Prime editing optimized RTT permits the correction of the c.8713C>T mutation in DMD gene

Abstract

Duchenne muscular dystrophy is a severe debilitating genetic disease caused by different mutations in the DMD gene leading to the absence of dystrophin protein under the sarcolemma. We used CRISPR-Cas9 prime editing technology for correction of the c.8713C>T mutation in the DMD gene and tested different variations of reverse transcription template (RTT) sequences. We increased by 3.8-fold the editing percentage of the target nucleotide located at +13. A modification of the protospacer adjacent motif sequence (located at +6) and a silent mutation (located at +9) were also simultaneously added to the target sequence modification. We observed significant differences in editing efficiency in interconversion of different nucleotides and the distance between the target, the nicking site, and the additional mutations. We achieved 22% modifications in myoblasts of a DMD patient, which led to dystrophin expression detected by western blot in the myotubes that they formed. RTT optimization permitted us to improve the prime editing of a point mutation located at +13 nucleotides from the nick site to restore dystrophin protein.

Keywords: CRISPR-Cas9; DMD gene; Duchenne muscular dystrophy; MT: RNA/DNA editing; RTT; c.8713C>T mutation; prime editing.

Graphical abstract

Figure 1

PE2 and PE3 editing of DMD exon 59 using SpCas9 and variants (A) Editing efficiency using the initial three pegRNAs for the PE2 and PE3 strategies (using an sgRNA inducing a nick at position +62) to induce c.8713C>T mutation in exon 59 of DMD gene. The differences between pegRNA1, pegRNA2, and pegRNA3 for PE2 and PE3 were statistically significant (∗∗∗p < 0.001). (B) Results obtained with three different pegRNAs (a, b, and c) designed individually for each nuclease variant recognizing the NGG PAM (for SpCas9), the NGAN PAM (for SpCas9-VQR), and the NNN PAM (for SpCas9-RY). ns indicates that the differences between the pegRNAs of these Cas9 variants were not significant. The asterisks indicate that the differences were statistically significant (∗∗∗p < 0.001). (C) Editing efficiency for PE2 and PE3 strategies using three pegRNAs containing two mutations each: the target mutation and the mutation in the PAM sequence (PM). The difference of mutation at the target site was significant only for pegRNA1 used for PE2 and PE3. (D) Partial sequence of DMD exon 59 carrying a nonsense mutation to be corrected (TGA sequence shown by the red square at the position +13). The red square is the TGA stop codon to be corrected to a CGA codon which is an arginine (R). The orange square contains the sequence of the PAM CGG to be modified to CGT to increase the editing efficiency of the nonsense mutation at the position +13. The numbers +1 and +13 represent different positions from the nick site, and the blue arrow is the 20-nt spacer sequence. These experiments were done in independent triplicates (n = 3). The non-parametric Mann-Whitney U test was performed to calculate the p values.

Figure 2

Influence of the PAM nucleotide or other nucleotides in the target (A) Results obtained when the guanine nucleotide at +6 from the PAM is modified into a thymine simultaneously with the modification of one nucleotide located at positions spanning +1 to +13 from the nick induced by the SpCas9n. Position +13 is indicated in the red square in Figure 1D. At this position 13, the CGA codon is to be changed to a TGA codon. (B) Results obtained when modifications are done from +1 to +19 while modifying simultaneously the target nucleotide at +13. These experiments were done in independent triplicates (n = 3). All the editing percentages at different targets were compared with the modification at +13. The p values were calculated using the non-parametric Mann-Whitney U test. ∗∗∗p < 0.001; ∗∗p = 0.01; ns, non-significant difference.

Figure 3

The type of nucleotide to be changed at the target This figure shows the difference in editing efficiency to induce c.8713C>T mutation in exon 59 of DMD gene while also changing one nucleotide of the PAM sequence. The PAM sequence CGG is changed respectively to CGT, CGA, and CGC, which are all coding for the arginine amino acid. Each of these modifications was done simultaneously with the modification of the target at +13 changing C to T. These experiments were done in independent triplicates (n = 3). The differences in editing efficiency at the target for the different modifications in PAM sequences were not statistically significant (ns).

Figure 4

Influence of the RTT length on the target (A and B) The PE2 (A) and PE3 (B) results for the introduction of c.8713C>T mutation in exon 59 of DMD gene when the RTT length varies from 13 (RTT 13) to 42 (RTT 42). The difference was not statistically significant (ns) either for PE2 or PE3 using the Kruskal-Wallis test. (C and D) The PE2 (C) and PE3 (D) results when the RTT length varies from RTT 13 to RTT 42. The modification at the target (green) is done simultaneously with the modification of the PAM sequence (purple). The experiments were done in triplicates (n = 3). The p values were calculated using the Kruskal-Wallis test. The editing percentages were compared between RTT13 and other RTTs for the mutation at the target site. ∗∗∗p = 0.001; ∗∗p = 0.01; ∗p < 0.05; ns, non-significant difference.

Figure 5

Influence of simultaneous additional mutations on the target (A and B) Variations in editing efficacy for PE2 (A) and PE3 (B) strategies for the introduction of c.8713C>T mutation in exon 59 of DMD gene when the PAM sequence and a third additional nucleotide (ADD MUT) are simultaneously modified. The three mutations are all introduced by a pegRNA. The third mutation is introduced at different positions (from +1 to +15) in RTT19 and RTT31. (C) Illumina deep sequencing and Sanger sequencing results for PE3 strategy (shown in B) when the modification at the target is done simultaneously with the modification in the PAM sequence and the introduction of the third additional silent mutations at different positions of RTT19 and RTT31. ∗p = 0.01 using the non-parametric Mann-Whitney U test. ns, non-significant difference. (D) Individual results for the combination of five different mutations at different positions of the RTT19 sequence. The experiments were done in independent triplicates (n = 3).

Figure 6

Influence of the type of nucleotide to be changed at the target This figure shows the PE3 results with different pegRNAs having RTT19 (A) and RTT31 (B). Here, the C nucleotide at the target site is changed either to T (C>T), G (C>G), or A (C>A). Each time these mutations are done, the PAM sequence at the position +6 and the third additional nucleotide (ADD MUT) at the position +3 are simultaneously changed as indicated in the x axis of the graph. The experiments were done in independent triplicates (n = 3). The p values were calculated using the non-parametric Mann-Whitney U test . The C>T groups were compared with C>G and C>A groups. ∗∗∗p = 0.0001, ∗∗p = 0.001, ∗p = 0.01, and p > 0.05 (ns) for 5% confidence interval.

Figure 7

Correction of DMD c.8713C>T mutation (A) Editing percentage for PE3 and PE5 strategies in human myoblasts for the correction of c.8713C>T mutation in exon 59 of DMD gene. The RTT19 and RTT31 used here permitted us to induce the desired modification (T>C) at +13 while modifying simultaneously the PAM sequence (G>T) at +6 and the additional nucleotide (T>C) at +9. The experiments were done in triplicates (n = 3). The mean editing percentage at the target site was statistically significant with (∗∗∗p < 0.001) using the non-parametric Mann-Whitney U test. (B) Western blot resulting from 20 μg of total protein obtained by the lysis of myotubes from the culture plate. It indicates the molecular weight marker (460 kDa), the negative control sample (Ctrl−), which used myoblasts with point mutation in exon 59, the positive controls (Ctrl+), which were healthy human myoblasts, and the samples treated with the pegRNAs described in (A).