Optimised genome editing for precise DNA insertion and substitution using Prime Editors in zebrafish

Reviewer #1 (Public review):

Ono et al. compared the activity of prime editor Nickase PE2 and prime editor nuclease PEn in introducing SNPs and short exogenous DNA sequences into the zebrafish genome to model human disease variants. They find the nickase PE2 prime editor had a higher rate of precise integration for introducing single-nucleotide substitutions, whereas the nuclease PEn prime editor showed improved precision of integration of short DNA sequences. In somatic tissue, the percentage of SNP variant precision edits improved when using PE2 RNP injection instead of mRNA injection, but increased precision editing correlated with elevated indel formation. While PEn overall had higher rates of precision edits, the indel rate was also elevated. Similar rates were observed when introducing a 3 bp stop codon into the ror gene using a standard pegRNA with a 13-nucleotide homology arm, or a springRNA lacking the homology arm that drives integration via NHEJ. Inclusion of an abasic sequence in the springRNA prevented imprecise edits caused by scaffold incorporation, but did not improve the overall percentage of precise edits in somatic tissue. Recovery of a germline ror-TGA integration allele using PEn with RNP was robust, resulting in 5 out of 10 founders transmitting a precise allele. Lastly, the authors demonstrate that PEn was effective at the integration of a 30 bp nuclear localization signal into the 5' end of GFP in an existing muscle-specific reporter line. However, the undefined number of cassettes in this multicopy transgene complicates accurate measurements of editing frequency. Integration of the NLS or other longer sequences at an endogenous locus would demonstrate the broad utility of this approach. From the work presented, it is unclear how prime editing could be used to transiently model human pathogenic variants, given the low frequency of precision edits in somatic tissue, or to isolate stable germline alleles of variants that are potentially dominant negative or gain-of-function in nature. Without a direct comparison with CRISPR/Cas9 nuclease HDR-based methods that use oligonucleotide templates to introduce edits, the advantage of prime editing is unclear. A cost comparison between prime editing and HDR methods would also be of interest, particularly for integration of longer DNA sequences.

The conclusions of the paper are mostly well supported, but some changes to the text and additional analyses would strengthen the conclusion that PE2 vs. PEn is preferred for introducing variants, short or long DNA sequences.

(1) In Figure 3, the data indicate a significant increase in precise edits of the 3 bp TGA using PE2 RNP (11.5%) vs. PE2 mRNA (1.3%). At the adgrf3b locus, only PEn mRNA was tested for introducing the 3 bp and 12 bp insertions. The previous study testing PE2 for 3 and 12 bp insertions was mentioned, but the frequency was not listed, and the study wasn't cited (lines 204 - 207). A comparison of germline transmission rates using PE2 vs. PEn would support the conclusion that PEn allows precise integration of longer templates and recovery of germline integration alleles.

(2) Figure 4 shows the results of introducing a TGA stop codon that is predicted to result in nonsense-mediated decay. Testing the ability to also isolate different substitution mutations in the germline would be useful information for identifying the most effective approach for generating human disease variant models.

(3) A comparison with the prime editing variant knock-in frequencies reported in the recent publication by Vanhooydonck et al., 2025, Lab Animal should be included in the Discussion.

Reviewer #2 (Public review):

Summary:

The manuscript provides a comparison of nickase-based (PE2) and nuclease-based (PEn) Prime Editors in zebrafish, evaluating their efficiencies for substitutions, short insertions (3-30 bp), and germline transmission.

Strengths:

The manuscript has demonstrated for the first time that nuclease-based PEn more efficiently inserts nucleotide sequences up to 30 bp (nuclear localization sequence) than PE2, providing an improvement for the application of gene editing in functional genetics research. Additionally, the demonstration of stable zebrafish lines with edited ror2 and smyhc1:gfp loci is well-supported by sequencing and phenotypic data, confirming functional consequences of edits.

