Prime editing is a versatile and precise genome-editing tool. Most prime editors (PEs) rely on reverse transcriptase (RT) derived from Moloney murine leukemia virus (MMLV). Here, we established a PE, pvPE, using a RT derived from a porcine endogenous retrovirus (PERV) from a Bama mini-pig. Through various optimization strategies, including RT engineering, structural modifications, and La protein fusion, we gradually upgraded to pvPE-V4. This version achieved 24.38-101.69-fold higher efficiency compared with pvPE-V1 and up to 2.39-fold higher efficiency than another upgraded PE, PE7, with significantly fewer unintended edits across multiple mammalian cell lines. We further show that nocodazole (Noc) significantly enhanced pvPE efficiency by 2.25-fold on average. Using our pvPE system, we efficiently modified three genes simultaneously in porcine fibroblasts and subsequently generated cloned pigs that could serve as valuable models for Alzheimer's disease (AD) in humans. Our results highlight the broad application prospects of pvPE systems in mammalian genome editing.

Mucopolysaccharidosis type IIIB (MPS IIIB) is a rare genetic disorder caused by loss-of-function mutations in the NAGLU gene. Pigs are an ideal large animal model for human diseases; however, a porcine model of MPS IIIB has not been reported. We have previously generated a heterozygous NAGLU-deficient (NAGLU^+/-) Large White boar via a transgenic approach. Here we characterized phenotypes of the F₁ offspring of this founder to establish a pig model for MPS IIIB. qRT-PCR revealed that the NAGLU expression level was significantly decreased in a variety of tissues in NAGLU^+/− pigs. ELISA assays showed obvious deficiency of NAGLU and higher (P<0.05) glycosaminoglycan levels in multiple tissues from NAGLU^+/− pigs. NAGLU^+/− pigs grew at a significantly (P<0.05) slower rate than control animals (NAGLU^+/+). Death, mostly sudden death, occurred at all ages in NAGLU^+/− pigs, most of which died within two years. Necropsy findings included pleural adhesions, lung shrinkage and abnormalities in the pericardium and mild hepatomegaly in NAGLU^+/− pigs. Notable pathological changes were observed in the sections of brain, liver, spleen and kidney from NAGLU^+/− pigs. Brain atrophy, ventriculomegaly, cerebellar atrophy and abnormalities in the intracerebral capsule, parietal lobes and the thalamus were also evident in NAGLU^+/− pigs. Together, NAGLU^+/− pigs show typical symptoms of human MPS IIIB patients and thus represent a novel large animal model for the disease. This article has an associated First Person interview with the first author of the paper.

Objective: Copy number variations (CNVs) are a major source of genetic diversity complementary to single nucleotide polymorphism (SNP) in animals. The aim of the study was to perform a comprehensive genomic analysis of CNVs based on high density whole-genome SNP markers in Chinese Dongxiang spotted pigs. Methods: We used customized Affymetrix Axiom Pig1.4M array plates containing 1.4 million SNPs and the PennCNV algorithm to identify porcine CNVs on autosomes in Chinese Dongxiang spotted pigs. Then, the next generation sequence data was used to confirm the detected CNVs. Next, functional analysis was performed for gene contents in copy number variation regions (CNVRs). In addition, we compared the identified CNVRs with those reported ones and quantitative trait loci (QTL) in the pig QTL database. Results: We identified 871 putative CNVs belonging to 2,221 CNVRs on 17 autosomes. We further discarded CNVRs that were detected only in one individual, leaving us 166 CNVRs in total. The 166 CNVRs ranged from 2.89 kb to 617.53 kb with a mean value of 93.65 kb and a genome coverage of 15.55 Mb, corresponding to 0.58% of the pig genome. A total of 119 (71.69%) of the identified CNVRs were confirmed by next generation sequence data. Moreover, functional annotation showed that these CNVRs are involved in a variety of molecular functions. More than half (56.63%) of the CNVRs (n = 94) have been reported in previous studies, while 72 CNVRs are reported for the first time. In addition, 162 (97.59%) CNVRs were found to overlap with 2,765 previously reported QTLs affecting 378 phenotypic traits. Conclusion: The findings improve the catalog of pig CNVs and provide insights and novel molecular markers for further genetic analyses of Chinese indigenous pigs.

