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.