Optimised genome editing for precise DNA insertion and substitution using prime editors in zebrafish
- Living Systems Institute, Biosciences, University of Exeter, United Kingdom
- Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Sweden
- Biosciences, University of Exeter, United Kingdom
- School of Physiology, Pharmacology, and Neuroscience, University of Bristol, United Kingdom
- Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, United Kingdom
Figures
Figure 1 with 1 supplement
Prime editing substitution in the zebrafish crbn gene, comparing Cas9-nickase-based (PE2) and Cas9-nuclease-based Prime Editors (PEn).
(A) Schematic illustration of the functioning of prime editing by the PE2 and the PEn. (B) Schematic illustration of the strategy for prime editing substitution in crbn gene. The nucleotides for substitution are indicated in red. The guide RNA (gRNA) target sequence is underlined. (C–E) Comparison between PEn and PE2 in prime editing substitution in the crbn gene with the prime editing guide RNA (pegRNA) refolding procedure. Proportions of editing outcomes in individual injected embryos are shown by amplicon sequencing (C), alongside quantitative analyses of precise prime edits (D) and indels (E) comparing experimental conditions (n=10 per group). p-Values were determined using one-way ANOVA with Tukey’s multiple comparison test in (D), and the Kruskal-Wallis test with Dunn’s multiple comparison test in (E). Error bars in the bar graphs represent the mean and standard deviation, and individual data points indicate values from single injected embryos.
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Figure 1—source data 1
Proportion of edits in amplicon sequencing crbn gene.
- https://cdn.elifesciences.org/articles/107475/elife-107475-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Prime editing substitution in the zebrafish crbn gene.
Quantitative analyses of precision scores comparing between Cas9-nuclease-based Prime Editor (PEn) and Cas9-nickase-based Prime Editor (PE2) in prime editing substitution in the crbn gene with the prime editing guide RNA (pegRNA) refolding procedure (n=10 per group). p-Values were determined using Welch’s one-way ANOVA with Dunnett T3 multiple comparison test. Error bars in the bar graphs represent the mean and standard deviation, and individual data points indicate values from single injected embryos.
Figure 2 with 1 supplement
Prime editing insertion in zebrafish ror2 gene using Cas9-nuclease-based Prime Editor (PEn).
(A) Schematic illustration of the functional domains in the Ror2 protein and alignment of partial amino acid sequences within the tyrosine kinase domain. Sequences from multiple species, including those related to Robinow syndrome (RS) in humans W720X and the corresponding zebrafish W722X mutant, are aligned. The conserved tyrosine residue is highlighted. (B) Schematic illustration of guide RNA (gRNA) designs for prime editing insertion in ror2. (C) Schematic illustration of prime editing insertion by PEn. An additional DNA fragment, reverse-transcribed at the target cleavage site, containing the programmed insertion, is integrated into the genome via homology-directed repair or non-homologous end joining. (D) Agarose gel images of genomic PCR products from embryos injected with Prime Editor mRNA and prime editing guide RNA (pegRNA)/single primed insertion gRNA (springRNA). PCR products of the ror2 target region (top) and those after digestion with T7 endonuclease I (T7E1, bottom). (E) Sequence alignment of the edits in the ror2 target site obtained from embryos injected with PEn/springRNA. Prime editing insertion (TGA) is outlined, and the gRNA target sequence is underlined. (F) Quantitative comparison of editing outcomes using different combinations of Prime Editor and gRNA. The proportion of sequence reads with each type of edit in amplicon sequencing is presented.
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Figure 2—source data 1
PDF file containing original agarose gel image for Figure 2D, indicating the relevant DNA bands and experimental conditions.
- https://cdn.elifesciences.org/articles/107475/elife-107475-fig2-data1-v1.zip
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Figure 2—source data 2
Original file of agarose gel image used to prepare Figure 2D.
