# Nucleic Acid-Based Anti-CRISPR Inhibitors of Cas9

> **NIH NIH R01** · SOUTHERN ILLINOIS UNIVERSITY CARBONDALE · 2020 · $281,595

## Abstract

PROJECT SUMMARY
 Sophisticated, facile, and potent inhibitors of Cas enzymes that are simple to use are still a major
missing tool for CRISPR-based biomedical and therapeutic research. The rationale for CRISPR-Cas
inhibitors includes a need for more efficient generation of model cells and organisms, the threat of
dangerous off-target editing in CRISPR-based therapeutics, and the desire for more precise control over
Cas enzyme activity in synthetic biology and diagnostics applications.
 Methods and modified Cas enzymes have been developed to help address these issues and other
shortcomings of CRISPR-Cas systems, particularly Cas9 from Streptococcus pyogenes (SpCas9). These
include Cas9 mutants with lower off-target activity and Cas9 mutants and protein fusions that allow small
molecule or light-based activation of Cas9. However, these modifications typically have a concomitant
decrease in on-target activity and do not address the need for rapid and specific shut-down of Cas9.
Despite improvements to Cas enzymes, the need for specific, broadly applicable, and easy-to-use
inhibitors will remain a necessary tool that is not available. Small molecules that can inhibit Cas9 may take
significant effort to develop and potentially come with downsides, including their own off-target effects and
inadvertent impacts on the organism’s microbiota, possibly similar to antibiotic side-effects.
 Natural anti-CRISPR proteins have recently been discovered that bind Cas9 and other Cas proteins
with high affinity. While promising as inhibitors, they are too large to possess drug-like properties and their
minimization or optimization is not an obvious exercise. They must be genetically encoded for use in cells
or organisms. However, they provide inspiration for the design of a new class of CRISPR-Cas inhibitors.
 Here we propose to develop a new technology, nucleic acid-based (NAB) inhibitors of Cas9 enzymes.
These molecules are smaller than natural anti-CRISPR proteins but can bind with similar affinity, be
chemically synthesized, and be readily introduced into cells with common methods. In this project, we will
develop NAB inhibitor technology by optimizing their size, chemistry, binding affinity, inhibitory activity,
and cellular stability. We will further develop methods and molecules that facilitate direct, carrier-free
delivery and timed-release of NAB inhibitors. The resulting NAB inhibitors are expected to be broadly
applicable and straightforward to use for diverse biomedical research. We expect NAB inhibitors to
become valuable fail-safe inhibitors to overcome the critical safety hurdles in CRISPR-based therapeutics.

## Key facts

- **NIH application ID:** 9867347
- **Project number:** 1R01GM135646-01
- **Recipient organization:** SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
- **Principal Investigator:** Keith Thomas Gagnon
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $281,595
- **Award type:** 1
- **Project period:** 2020-01-03 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9867347

## Citation

> US National Institutes of Health, RePORTER application 9867347, Nucleic Acid-Based Anti-CRISPR Inhibitors of Cas9 (1R01GM135646-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9867347. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*