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Fig. 1 | Genome Biology

Fig. 1

From: Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor

Fig. 1

Improvement of prime-editing efficiency using chromatin-modulating peptides and proximal dsgRNA in mouse cells and embryos. a Schematic diagrams of the prime editor (PE), chromatin-modulating peptide-PE-Variant1 (CMP-PE-V1), and chromatin-modulating peptide-PE-Variant2 (CMP-PE-V2) constructs. PE consists of nCas9 (H840A) and M-MLV RT. CMP-PE-V1 has HN1 at the N-terminus of nCas9 and H1G at the C-terminus of nCas9 in the PE structure. CMP-PE-V2 has HN1 at the N-terminus of nCas9 and H1G at the C-terminus of M-MLV RT in the PE structure. HN1, high-mobility group nucleosome binding domain 1; H1G, histone H1 central globular domain. bg Comparison of the prime-editing efficiencies of PE3, PE3+dsgRNA, CMP-PE3-V1, and CMP-PE3-V1+dsgRNA at the Igf2 (b), Adamts20 (c), Casp1 (d), Hoxd13 (e), Angpt1 (f), and Ksr2 (g) target sites in NIH/3T3 and C2C12 cells. Data and error bars show the mean ± standard deviation (s.d.) of five independent biological replicates (n = 5). P-values were obtained using two-tailed Student’s t-tests. *P <0.05, **P <0.01, ***P <0.001. hm The components of the prime-editing system were injected into the pronucleus of the mouse zygote and analyzed 4 days after injection. The dots indicate the frequencies of the desired mutations in Igf2 (h), Adamts20 (i), Hoxd13 (j), Angpt1 (k), Ksr2 (l), and Ar (m) from each blastocyst treated with PE3, PE3+dsgRNA, CMP-PE3-V1, or CMP-PE3-V1+dsgRNA. The numbers above the dots in the graph represent the number of edited embryos/total embryos. The black horizontal line denotes the mean of the frequencies of the desired mutations. n Fractions of intact genomic DNA from the Igf2 and Adamts20 target loci were measured using real-time qPCR after a DNase I digestion assay. The gene located at Chr 3: 71,026,628–71,026,685 (mouse genome build mm9) was used as the negative control (closed chromatin) and Col6a1 was used as the positive control (open chromatin). Data and error bars show the mean ± s.d. of three independent experiments (n = 3). o After transfecting with plasmids encoding PE3, PE3+dsgRNA, CMP-PE3-V1, or CMP-PE3-V1+dsgRNA to C2C12, fractions of intact genomic DNA from the Igf2 target locus were measured using real-time qPCR after a DNase I digestion assay. Data and error bars show the mean ± s.d. of three independent experiments (n = 3)

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