# Increasing the efficiency and range of prime editing for disease modeling in zebrafish

> **NIH NIH R21** · WASHINGTON UNIVERSITY · 2024 · $194,375

## Abstract

Zebrafish is among the premier model organisms for modeling human birth defects and diseases. Single base
pair changes are common candidates for regulatory or protein altering mutations underlying many common and
rare diseases. However, editing the endogenous zebrafish genes to model missense mutations or other discrete
human variants remains challenging. Prime editing (PE) is a breakthrough Cas9-based application for the
creation of animal and cell-based models of disease and for developing treatments of human genetic diseases
because it enables precise genome editing such as base substitutions, small insertions, and deletions. Prime
editing enzyme (PE2) is directed by pegRNA to a complementary genomic locus that contains an appropriate
protospacer adjacent motif (PAM) recognition site, 5’-NGG-3’. Nickase activity of PE2 introduces a nick to the
non-target strand of the target sequence. The reverse transcriptase (RT) of PE2 then reads the RT template of
the pegRNA containing the desired edit and synthesizes the DNA strand. Whereas a recent study showed that
delivery of PE2 protein can induce PE in zebrafish, because the PE2 enzyme is not yet commercially available,
this approach remains inaccessible to many zebrafish laboratories. Moreover, the current method is not efficient
and editable PE range is a relatively small region encompassing 3 bp upstream to 29 bp downstream of the PAM
recognition site.
 To overcome these limitations of PE and make it broadly functional in zebrafish laboratories, in our
preliminary studies we showed feasibility in zebrafish of a modified PE method that uses the RNA forms of PE2
and Cas9-RT achieving editing in up to 20% of injected F0 embryos. Our Aim 1 is 1) to optimize conditions for
the modified prime editing method by injecting different doses of the three RNA components, PE2, Cas9-RT,
and pegRNA, into zebrafish and 2) to establish a PE transgenic line. Our Aim 2 is to expand the prime editing
range. To this end, we will first test whether Cas9 D10A nickase can be applied as a PE2 enzyme to access
sequences upstream of the PAM site.
Instead of using the Cas9 H840A nickase that cleaves the non-target
strand, we will test the Cas9 D10A mutant form, which cleaves the target strand. Nicking the target strand will
trigger hybridization of the primer binding site of pegRNA to the sequences near the PAM site. Consequently,
the RT template of the pegRNA will be located further upstream of the PAM site, bringing the upstream sequence
within editable range. In parallel, we will investigate whether
Cas9 from Streptococcus canis (ScCas9), which
requires a single guanine (G) nucleotide as a PAM, can substitute in PE2 the standard Cas9, which requires 5’-
NGG-3’. Through these lines of research, this project will optimize the modified method of prime editing in
zebrafish and expand its range to facilitate generation of accurate zebrafish models for improved diagnosis,
mechanistic studies and therapeutics screens.

## Key facts

- **NIH application ID:** 10774325
- **Project number:** 5R21OD033668-02
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** LILIANNA SOLNICAKREZEL
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $194,375
- **Award type:** 5
- **Project period:** 2023-02-15 → 2026-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10774325

## Citation

> US National Institutes of Health, RePORTER application 10774325, Increasing the efficiency and range of prime editing for disease modeling in zebrafish (5R21OD033668-02). Retrieved via AI Analytics 2026-06-13 from https://api.ai-analytics.org/grant/nih/10774325. Licensed CC0.

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