Figure 2. Improving prime editing efficiencies in zebrafish by pegRNA refolding and mutations in RTT.

(a–b) Pure PE frequencies of non-refolded and refolded substitution pegRNAs (a) and insertion or deletion pegRNAs (b) with PE2 in zebrafish. Target loci, PBS lengths (labeled as ‘P’ followed by the number of nucleotides), RTT lengths (labeled as ‘R’ followed by the number of nucleotides), and pegRNA-specified edits (denoted as the position of the edit followed by the edit) are shown at the top. Pure PE represents sequencing reads containing only the pegRNA-specified mutations. (c–d) Pure PE frequencies with refolded pegRNAs carrying additional RTT mutations (at +1,+2 or+3) and PE2 in zebrafish. Target loci, PBS, and RTT lengths are shown at the top and pegRNA-specified edits are shown at the bottom. All pegRNAs had 3 or 4 thymine (T) nucleotides at the 3’ end except for the ones labeled ‘A end’ for scn2b in which the terminal Ts were replaced with adenine (A) nucleotides. Dots represent individual data points (n=3 biologically independent replicates, 5–10 embryos per replicate), bars the mean and error bars ± s.e.m. *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed t-test with equal variance).

Figure 2—figure supplement 1. Purities of prime editing with PE2 and non-refolded or refolded pegRNAs in zebrafish.

(a–b) Frequencies of non-pure PE edits (labeled as other edits) mediated by PE2 with non-refolded and refolded substitution pegRNAs (a) and insertion or deletion pegRNAs (b). (c–d) PE purities (%) calculated as pure PE over total edit (pure and non-pure PE) frequencies for substitution pegRNAs (c) and insertion or deletion pegRNAs (d). Target loci, PBS lengths (labeled as ‘P’ followed by the number of nucleotides), RTT lengths (labeled as ‘R’ followed by the number of nucleotides), and pegRNA-specified edits are indicated at the top. Dots represent individual data points (n=3 biologically independent replicates, 5–10 embryos per replicate), bars the mean and error bars ± s.e.m. Results of unpaired two-tailed t-test with equal variance are shown in *p<0.05, **p<0.01, ***p<0.001.

Figure 2—figure supplement 2. Purities of prime editing with PE2 and refolded pegRNAs carrying additional RTT mutations in zebrafish.

(a–b) Frequencies of non-pure PE edits (labeled as other edits) mediated by PE2 with refolded pegRNAs with or without an additional mutation in the RTT (at +1,+2 or+3). (c) PE purities calculated as pure PE over total edit (pure and non-pure PE) frequencies. Target loci, PBS lengths (labeled as ‘P’ followed by the number of nucleotides), and RTT lengths (labeled as ‘R’ followed by the number of nucleotides) are shown at the top. pegRNA-specified edits (denoted as the position of the edit followed by the edit) are shown at the bottom. Dots represent individual data points (n=3 biologically independent replicates, 5–10 embryos per replicate), bars the mean, error bars ± s.e.m. Results of unpaired two-tailed t-test with equal variance are shown in *p<0.05, **p<0.01, ***p<0.001.

Figure 2—figure supplement 3. Indel frequencies in zebrafish induced by SpCas9 complexed with refolded pegRNAs with or without RTT mutation at +2 position.

SpCas9 protein and pegRNA were combined at a molar ratio of 1:2. (1.8 µM of pegRNA). Target loci, PBS lengths (labeled as ‘P’ followed by the number of nucleotides), and RTT lengths (labeled as ‘R’ followed by the number of nucleotides) are shown at the top. pegRNA-specified edits (denoted as the position of the edit followed by the edit) are shown at the bottom. Dots represent individual data points (n=3 biologically independent replicates, 5–10 embryos per replicate), bars the mean and error bars ± s.e.m. Results of unpaired two-tailed t-test with equal variance are shown in **p<0.01.