P53 is the most frequently mutated gene in human cancers, with over half of all tumors harboring mutation at this locus. R248 and R249 (corresponding to porcine R241 and R242), are among the hotspot mutations frequently mutated in liver, lung, breast, and some other cancers. In this study, p53 gene was knocked out or point-edited (R241 and R242 were converted to 241W and 242S) in porcine fetal fibroblast (PFF) cells via CRISPR-Cas9 technique. High throughput sequencing of miRNA and mRNA uncovered a total of 225 differentially expressed miRNAs (DEMs) and 738 differentially expressed genes (DEGs) in the p53 knockout (p53-KO) cells, and a total of 211 DEMs and 722 DEGs in the point-modified (p53-241W242S) cells. Totally 28 annotated DEMs were found to overlap between p53-KO/p53-WT and p53-241W242S/p53-WT miRNAs datasets, of which miR-34 c, miR-218, miR-205, miR-105-1, miR-105-2, miR-206, miR-224 and miR-429 play important roles in p53 regulatory network. Among the top 10 DEGs in p53-KO and p53-241W242S cells, most genes were reported to be involved in tumors, cell proliferation or cell migration. p53-KO and p53-241W242S cells showed a significantly higher (P < 0.01) proliferation rate compared with p53-WT cells. In conclusion, genetic modifications of p53 gene significantly affect the expression levels of a large number of genes and miRNAs in the PFF cells. The p53-edited PFF cells could be used as non-tumor cell models for investigating the p53 signaling network, and as donor cells for somatic nuclear transfer, with the aim to develop porcine models with the corresponding p53 mutations. Abbreviations: CRISPR-Cas9: Clustered regularly interspaced short palindromic repeats-associated protein 9; PFF: porcine fetal fibroblasts; SCNT: somatic cell nuclear transfer; RNA sequencing: small RNA sequencing and mRNA sequencing; DEGs: differentially expressed mRNAs; DEMs: differentially expressed miRNAs.

Cytosine base editors (CBEs) and adenine base editors (ABEs) are recently developed CRISPR-mediated genome-editing tools that do not introduce double-strand breaks. In this study, five ABEs, ABE7.10, ABEmax, NG-ABEmax, ABE8e and NG-ABE8e, were used to generate A-to-G (T-to-C) conversions in five genome loci in porcine fetal fibroblasts (PFFs). Variable yet appreciable editing efficiencies and variable activity windows were observed in these targeting regions via these five editors. The strategy of two sgRNAs in one vector exhibited superior editing efficiency to that of using two separate sgRNA expression vectors. ABE-mediated start-codon mutation in APOE silenced its expression of protein and, unexpectedly, eliminated the vast majority of its mRNA. No off-target DNA site was detected for these editors. Substantial off-target RNA events were present in the ABE-edited cells, but no KEGG pathway was found to be significantly enriched. Our study supports that ABEs are powerful tools for A-to-G (T-to-C) point-mutation modification in porcine cells.

Although massive cell atlases are already available, it is still a challenge to obtain an atlas of all tissues within a single body, especially in fetuses and pregnant mothers. We present a transcriptomic atlas of 2.56 million single cells covering 115 and 119 tissues from one fetal pig and its pregnant mother, respectively. We found that a cluster of heart capillary endothelial cells with enhanced fatty acid transit capability was enriched for pregnancy but restored after gestation. We also deciphered that l -leucine transport insufficiency in the trophoblast causes fetal growth restriction by reducing a muscle type II myofiber subcluster. Our “all-from-one” strategy enabled the identification of tissue cell type–specific transcription factors and provided insights into pregnancy heart adaptation and fetal growth restriction.