With the aim to demonstrate the potential of this technology for animal breeding we selected RELA as the target locus. We have previously argued for a role of porcine RELA in the pathology associated with African Swine Fever Virus infection in pigs. Specifically, polymorphic sequence variation within the transactivation domains of porcine RELA parallels the differing severity associated with African Swine Fever Virus infection observed in domestic pigs in contrast to African pigs such as warthogs14. Therefore we designed genome editing tools to target mutation to this region of the porcine RELA gene (Fig. 1A). The TALENs were designed using the TALE-NT software13 and assembled to target a region located at 1458 to 1505 bp relative to the translational start site in the porcine RELA cDNA sequence (NM_001114281).

Figure 1 A. Location of TALEN, ZFN and PCR primers for sequencing amplicon. Porcine RELA sequence 769417-769110 accession number NW_003609513.1. B. Example Cel1 Assay. Lanes A2, A12, B6, B14 and B20 were identified as having cleavage products consistent with editing events at the target site. Direct sequencing of the same PCR products confirmed this interpretation and identified B12, B16, B17 and B18 as having only wild-type sequence. Full size image

Studies in rats demonstrated that zygote cytoplasmic injection was as effective as pronuclear injection for the delivery of genome editors. Given the difficulty associated with visualization of the pronucleus in porcine zygotes, we elected to perform cytoplasmic injection. Oocytes were harvested from slaughterhouse material for in vitro studies and superovulated artificially inseminated sows were used for embryos destined for transfer into recipients. Initially 208 zygotes were subjected to a single 10 pl cytoplasmic injection of a solution composed 20 ng/μl RELA TALEN mRNA with 5 ng/μl EGFP mRNA (to enable visual identification and isolation of embryos that functionally translated the injected mRNA). After approximately 3 days of in vitro development, GFP fluorescence was detected in 36% of embryos. The presence of non-expressing embryos may reflect as yet unknown species differences in the ability to process introduced RNA since it is not generally seen in rodents nor in bovine13 but in our hands is reproducible in pigs. We conclude that mRNA injected into the cytoplasm of pig zygotes translates to functional protein in a proportion of embryos (Table 1).

GFP positive embryos were screened for editing events by Cel1 surveyor assay (Fig. 1B) and sequencing of PCR amplified fragments (Supplementary Fig. 2). We detected 16 editing events in 46 GFP-positive embryos analyzed (35%). In a second experiment we tested 34 embryos injected with 2 pl of 20 ng/μl RELA TALEN mRNA but without selection for GFP activity and detected 2 editing events (6%). In two further experiments where 2 pl of 10 ng/μl and 2 ng/μl RELA TALEN mRNA was injected, 0% and 18% editing frequency, respectively, were observed. We suspect the lack of cutting observed for the 10 ng/μl dose may simply reflect the small tested sample size (n = 3). In total we identified 21 editing events in porcine embryos in vitro (21% of tested embryos: Table 1) and a high frequency of these editing events were biallelic in nature (33% of edited embryos: Table 1). Calculating this as a frequency of tested embryos, we achieved a biallelic editing frequency of 7%. Since both the highest and lowest tested concentrations of RELA TALEN mRNA produced edited embryos in vitro we elected to transfer zygotes injected at each dosage into recipient sows and allow pregnancies to develop to term. Pregnancy rates for the higher concentrations of RELA TALEN mRNA were poor; 0 out of 2 transfers and 1 out of 2 transfers for embryos injected with 20 ng/μl or 10 ng/μl, respectively. This poor pregnancy rate reflected the visual observation that 2 ng/μl RELA TALEN mRNA injected embryos developed better in vitro than those injected with higher concentrations of TALEN mRNA; the injections carried out using lower concentrations of mRNA generally showed better development, with 7.3% developing to early morula at day three of culture following injection with 2 ng/μl, as opposed to 3.2% at 10 ng/μl and only 0.7% at 25 ng/μl. The one pregnancy from 10 ng/μl delivered 7 piglets, none of which harbored RELA editing events by direct sequencing of PCR amplified products. We did not pursue transfers of embryos with these higher TALEN mRNA concentrations any further.

In contrast, transfer of embryos injected with 2 ng/μl RELA TALEN RNA resulted in 5 pregnancies from 7 recipients. One subsequently aborted at 15 weeks of pregnancy just prior to parturition; analysis of the 9 foetuses carried revealed 3 to have editing events. In total from the remaining 4 farrowings, 39 piglets were produced of which 8 carried editing events (21%). Of the 8 edited animals, two were stillborn, another died neonatally due to being crushed by the mother; leaving 5 live piglets.

In parallel we tested a pair of ZFNs with a target location of 1496 to 1532 bp relative to the translational start site in porcine RELA cDNA sequence (NM_001114281). One transfer of embryos injected with RELA ZFN mRNA at 10 ng/μl failed to generate a pregnancy while the two transfers of embryos injected with RELA ZFN mRNA at 2 ng/μl both became pregnant resulting in the birth of 9 piglets. Of these 9 piglets, one carried an editing event at the ZFN target site (11%), comparable in frequency to our observed TALEN editing efficiency (Table 1).

Direct sequencing of PCR products revealed a variety of editing events in piglets derived from TALEN and ZFN injected embryos (Supplementary Fig. 3). Analysis of ear biopsy isolated genomic DNA identified both deletions and insertions at the target sites. Sequence data from 2 animals constituted multiple overlapping traces indicating two or more editing events; this was subsequently confirmed by sequencing of multiple cloned PCR products. Presuming that in these cases of multiple editing the frequency of events detected in ear biopsy reflects frequency in the early embryo, designer nuclease editing can remain active beyond the 2-cell stage (i.e. some events display low representation in the PCR pool and are therefore only present in a subset of cells). In total, 5 biallelic events were identified from 9 edited piglets (56%; 9% of piglets born); 4 from TALEN and 1 from ZFN mRNA injections. Of these bialleleic events 2 were homozygous, with 3 displaying different indels on each allele. While both piglets carrying homozygous biallelic event survived farrowing (milk in stomach post mortem), they were both accidentally killed by their mother within the first 24 hours of life.