- https://cdn.elifesciences.org/articles/107475/elife-107475-fig2-data2-v1.zip
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Figure 2—source data 3
Proportion of edits in prime editing insertion in ror2 gene.
- https://cdn.elifesciences.org/articles/107475/elife-107475-fig2-data3-v1.xlsx
Figure 2—figure supplement 1
Analysis of prime editing insertion in zebrafish ror2 gene by sequencing the clones.
(A) Sequence alignment of the edits in the ror2 target site obtained from randomly selected bacterial clones comparing prime editing outcomes using Cas9-nickase-based Prime Editor (PE2)/prime editing guide RNA (pegRNA) (top) and Cas9-nuclease-based Prime Editor (PEn)/pegRNA (bottom) combinations. The guide RNA (gRNA) target sequence is indicated by red arrows. (B) Quantitative comparison of editing outcomes using different combinations of Prime Editor and gRNA. The proportion of the clones with each type of edit is presented.
Figure 3 with 1 supplement
Comparing different prime editing approaches in zebrafish embryos.
(A and B) Comparison between Cas9-nuclease-based Prime Editor (PEn) and Cas9-nickase-based Prime Editor (PE2) in prime editing insertion into ror2 with various delivery methods. The proportion of editing outcomes in individual injected embryos was assessed through amplicon sequencing (A), along with a quantitative analysis of precise prime edit (B) and indels (D) across experimental conditions (n=10 per group). (C) Proportion of reads with precise homology-directed repair (HDR)-mediated insertion in ror2 using single-stranded sense or antisense donor DNAs with 40 bp homology arms. (D) Proportion of reads with indels comparing PEn and PE2 in prime editing insertion into ror2. (E) Sequence alignment of edits in prime editing insertion in ror2 via PE2 mRNA/prime editing guide RNA (pegRNA) (top), PEn mRNA/pegRNA (middle), and PEn mRNA/single primed insertion gRNA (springRNA) (bottom) combinations. (F) Schematic illustration of springRNA design featuring an abasic RNA spacer for prime editing insertion in ror2. (G and H) Proportion of reads with scaffold incorporation (G) and precise prime edit (H) as determined by amplicon sequencing of prime editing insertion in ror2 using control and abasic springRNA (n=10 per group; one sample of the PEn ribonucleoprotein [RNP]/abasic springRNA combination was excluded from analysis due to low read count). (I and J) Prime editing insertion in the adgrf3b gene using PE2 and PEn. The proportion of reads with a precise 3 bp (I) and 12 bp insertion (J) was evaluated (n=10 per group). p-Values were determined by Welch’s one-way ANOVA with Dunnett T3 multiple comparison test in B, D, and J, and Kruskal-Wallis test with Dunn’s multiple comparison test in G. Error bars in bar graphs represent the mean and standard deviation, and each individual data point indicates the value from a single injected embryo.
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Figure 3—source data 1
Proportion of edits in amplicon sequencing ror2 and adgrf3b genes.
- https://cdn.elifesciences.org/articles/107475/elife-107475-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Amplicon sequencing analysis of prime editing insertion in zebrafish ror2 and agdrf3b genes.
