35** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2021 · $377,976

## Abstract

Project Summary
Prokaryotic horizontal gene transfer (HGT) underlines the spread of antibiotic resistance and pathogenic traits.
The battle against antibiotic resistance must be fought on multiple fronts, including the understanding of natural
barriers that microbes use to restrict HGT. Most bacteria rely on the CRISPR-Cas system to establish adaptive
immunity against mobile genetic elements. DNA pieces from these invaders' genome can be captured and
stored as immunological memories termed spacers, at the CRISPR loci. Small, antisense RNAs produced from
CRISPR (crRNAs) will guide Cas enzymes to destroy invaders with a matching target site. In the past decade,
much progress has been made in understanding the CRISPR interference enzymes and their applications in
genetic engineering. However, how microbes acquire their CRISPR memories remains very poorly understood.
In this proposal, we aim to uncover the molecular basis for CRISPR memorization (i.e. spacer adaptation). We
use the gram-negative pathogen Neisseria meningitidis (Nme) as a model organism, due to of its clinical
importance and tractable genetics. Current knowledge about spacer adaptation mostly comes from studies of
the type I CRISPR native to E. coli; products of its conserved cas1-cas2 integrase genes can create functional
memories independently of the interference enzymes. Our recent preliminary
findings
suggest that the type II
CRISPR of N. meningitidis creates memory by a distinct mechanism. The interference genes, Nmecas9 and
tracrRNA co-factor, play important but non-conventional roles in the acquisition of functional spacers. We will
use molecular genetic, genomic and biochemical approaches to address fundamental questions, including:
What are the molecular roles of Cas9 and the CRISPR-encoded tracrRNA in spacer acquisition? What are the
rules governing memory DNA selection? How does Cas9/tracr cooperate with the Cas1-2 integrase? And
finally, how would the anti-CRISPR proteins affect the memorization process?
The proposed research will illuminate the interplay between pathogenic bacteria, their CRISPR systems, and
HGT. This work also promises to guide technology advances, including CRISPR-based novel antimicrobials
that kill specific bacterial pathogens, and Cas9-Cas1-Cas2 based genome-tagging devices that help record
cellular/disease history.

## Key facts

- **NIH application ID:** 10227166
- **Project number:** 5R35GM137883-02
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Yan Zhang
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $377,976
- **Award type:** 5
- **Project period:** 2020-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10227166

## Citation

> US National Institutes of Health, RePORTER application 10227166, Generation of DNA memory by bacterial CRISPR-Cas9 systems (5R35GM137883-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10227166. Licensed CC0.

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