# Enhancing CRISPR-mediated homology-directed repair using anti-CRISPR proteins

> **NIH NIH R43** · ACRIGEN BIOSCIENCES, INC. · 2022 · $256,580

## Abstract

Abstract
Genetic disorders take a significant toll on individuals, families, and communities. The cause of many of these
diseases is a single point mutation in the genome. Despite advances in the diagnosis and underlying genetic
foundation for these disorders, curative treatments have remained elusive. The discovery of the CRISPR-Cas9
system and its ability to edit human genomes has brought renewed hope for curative therapies. However,
correcting point mutations in the genome requires precise repair of the DNA break induced by Cas9 through the
homology-directed repair (HDR) pathway. Unfortunately, this repair pathway is inefficient, leading to low genome
correction frequencies. Acrigen Biosciences, Inc. is pioneering the use of anti-CRISPR (Acr) proteins to enhance
genome correction by increasing the efficacy of HDR. This Phase I project will use a recently discovered Acr to
transiently tether repair DNA to the Cas9 nuclease, increasing the local concentration of repair template to the
Cas9 DNA cleavage site. This will increase the efficiency of HDR and subsequent correction of the disease
causing mutation. The Phase I proposal has the following Aims: 1) Establish a human cell reporter system to
assess the efficiency of homologous recombination. Acrigen will design CRISPR-Cas9 guide RNAs and donor
DNA constructs to convert the fluorescent reporter EGFP to BFP through HDR. The reporter system will be
validated in human cells. 2) Increase reporter HDR efficiency using Acr technology. Acrigen will validate binding
of Acr to both Cas9 and the HDR DNA donor. Acrigen will then use Acr to transiently tether the donor DNA to
Cas9 and assess the efficiency of homologous recombination in the fluorescent reporter system. 3) Validate Acr-
enhanced HDR against SMN2. Acrigen will design Cas9 guide RNAs and donor DNA templates to convert
truncated SMN protein to full-length SMN by editing a single point transition in the SMN2 gene. Finally, Acrigen
will then use Acr to increase HDR efficiency and SMN conversion in spinal muscular atrophy (SMA) patient-
derived cells. At the end of Phase I, we will have developed a new approach to increase HDR efficiency in
cells and will have validated this technology to enhance conversion of SMN as a potential cure for SMA.

## Key facts

- **NIH application ID:** 10383623
- **Project number:** 1R43GM145002-01
- **Recipient organization:** ACRIGEN BIOSCIENCES, INC.
- **Principal Investigator:** David Rabuka
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $256,580
- **Award type:** 1
- **Project period:** 2022-02-15 → 2022-11-14

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10383623

## Citation

> US National Institutes of Health, RePORTER application 10383623, Enhancing CRISPR-mediated homology-directed repair using anti-CRISPR proteins (1R43GM145002-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10383623. Licensed CC0.

---

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