# Engineering improved delivery and immune profiles of Cas9 orthologues for gene therapy

> **NIH NIH F32** · UNIVERSITY OF CALIFORNIA BERKELEY · 2022 · $58,310

## Abstract

Project Summary / Abstract
The current gold standard to deliver gene therapies, adeno-associated virus (AAV), has shown safe and stable
transgene expression in many applications. However, the combination of AAV with CRISPR introduces unique
risks of genotoxic side effects from long-term nuclease expression and integration of viral DNA into sites of DNA
breaks in the host genome. Furthermore, the ssDNA genome of AAV limits its packaging capacity so that two
AAVs must be used to deliver Cas9 from Streptococcus pyogenes (SpyCas9) and its guide RNA, limiting
efficiency and increasing costs. Interest has switched to using a smaller Cas9 orthologue derived from
Staphylococcus aureus (SauCas9), although several enzyme features have been shown to be significantly
different. Furthermore, pre-existing immunity to AAV capsids, as well as SpyCas9 and SauCas9, has been
identified in humans, potentially limiting the therapeutic use of these molecules. While the host response to the
two orthologues is dependent on previous exposure, SauCas9 was found to elicit a stronger immune response
than SpyCas9 in human subjects when measured by immunoblot, ELISA, and ELISpot assays. Therefore,
although SauCas9 is a smaller nuclease that enables delivery by AAV, there are several questions about safety
that must be addressed. Previous work in the Doudna laboratory has shown that the SpyCas9 endonuclease
can be engineered with cationic residues to make the ribonucleoprotein (RNP) inherently cell-penetrating in
neural precursor cells in vitro and in neurons in vivo by non-viral delivery. I have now engineered SauCas9 to
also act as a cell-penetrating RNP. The purpose of this research proposal is to evaluate the outcomes of
engineered Cas9 orthologues delivered as RNPs or AAVs in the mammalian brain. Aim 1 employs an unbiased
protein engineering and screening strategy to make small deletions across Cas9 that improve the cellular host
immune response to the protein. Aim 2 will test these variants as ribonucleoproteins or as adeno-associated
viruses delivered by stereotaxic injection into the striatum of a fluorescent reporter animal model to assess
genome editing outcomes and the host immune response; and Aim 3 will apply these findings to genetically
correcting SOD1 mutations in an animal model of amyotrophic lateral sclerosis. Taken together, developing a
cell-penetrating and immune-stealthy Cas9 RNP for transient and local genome editing would improve the safety
of CRISPR therapies and accelerate the pace of clinical trials that could immediately benefit patients.

## Key facts

- **NIH application ID:** 10444901
- **Project number:** 5F32GM140637-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA BERKELEY
- **Principal Investigator:** Elizabeth Stahl
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $58,310
- **Award type:** 5
- **Project period:** 2021-08-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10444901

## Citation

> US National Institutes of Health, RePORTER application 10444901, Engineering improved delivery and immune profiles of Cas9 orthologues for gene therapy (5F32GM140637-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10444901. Licensed CC0.

---

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