Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurrently limiting on-target non-homologous end joining (NHEJ)-based HBB disruption. In this study, we directly compared the relative efficiency of co-delivery of a novel CRISPR/Cas9 ribonucleoprotein targeting HBB in association with recombinant adeno-associated virus 6 (rAAV6) versus single-stranded oligodeoxynucleotides (ssODNs) to introduce the sickle mutation (GTC or GTG; encoding E6V) or a silent change (GAA; encoding E6optE) in human CD34 + mobilized peripheral blood stem cells (mPBSCs) derived from healthy donors. In vitro , rAAV6 outperformed ssODN donor template delivery and mediated greater HDR correction, leading to both higher HDR rates and a higher HDR:NHEJ ratio. In contrast, at 12–14 weeks post-transplant into recipient, immunodeficient, NOD, B6, SCID Il2rγ −/− Kit(W41/W41) (NBSGW) mice, a ∼6-fold higher proportion of ssODN-modified cells persisted in vivo compared to recipients of rAAV6-modified mPBSC s . Together, our findings highlight that methodology for donor template delivery markedly impacts long-term persistence of HBB gene-modified mPBSCs, and they suggest that the ssODN platform is likely to be most amenable to direct clinical translation.

In the past few years, new technologies have arisen that enable higher efficiency of gene editing. With the increase ease of using gene editing technologies, it is important to consider the best method for transferring new genetic material to livestock animals. Microinjection is a technique that has proven to be effective in mice but is less efficient in large livestock animals. Over the years, a variety of methods have been used for cloning as well as gene transfer including; nuclear transfer, sperm mediated gene transfer (SMGT), and liposome-mediated DNA transfer. This review looks at the different success rate of these methods and how they have evolved to become more efficient. As well as gene editing technologies, including Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the most recent clustered regulatory interspaced short palindromic repeats (CRISPRs). Through the advancements in gene-editing technologies, generating transgenic animals is now more accessible and affordable. The goals of producing transgenic animals are to 1) increase our understanding of biology and biomedical science; 2) increase our ability to produce more efficient animals; and 3) produce disease resistant animals. ZFNs, TALENs, and CRISPRs combined with gene transfer methods increase the possibility of achieving these goals.

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In cattle, a mutation in the NHL-repeat containing 2 genes causes a heritable abnormality referred to as developmental duplications. Calves homozygous for this mutation are affected with a broad range of phenotypes resulting from abnormal neural crest cell migration, most commonly manifested as polymelia, the presence of additional limbs. This mutation has become highly prevalent in Angus beef cattle, as lines of cattle with high genetic merit have been shown to have an increased allele frequency of the mutation. The mutation has been identified as a single nucleotide polymorphism resulting in a valine to alanine substitution in a highly conserved protein-coding region of the gene. CRISPR/Cas9 genome editing technology has been shown to induce changes in the genome by using a guide RNA to target a specific site paired with a Cas-9 protein to create a break in the DNA. These breaks are repaired by either nonhomologous end joining or homology-directed repair. The aim of this preliminary study was to determine the editing efficiency of CRISPR/Cas-9 proteins paired with site-specific guide RNA using cell lines derived from animals homozygous and heterozygous for the NHLRC2 mutation. Bovine fetal fibroblasts of both genotypes were grown in DMEM/F10 media supplemented with 10% fetal bovine serum, 0.01 µg mL−1 of basic fibroblast growth factor, 1 mL L−1 penicillin streptomycin, and 1 mL L−1 of amphotericin B. Cells were plated in a 6-well plate at 80,000 cells/well 48 h before transfection. Two 20-nucleotide guide RNA targeting the genome near the developmental duplications mutation were designed and ligated into pSpCas9(BB)-2A-GFP CRISPR plasmids. Six microliters of Fugene 6 was added to 150 µL of Opti-MEM followed by 2 µg of plasmid DNA and complexed at 37°C for 15 min before being added to each well. At 24 h after transfection, cells were detached with trypsin and sorted by fluorescence-activated cell sorting. When wells were confluent, DNA was extracted using 65 µL of QuickExtract DNA extraction solution. The 500-bp fragment surrounding the mutation was amplified and subjected to a restriction enzyme digest. Fragments with the exact sequence of the mutation were cleaved, whereas normal genotypes or edited genotypes without the mutation sequence remain uncleaved. Fragments were size separated on a 2% agarose gel. Band intensity under ultraviolet illumination was calculated with GelReader and the ratios of cleaved versus uncleaved fragments were compared with the ratio of control (unedited cell lines) for both guide RNA and both heterozygous and homozygous cell lines. Based on this preliminary data, CRISPRs with guide RNA 1 edited the genome at the target site at 13.3 and 12.2% for heterozygous and homozygous cell lines, respectively, and guide RNA 2 affected the target site at 2.5 and 4.1% for heterozygous and homozygous, respectively. These data show that the designed guide RNA paired with CRISPRs are able to elicit changes at the desired locus. In order to understand what repair mechanisms were employed at these loci, the next step is to subclone and sequence PCR products.

