# Engineering Synthetic Guide RNAs and Compact Base Editors for Enhanced In Vivo Delivery

> **NIH NIH F31** · UNIV OF MASSACHUSETTS MED SCH WORCESTER · 2022 · $33,910

## Abstract

Project Summary
CRISPR-Cas9 technology has profoundly advanced genetic research and gene therapy by enabling rapid,
precise, and programmable genome editing to study gene function or correct mutations in cells and in vivo.
Among CRISPR-mediated gene editing approaches, cytidine and adenine base editors (CBEs and ABEs) enable
efficient and precise single-base transitions in dividing and non-dividing cell types. Base editors comprise a
catalytically-impaired nickase Cas9 (nCas9) fused to a cytosine deaminase (CBE) or adenine deaminase (ABE)
that is guided to a target site by a “guide RNA”. With the potential to enable all four nucleotide transitions in the
context of a base-pair (C:G→T:A or A:T→G:C), base editors have the potential to cure a wide range of genetic
disorders. Realizing this hope requires efficient and safe in vivo delivery methods.
In vivo delivery of base editors relies on adeno-associated virus (AAV) vectors. Due to the limited packaging
capacity of AAVs and the large size of nCas9 orthologs (e.g., SpyCas9 from Streptococcus pyogenes), delivery
requires two AAVs encoding an intein-split nCas9-BE that fuses into a functional complex when co-expressed
in cells. Dual AAVs encoding intein-split SpyCas9-BEs achieve therapeutically-relevant levels of base editing in
pre-clinical disease models, including in the central nervous system (CNS). Nonetheless, dual AAV SpyCas9-
BE delivery suffers from several limitations, including: toxicity from increased viral load; high vector production
costs; immune response and off-target editing caused by sustained expression of nCas9-BE components; and
limited ability of guide RNA to specify multiplexed edits.
Under guidance from Drs. Erik Sontheimer (CRISPR), Miguel Sena Esteves (AAV delivery), Anastasia Khvorova
(oligonucleotide chemistry), and Athma Pai (sequencing, bioinformatics), this proposal aims to develop flexible
delivery approaches for efficient and safe base editing in vivo. This project will take advantage of established
gene therapy modalities, including a single AAV vector encoding a compact Cas9 from N. meningitidis
(Nme2Cas9), and chemically-modified oligonucleotides. Aim 1 will optimize and validate an all-in-one AAV
encoding a compact Nme2Cas9-ABE and its guide RNA for efficient in vivo base editing in mice. The use of a
compact ABE for AAV delivery will decrease viral load, production costs and may increase delivery efficiency.
Aim 2 will develop chemically-modified Nme2Cas9 crRNA (target-specify portion of guide RNA) for co-delivery
separate from an AAV encoding Nme2Cas9-ABE and tracrRNA (invariant portion of guide RNA). This approach
will provide a way to control nCas9-BE expression and streamline multiplexed base editing via delivery of multiple
crRNA. Although these delivery approaches are applicable to variety of tissues, this study will focus on the CNS,
where AAV- and oligonucleotide-based therapies have shown some success, but a dire need for transformative
therapeutics rem...

## Key facts

- **NIH application ID:** 10541817
- **Project number:** 5F31GM143879-02
- **Recipient organization:** UNIV OF MASSACHUSETTS MED SCH WORCESTER
- **Principal Investigator:** Nathan Bamidele
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $33,910
- **Award type:** 5
- **Project period:** 2021-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10541817

## Citation

> US National Institutes of Health, RePORTER application 10541817, Engineering Synthetic Guide RNAs and Compact Base Editors for Enhanced In Vivo Delivery (5F31GM143879-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10541817. Licensed CC0.

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