# Advancing programmable RNA-targeting tools for research and therapeutics

> **NIH NIH R01** · BROAD INSTITUTE, INC. · 2022 · $778,909

## Abstract

PROJECT SUMMARY
We previously developed a suite of tools for modulating and studying RNA based on the RNA-targeting CRISPR-
Cas13 system, which has been adopted or extended by many researchers in the life sciences. This proposal
seeks to discover and characterize additional programmable RNA binding proteins and develop them
for use as molecular technologies. In particular, we are focusing on identifying ultra-small Cas13 proteins,
which can be fused to RNA editing effectors to create compact platforms for precision, single-base transcript
editing. RNA editing has significant therapeutic potential across a spectrum of conditions, including genetic
diseases where it is not possible or too risky to edit the genome as well as acute insults where transient genetic
changes are desirable. Thus, another aim is to demonstrate the feasibility of using RNA editing therapeutically.
Beyond RNA editors, we will also use the new proteins we identify to develop transcriptional state sensors, which
can be used to mark specific cell sub-types within a heterogenous population, either for imaging, isolation, or
functional outcomes.
To achieve these goals, we will leverage our previous experience to discover and characterize new RNA
targeting CRISPR systems, with a focus on identifying small enzymes that support RNA editing activity. We will
also explore the possibility of using novel RNA deaminase enzymes in our RNA editing constructs. In addition to
creating RNA editing constructs, we will also fuse the RNA targeting enzymes to GFP or Cre to create
transcriptional sensors. A critical aspect of our work will be protein engineering. Candidate enzymes (or their
RNA components) may need to be modified for efficient, specific activity in mammalian cells. We will use protein
engineering to increase the specificity and activity of RNA deaminases, as well as extend the substrate base
preference of these enzymes. For our transcriptional state sensors, protein engineering will be central to
successfully generating sensors that afford high specificity and high signal-to-noise ratios. All of these efforts will
be guided by structural and biochemical studies of the relevant enzymes.
Finally, we will apply the compact, high-specificity RNA editors in a mouse model of acute liver damage to
demonstrate their therapeutic potential as short-lived, reversible treatments. In parallel, we will demonstrate the
feasibility of using RNA editing to correct a mutation that causes the neurodevelopmental disorder Rett syndrome
using a previously established mouse model of this disease.
This work will substantially advance RNA editing toward clinical use, as well as uncover new biology about
CRISPR systems and other microbial defense systems.

## Key facts

- **NIH application ID:** 10475182
- **Project number:** 5R01HG009761-06
- **Recipient organization:** BROAD INSTITUTE, INC.
- **Principal Investigator:** Feng Zhang
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $778,909
- **Award type:** 5
- **Project period:** 2017-08-17 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10475182

## Citation

> US National Institutes of Health, RePORTER application 10475182, Advancing programmable RNA-targeting tools for research and therapeutics (5R01HG009761-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10475182. Licensed CC0.

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