Bovine leukosis virus (BLV) is a pathogen that affects the bovine immune system and leads to lymphosarcoma, leukemia, decreased milk production, and increased culling rates in cattle. BLV-infected cattle herds can be found worldwide; in the United States, specifically, 38% of beef herds, 84% of all dairy herds, and 100% of large-scale dairy operation herds are infected (Buehring et al. 2014 Emerg. Infect. Dis. 5, 772–782). The main transmission between cattle in herds is affected leukocytes in blood. Several farm practices, such as dehorning, rectal palpation, and vaccinating can lead to the pathogen transmission. Due to international trade laws and biosecurity concerns, semen from a BLV-positive bull is illegal to sell within certain countries. Prior studies have looked at use of seropositive bulls in AI with little risk in affecting the dam (Burger et al. 2000 AVJR 60, 819). Other studies used semen that was artificially infected with the virus then used for IVF (Bielanski et al. 2000 Vet. Rec. 146, 255–256). The aim of this research was to evaluate naturally infected BLV donor semen using abattoir-derived oocytes and the possible contamination of in vitro-produced (IVP) embryos. Semen was collected and frozen by a private company. Three seropositive bulls and 1 negative control bull were selected. All positive bulls were selected based on availability of seropositive BLV status. Prior to the experiment, all bulls used were evaluated for motility, concentration, and morphology. The negative control was used in prior IVF experiments that produced acceptable results for use in this experiment. Frozen sperm were thawed at 37°C for 40 s and pelleted by centrifugation (25 min at 300 × g) on a Percoll discontinuous gradient (45–80% in Tyrode’s modified medium without glucose and BSA). The matured oocytes were purchased from DeSoto Biosciences (Seymour, TN, USA) and were IVF according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Using 200 oocytes per replicate, the 3 positive bulls and 1 control bull were allocated 50 oocytes per bull in each replicate. After 20 to 22 h of gametes co-incubation, zygotes were denuded and cultured for 7 days in SOF, followed by the evaluation of embryos (from tight morula until hatching blastocyst). Positive bull #1 produced and tested 48 embryos. Positive bull #2 produced and tested 41 embryos. Positive bull #3 produced and tested 46 embryos. The negative control produced and tested 55 embryos. Embryonic DNA extraction was performed using standard procedures (Sattar et al. 2011 Reprod. Domest. Anim. 46, 1090–1097). Nested PCR followed the Fechner evaluations methods (Fechner et al. 1996 J. Vet. Med. B 43, 621–630). To detect BLV presence, electrophoresis was used with a 2% agarose gel containing 0.1% ethidium bromide. A total of 190 embryos were evaluated that were produced in 3 replicates. All samples analysed showed no evidence of BLV. In conclusion, use of BLV seropositive donor semen showed no transmission of the virus upon IVF of the oocytes.