Weaknesses:

The study lacks conceptual innovation, as the central methodology-RNP-based Prime Editor delivery in zebrafish-was previously established by Petri et al. (2022). The present study extends this by testing longer insertions (30 bp) with nuclease-based PEn, but this incremental advance does not substantially shift the field's understanding or capabilities. The manuscript does not sufficiently differentiate its contributions from these precedents.

The comparative analysis between PE2 and PEn systems suffers from limited evidentiary support. The comparison relies on single loci for substitutions (crbn) and insertions (ror2), raising concerns about generalizability. Additional validation across multiple loci is necessary to support broad conclusions about PE2/PEn performance.

Reviewer #3 (Public review):

The manuscript by Ono et al describes the application of prime editors to introduce precise genetic changes in the zebrafish model system. Probably the most important observation is that, compared to the "standard" PE2, the prime editor with full nuclease activity appears to be more efficient at introducing insertions into the genome. Although many laboratories around the world have successfully used oligonucleotide-mediated HDR to insert short exogenous sequences such as epitope tags or loxP sites into the zebrafish genome, the method suffers from a high frequency of indels at the edit site. Thus, additional tools are badly needed, making this manuscript very important. Length of the longer reported insertion (+30) is quite close to the range of V5 (14 amino acids) and ALFA (12 amino acids without "spacer" prolines) epitope tags, as well as loxP site (34 nucleotides). Conclusions drawn in the paper are supported by compelling evidence. I only have a few minor comments:

(1) The logic for introducing two nucleotide changes (at +3 and +10) to change a single amino acid (I378) should be explicitly explained in the main body of the manuscript. It is indeed self-explanatory when looking at Supplementary Figure 1. One way of doing it could be to include Supplementary Figure 1a in Figure 1.

(2) It is not clear why a 3-nucleotide insertion was used to generate W722X. The human W720X is a single-nucleotide polymorphism, and it should be possible to make a corresponding zebrafish mutant by introducing two nucleotide changes.

(3) Lines 137-138: T7 Endonuclease assay used in Figure 2d detects all polymorphisms, both precise changes and indels. Thus, if this assay were performed on embryos shown in Figure 1c-d, the overall percentage of modified alleles would be similarly higher for PEn over PE2 (add up precise prime edits and indels). The conclusion in the last sentence of the paragraph is, therefore, incorrect, I believe.

(4) Use of terminology. "Germline transmission" is typically used to refer to the fraction of F0s transmitting desired changes (or transgenes) to their progeny, while "germline mosaicism" refers to the fraction of F1s with the desired change in the progeny of a given F0. "Germline transmission" in line 217 should be replaced with "germline mosaicism".

(5) Lines 253-255: The fraction of injected embryos that had mosaic nuclear expression of GFP, indicative of NLS insertion, should be clarified. It should also be clarified whether embryos positive for nuclear GFP were preselected for amplicon sequencing and germline transmission analyses. This is extremely important for extrapolation to scenarios like epitope tagging, where preselection is not possible.

(6) Statistical analyses. It would be helpful to clarify why different statistical tests are sometimes used to assess seemingly very similar datasets (Figures 1c, 1d, 2b, 2c, 2f).

(7) Discussion. Since authors suggest that PEn might be especially beneficial for insertion of additional sequences, it is important to stress locus-to-locus variability of success. While the precise +3 insertion was indeed tremendously efficient at both tested loci (ror2 and adgrf3b), +12 addition into adgrf3b was over 10 times less efficient (lines 193-194). In contrast, +30 into smyhc:GFP using the shorter pegRNA was highly efficient again with an average of 8.5% of sequence reads indicating precise integration (line 257, Figure 5c). Longer pegRNA did not work nearly as well (Figure 5c), but was still much better than +12 into adgrf3b. As dangerous as it is to extrapolate from small datasets, perhaps these observations indicate that optimization of RT template and PBS may be needed for each new locus in order to significantly outperform oligonucleotide-mediated HDR? If so, would the cost of ordering several pegRNAs and the effort needed to compare them factor in when deciding which method to use? Reported germline transmission rates for both ror2 W722X (+3, Figure 4a) and smyhc:NLS-GFP (+30, Figure 5f) are tantalizingly high.