# Discovery, Mechanism and Function of Type-V CRISPR-Cas Inhibitors

> **NIH NIH F32** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2020 · $65,310

## Abstract

PROJECT SUMMARY
CRISPR-Cas12a has recently emerged as a powerful gene editing tool with great potential to ameliorate wide-
ranging diseases through gene therapy. Cas12a, like Cas9, is a nuclease that can be programmed to cut
genomes at specific sequences with high efficiency, but it is better for targeting AT-rich sequences and multiple
genes simultaneously. The safe implementation of Cas12a, however, requires the development of inhibitors
that can enable regulation and prevent editing at off-target sites. It also requires improved understanding of
Cas12a biology and cleavage activity in cells, for which a paucity of data exists. For example, Cas12a has
been shown to indiscriminately cleave single-stranded DNA (i.e. perform trans-cleavage) after binding to its
target DNA in vitro, but it is not known if this occurs in cells.
The long-term objectives of this proposal are to identify and develop Cas12a inhibitors and determine if Cas12
trans-cleavage occurs in vivo. Using bioinformatics and in vivo assays, we have recently discovered the first
three proteins (acrVA1-3) that inhibit Cas12a cleavage in bacteria and in human cells. These proteins are
encoded in a phage (virus) infecting bacteria, where they inhibit phage cleavage by Cas12a. These inhibitors
stand to provide useful tools for Cas12a regulation, but their successful implementation requires insight into
their mechanisms of inhibition. Preliminary evidence suggests that each AcrVA protein functions by a distinct
mechanism, which will be elucidated using a variety of in vitro and in vivo assays that determine their effect on
Cas12a expression and target DNA binding. Next, we will identify AcrVA proteins that optimally inhibit different
Cas12a variants commonly used in gene editing. This will be achieved by mutagenizing acrVA1 and selecting
for optimized inhibitors using bacterial selection screens as well as by exploring natural acrVA diversity using
bioinformatics and in vivo inhibition assays. Finally, the existence of indiscriminate Cas12a trans-cleavage in
vivo and its susceptibility to inhibition by acrVA1-3 will be determined using phage infection experiments in
bacteria. Overall, this work will illuminate fundamental Cas12a biology and develop these novel inhibitors into
powerful tools that can regulate Cas12a activity. In doing so, it will significantly improve the safety and utility of
Cas12a in correcting genetic disorders. This work will be performed at UCSF, which hosts world-class facilities
and a highly intellectual and collaborative research community. It will also provide me with the expertise in
bacterial-phage biology, biochemistry, and gene editing that I need to fulfill my postdoctoral training goals and
pioneer an independent research program in bacterial-phage counter-immunity.

## Key facts

- **NIH application ID:** 9925050
- **Project number:** 5F32GM133127-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Nicole D. Marino
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $65,310
- **Award type:** 5
- **Project period:** 2019-05-01 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9925050

## Citation

> US National Institutes of Health, RePORTER application 9925050, Discovery, Mechanism and Function of Type-V CRISPR-Cas Inhibitors (5F32GM133127-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9925050. Licensed CC0.

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