Efficient Generation of P53 Biallelic Mutations in Diannan Miniature Pigs Using RNA-Guided Base Editing

Abstract

The base editing 3 (BE3) system, a single-base gene editing technology developed using CRISPR/Cas9n, has a broad range of applications for human disease model construction and gene therapy, as it is highly efficient, accurate, and non-destructive. P53 mutations are present in more than 50% of human malignancies. Due to the similarities between humans and pigs at the molecular level, pig models carrying P53 mutations can be used to research the mechanism of tumorigenesis and improve tumor diagnosis and treatment. According to pathogenic mutations of the human P53 gene at W146* and Q100*, sgRNAs were designed to target exon 4 and exon 5 of the porcine P53 gene. The target editing efficiencies of the two sgRNAs were 61.9% and 50.0%, respectively. The editing efficiency of the BE3 system was highest (about 60%) when C (or G) was at the 5th base. Puromycin screening revealed that 75.0% (21/28) and 68.7% (22/32) of cell colonies contained a P53 mutation at sgRNA-Exon5 and sgRNA-Exon4, respectively. The reconstructed embryos from sgRNA-Exon5-5# were transferred into six recipient gilts, all of which aborted. The reconstructed embryos from sgRNA-Exon4-7# were transferred into 6 recipient gilts, 3 of which became pregnant, resulting in 14 live and 3 dead piglets. Sequencing analyses of the target site confirmed 1 P53 monoallelic mutation and 16 biallelic mutations. The qPCR analysis showed that the P53 mRNA expression level was significantly decreased in different tissues of the P53 mutant piglets (p < 0.05). Additionally, confocal microscopy and western blot analysis revealed an absence of P53 expression in the P53 mutant fibroblasts, livers, and lung tissues. In conclusion, a porcine cancer model with a P53 point mutation can be obtained via the BE3 system and somatic cell nuclear transfer (SCNT).

Keywords: BE3 system; P53 gene; SCNT; cancer; point mutation.

Figure 1

Conversion of C-to-T by the BE3 system. (A) Schematic diagram of the target site at the P53 locus. sgRNA sequences are presented in black. PAM sequences are highlighted in green. The BE3-mediated nucleotide substitutions are marked in red and underlined. (B) Diagrammatic representation of the mutations associated with P53 in humans and pigs.

Figure 2

Targeting efficiency of P53-sgRNA-Exon4 and P53-sgRNA-Exon5. (A) Detection of sgRNA-Exon4 and sgRNA-Exon5: BE3-mediated base editing of P53 by PCR and T7EN1 cleavage assay. M, DNA marker; sgRNA-Exon4, P53sgRNA-Exon4; sgRNA-Exon5, P53sgRNA-Exon5; C, control. (B) Alignments of mutant sequences from targeted sequencing. PAM site and substitutions are shown in green and red, respectively. Relevant codons at the target site are underlined. The column on the right indicates amino acid mutation type, deletions (−), insertions (+), and proportion of positive colonies out of all sequenced colonies.

Figure 3

The predicted editing bar plot based on Sanger sequencing. (A) Base position in sgRNA-Exon4. (B) Base position in sgRNA-Exon5. (C) Synthetic 86-mers with sequences matching P53 gene sites were incubated with sgRNA-Exon4 and then analyzed for base editing by high-throughput DNA sequencing (HTS). Synthetic 84-mers with sequences matching P53 gene sites were incubated with sgRNA-Exon5 and then analyzed for base editing by HTS. The sequence of the protospacer is indicated to the right of the name of the site, with the PAM highlighted in blue. Underneath each sequence are the percentages of total DNA sequencing reads with the corresponding base.

Figure 4

BE3-mediated mutations in the cells clones of P53-sgRNA-Exon4 and P53-sgRNA-Exon5. (A) The sequences of P53 mutant cell lines. The WT sequence is shown above. PAM site and substitutions are shown in green and red, respectively. Relevant codons at the target site are underlined. The column on the right indicates amino acid mutation type. (B) Representative sequencing chromatograms at the P53-sgRNA-Exon4 and P53-sgRNA-Exon5 targets of WT and edited pig cell clones. Target sequence (black), PAM region (green), target sites (red), mutant amino acids (underlined), and amino acid mutation types are indicated. The relevant codon identities at the target site are presented beneath the DNA sequence.

Figure 5

BE3-mediated P53 mutations in piglets. (A) The P53 mutant piglets (P1, P2, P6, P9, P12, and P15). (B) The WT sequence is shown above. PAM site and substitutions are shown in green and red, respectively. Relevant codons at the target site are underlined. The column on the right indicates the amino acid mutation type. (C) Sanger sequencing chromatograms of WT and mutant founder piglets. Red box indicates mutation. The piglets with monoallelic mutations are shown in red. WT: wild-type.

Figure 6

The detection of P53mRNA and proteins in piglets. (A) The relative expression levels of P53 mRNA in the different tissues from P53 mutant and WT piglets. (B) P53 protein expression in lung and liver. (C) Quantification of P53 protein expression in different organs in WT and P53 mutant pigs. β-actin was used as an internal control. (D) The intracellular localization of P53 was analyzed using fluorescence microscopy. The livers and lungs from P53 mutant piglets and WT piglets were stained with DAPI (blue) and an anti-P53 antibody (red). (E) The fibroblast cells were treated with DOX and DMSO and protein expression levels were examined by western blotting. (F) Quantification of P53 protein expression in different organs in fibroblast cells treated with DOX and DMSO. β-actin was used as an internal control. (G) The intracellular localization of P53 was analyzed using fluorescence microscopy. The fibroblast cells from P53 mutant piglets and WT piglets were treated with DOX for 24 h and stained with Hoechst 33,342 (blue) and an anti-P53 antibody (red). (H) Lung samples from a P53 mutant and a WT piglet after autopsy.