Traditional pig breeding is limited by the long breeding cycle and insufficient genetic resources, highlighting the potential value of genetic modification that can significantly improve a specific heritable production trait in pigs in one generation. Using gene modification techniques, researchers have created a variety of genetically modified pigs with excellent production traits, resulting in significant improvements in feed utilization, lean meat percentage, disease resistance, and healthy fatty acid composition (Petersen 2017; Zhao. et al. 2019; Han et al. 2020; Xu et al. 2020a; Zhu et al. 2020b).
In addition to the potential applications in agriculture, genetically modified pigs have significant biomedical uses as ideal animal models (Petersen 2017; Perleberg et al. 2018; Yan et al. 2018; Zhu et al. 2018; Zhao et al. 2019; Koppes et al. 2020). Pigs have many advantages over other mammals (e.g., rodents and ruminants). Firstly, pigs possess many similarities to humans in terms of body size, physiology, organ development, disease process, gene sequences, and chromosome structure; pigs are thus better models for human diseases. Secondly, pigs are highly fecund, with early sexual maturation (5–8 months), short generation interval (10–12 months), large litters (10–12 piglets per litter on average), and year-round estrus, allowing rapid propagation of experimental materials. Finally, with the use of efficient gene editing techniques represented by CRISPR/Cas9 in pigs, it has become easier and simpler to construct genetically modified pigs that accurately model human diseases (Perleberg et al. 2018; Zhao. et al. 2019).
The mechanism of CRISPR/Cas9 involves Cas9 protein binding to a single guide RNA (sgRNA), cleaving DNA at the locus targeted by the sgRNA and creating a DNA double-strand break (DSB) (Doudna 2020). This activates two DNA repair mechanisms: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is a rapid DSB repair mechanism that ligates the ends of broken DNA double strands while inserting or deleting a certain number of base pairs at the break to introduce indels (insertions and deletions). An indel of length other than an integer multiple of 3 induces a frameshift mutation that causes loss of the functional protein (gene knockout). HDR is a more accurate DSB repair mechanism in the presence of a homologous template and introduces foreign gene sequences into the genome (gene knock-in). CRISPR/Cas9 gene editing has enabled efficient genetic modification of pigs and many other large animal species. However, recent studies have shown that the DSB caused by CRISPR/Cas9 may lead to unexpected changes in gene-edited cells such as uncontrolled indels by NHEJ, off-target effects, large genomic deletions or rearrangements, and tumor suppressor p53-mediated DNA damage (Haapaniemi et al. 2018; Ihry et al. 2018; Kosicki et al. 2018).
To avoid the side effects of DSB, researchers have developed gene editing tools that are independent of DSB and that induce single base mutations. Adenine base editor (ABE), for example, fuse Cas9 protein (Nickase Cas9 or nCas9) that cleaves single-stranded DNA with Escherichia coli RNA adenine deaminase TadA (Gaudelli et al. 2017). Under the guidance of sgRNA, the A·T base pairs within positions 4–8 are converted to G·C base pairs, enabling targeted repair of genomic mutations. Due to the absence of DSB, ABE effectively avoid the side effects of CRISPR/Cas9 and achieve specific single base conversion. ABE have been shown to have high accuracy in gene editing at the DNA and RNA levels; therefore, they hold significant potential for applications in agriculture and medicine (Anzalone et al. 2020; Porto et al. 2020). The use of ABE in pig gene editing allows target gene modification with minimal DNA changes, and thus high accuracy and biosafety in genetic improvement and disease model construction (Xie et al. 2020).
In the presence of protospacer adjacent motif (PAM) sequences, ABE can be used to convert A·T to G·C at specific positions in the pig genome, thereby modeling genetic diseases caused by single-base mutations (Anzalone et al. 2020; Porto et al. 2020). However, gene knockout requires conversion of the start codon ATG to GTG (or ATG to ACG in reverse complement sequences) to inhibit translation or induce frameshift mutations at the RNA level (Anzalone et al. 2020; Porto et al. 2020). Since ABE depend on NGG PAM and an editing window at positions 4–8 of sgRNA, the probability of a PAM sequence suitable for the start codon ATG is 62.5% (10/16). Specifically, nearly 40% of genes are unsuitable for ABE-mediated gene knockout, and this limits the application of ABE in pig genetic modification.