(A) Proportion of reads with indels in homology-directed repair (HDR)-mediated knock-in in ror2 using single-stranded sense or antisense donor DNAs with 40 bp homology arms. (B) Quantitative analyses of precision scores comparing various experimental conditions for prime editing insertion in ror2 gene using prime editing guide RNA (pegRNA). (C–E) Comparison between Cas9-nuclease-based Prime Editor (PEn) and Cas9-nickase-based Prime Editor (PE2) in prime editing substitution in ror2 to generate the W722X allele. The proportion of editing outcomes in individual injected embryos was assessed through amplicon sequencing (C), along with a quantitative analysis of precise prime edit (D) and indels (E) across experimental conditions. (F) Comparison of potential off-target sequences of ror2 pegRNA and single primed insertion gRNA (springRNA). Mismatched bases and a gap are indicated in bold red and primer binding site (PBS) is highlighted in blue. PAM sequences are underlined. (G) Proportion of overall editing in each off-target site comparing HDR-mediated knock-in and various prime editing conditions. (H and I) Precision scores in prime editing insertion in ror2 gene using springRNA (H), and in adgrf3b gene using PEn mRNA (I). (J and K) Proportion of reads with scaffold incorporation in 3 bp (J) and 12 bp (K) prime editing insertion in adgrf3b using springRNAs with an RNA (J) or DNA (K) spacer. The sample size is n=10 per group, and one sample of the PEn ribonucleoprotein (RNP)/abasic springRNA combination was excluded from analysis in H due to low read count. p-Values were determined by Welch’s one-way ANOVA with Dunnett T3 multiple comparison test in B, and Kruskal-Wallis test with Dunn’s multiple comparison test in H and I. To evaluate differences between the two groups, p-values were determined by unpaired t-test in D and E. Welch’s t-test and the Mann-Whitney U test were employed in J and K, respectively. Error bars in bar graphs represent the mean and standard deviation, and each individual data point indicates the value from a single injected embryo.
Figure 4 with 1 supplement
Generation and characterisation of stable ror2W722X mutant.
(A) Summary of the screening of F1 embryos. Sixteen embryos were genotyped per F0 founder. Each circle represents the genotype of a single embryo. The embryos were obtained by outcrossing the injected founder with wild-type fish; thus, all embryos with the mutation are heterozygous. (B–D) Lateral images of wild type (B), zygotic ror2W722X mutant (C), and maternal-zygotic (MZ) ror2W722X mutant (D) larvae at 5 days post-fertilisation (dpf). (E) Sanger sequencing chromatogram of wild type (top) and ror2W722X mutant (bottom) at the prime editing target site in ror2. Prime editing insertion (TGA) is highlighted. The target sequence of the guide RNA is underlined, and the cleavage site of Cas9 is indicated by a dotted line. (F) Quantitative analysis of total length comparing wild type, zygotic ror2W722X mutant, and MZ ror2W722X mutant at 5 dpf. Each data point on the graph represents the value from a single larva (n=10 per group). p-Values were determined by one-way ANOVA with a Tukey’s multiple comparisons test. Error bars represent the mean and standard deviation. (G and H) Lateral images of 1-year-old wild type (G) and MZ ror2W722X mutant (H). Nasal and maxillary barbels are indicated by black and white arrowheads, respectively. (I and J) Reconstructed computed tomography images of cranioskeletal morphologies in 1-year-old wild type (I) and MZ ror2W722X mutant (J). The images are ventral view and anterior is to the top. (K) Schematic illustration of mandibular bone from the ventral view and the location of geographical points for annotation to measure linear distances and calculate the aspect ratios. (L and M) Quantitative analysis of the aspect ratios in the mandible comparing wild type and MZ ror2W722X mutant. Aspect ratio of anterior part (L) and whole structure (M) of the mandible was analysed. The p-values were determined using unpaired t-test. Error bars represent the mean and standard deviation, while individual data points on the graph indicate values from single reconstructed image.
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Figure 4—source data 1
Morphological analysis of ror2W722X mutant.
- https://cdn.elifesciences.org/articles/107475/elife-107475-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Characterisation of stable ror2W722X mutant.
(A and B) Lateral images of 18-month-old wild type (A) and zygotic ror2W722X mutant (B). Nasal and maxillary barbels are indicated by black and white arrowheads, respectively.
Figure 5 with 2 supplements
Prime editing to insert a nuclear localisation signal sequence into the smyhc1:gfp transgene.