RNA splicing is a key step in the production of mature mRNAs in eukaryotes. Spliceosome identifies the 5’ end splice donors and 3’ end splice acceptors of introns in mRNAs, removes the introns, and joins exons to form mature mRNAs (Wilkinson et al. 2020). Eukaryotic genes are composed of various numbers of exons. Changes in RNA splicing patterns lead to diverse mature mRNAs and encoded proteins, thereby contributing to eukaryotic genetic diversity. In natural conditions, alternative RNA splicing creates new functional proteins and inactivates genes through frameshift mutation. For example, Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), which are common genetic diseases in humans, are caused by gene mutations that lead to abnormal RNA splicing and dysfunction of the encoded proteins (Montes et al. 2019). An important step in exon splicing is the recognition of highly conservative sequences (splice acceptors and donors) in exon-intron-junctions by spliceosome. Studies have shown that splice donors contain highly conservative GT sequence (at the 5’ end of the intron), and mutation leads to skipping of the upstream exon; splice acceptors contain highly conservative AG sequence (at the 3’ end of the intron), and mutation leads to skipping of the next downstream exon (Huang et al. 2019). Based on the mechanism of exon skipping, exons can be deleted at the mRNA level by base editing and mutation of the conservative bases in splice donors or acceptors. This strategy has been used to repair the coding sequences of the causative genes of DMD in animal models, demonstrating the potential of gene-editing for treating genetic diseases with high accuracy and safety (Long et al. 2018; Yuan et al. 2018).
ABE-mediated exon skipping creates new mRNAs, and if the length of the skipped exon is not an integer multiple of 3, the skipping induces a frameshift mutation and gene knockout (Yuan et al. 2018; Huang et al. 2019). Since eukaryotic genes are composed of several or even tens of exons, ABE-mediated exon skipping holds great promise for gene knockout. Mutation-induced exon skipping has not been used in the construction of genetically modified pigs. In this study, we used the growth hormone receptor (GHR) gene as an example to validate ABE-mediated exon skipping and gene knockout in pigs. This study aimed to provide a foundation for ABE-mediated exon skipping as a means to construct pig models for human diseases and related gene therapies. GHR is a membrane-bound receptor of growth hormones that triggers intracellular signals through binding to GHR to stimulate cell growth and division (Wang et al. 2019). Loss-of-function mutations in human GHR trigger Laron syndrome, and loss-of-function mutations in pig GHR cause phenotypes such as short stature and stunting that resemble Laron syndrome (Cui et al. 2015; Hinrichs et al. 2018; Yuan et al. 2020). Therefore, GHR knockout pigs are an ideal large animal model for human Laron syndrome.
In this study, we first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector was shown to induce efficient sgRNA-dependent A·T to G·C conversion in porcine cells during single-base editing at multiple endogenous gene loci. The ABE was designed to edit single adenine residues (thymine in reverse complement sequences) in the GHR gene at two sites: one was the conservative AG sequence of the splice acceptor at the 3’ end of the intron 5, and the other was the conservative GT sequence of the splice donor at the 5’ end of the intron 6. Efficient A·T to G·C (or T·A to C·G in reverse complement sequences) conversion was achieved at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor in the intron 5 of GHR were generated. RT-PCR showed exon 6 skipping at the mRNA level, while Western blotting confirmed the loss of GHR protein, and gene sequencing showed no sgRNA-dependent off-target effects in the genome. These results suggest that ABE-mediated exon skipping led to gene knockout in porcine cells. This work presents the first proof-of-concept study of ABE-mediated exon skipping and gene regulation in pigs, and the results provide a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.