(A) Schematic representation of the prime editing insertion of the nuclear localisation signal (NLS) sequence into the smyhc1:gfp transgene and the expected eGFP expression in slow-twitch muscle fibres. (B) Confocal microscopy image of the trunk muscle in smyhc1:gfp larvae at 4 days post-fertilisation (dpf) that were injected with the Cas9-nuclease-based Prime Editor (PEn) ribonucleoprotein (RNP) complex for the prime editing NLS insertion. Putative nuclear GFP expression is indicated by an arrowhead. Anterior is positioned to the left. (C) Quantitative analysis of the efficiency of precise NLS insertion via amplicon sequencing. Two prime editing guide RNAs (pegRNAs) of differing lengths are compared (n=10 per group, with one sample excluded from the analysis due to low read count). The p-value was determined using the Mann-Whitney U test. Error bars represent the mean and standard deviation, while individual data points on the graph indicate values from single injected larvae. (D) Confocal microscopy images of the trunk muscle of F1 larvae exhibiting nuclear GFP expression at 2 dpf, obtained from founder 1 (see panel F). The GFP fluorescence channel (left) and pseudo-colour (right) images are shown. Anterior is to the left. (E) Sequence alignment of the edits in the target site obtained from F1 embryos exhibiting nuclear GFP expression (founder 1). The NLS sequence is outlined in red. (F) Summary of F1 embryo screening. F1 embryos obtained from six founders are categorised based on the GFP expression pattern. The embryos were produced by outcrossing the heterozygous smyhc1:gfp founder injected with the PEn RNP complex to wild type; thus, half of the embryos are expected to be negative for the smyhc1:gfp transgene.
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Figure 5—source data 1
Proportion of edits in amplicon sequencing smyhc1:gfp transgene and wls gene, and F1 embryo screening.
- https://cdn.elifesciences.org/articles/107475/elife-107475-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Prime editing insertion of a nuclear localisation signal sequence into the smyhc1:gfp transgene.
(A) The expression of smyhc1:gfp transgene in 2 days post-fertilisation (dpf) larvae. Lateral images of whole larvae (left) and the trunk muscle (right). Anterior is to the left. (B) Confocal microscopy images of the trunk muscle of F1 larvae exhibiting nuclear GFP expression at 2 dpf, obtained from founder 2, 3, and 4 (see Figure 5F). (C) Sequence alignment of the edits in the target site obtained from F1 embryos exhibiting nuclear GFP expression (founder 2, 3, and 4). The nuclear localisation signal (NLS) sequences are outlined in red.
Figure 5—figure supplement 2
Prime editing insertion of a 46 bp attP sequence into zebrafish wls gene.
(A and B) Comparison between homology-directed repair (HDR)-mediated CRISPR/Cas9 knock-in and prime editing using Cas9-nuclease-based Prime Editor (PEn)/single primed insertion gRNA (springRNA) ribonucleoprotein (RNP) complex in inserting 46 bp attP sequence into wls gene. Two single-stranded donor DNAs with 40 bp homology arms in the same (+) or opposite (−) directions of the target spacer sequence of guide RNA were used. The proportion of editing outcomes in individual injected embryos was assessed through amplicon sequencing (A), along with a quantitative analysis of precise edit (B) across experimental conditions (n=10 per group). p-Values were determined using the Kruskal-Wallis test with Dunn’s multiple comparison test. Error bars in the bar graphs represent the mean and standard deviation, and individual data points indicate values from single injected embryos.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/107475/elife-107475-mdarchecklist1-v1.docx
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Supplementary file 1
The sequences of the pegRNA, springRNA and donor DNA used in the study.
- https://cdn.elifesciences.org/articles/107475/elife-107475-supp1-v1.xlsx
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Supplementary file 2
Primer sequences for amplicon sequencing.
- https://cdn.elifesciences.org/articles/107475/elife-107475-supp2-v1.xlsx
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Supplementary file 3
Parameters of the CRISPResso analysis.
- https://cdn.elifesciences.org/articles/107475/elife-107475-supp3-v1.xlsx
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Optimised genome editing for precise DNA insertion and substitution using prime editors in zebrafish
eLife 14:RP107475.
https://doi.org/10.7554/eLife.107475.3
