Alan Mileham

Use of the CRISPR/Cas9 System to Produce Genetically Engineered Pigs from In Vitro-Derived Oocytes and Embryos

ABSTRACT Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications.

Genome edited sheep and cattle.

Genome editing tools enable efficient and accurate genome manipulation. An enhanced ability to modify the genomes of livestock species could be utilized to improve disease resistance, productivity or breeding capability as well as the generation of new biomedical models. To date, with respect to the direct injection of genome editor mRNA into livestock zygotes, this technology has been limited to the generation of pigs with edited genomes. To capture the far-reaching applications of gene-editing, from disease modelling to agricultural improvement, the technology must be easily applied to a number of species using a variety of approaches. In this study, we demonstrate zygote injection of TALEN mRNA can also produce gene-edited cattle and sheep. In both species we have targeted the myostatin (MSTN) gene. In addition, we report a critical innovation for application of gene-editing to the cattle industry whereby gene-edited calves can be produced with specified genetics by ovum pickup, in vitro fertilization and zygote microinjection (OPU-IVF-ZM). This provides a practical alternative to somatic cell nuclear transfer for gene knockout or introgression of desirable alleles into a target breed/genetic line.

Live pigs produced from genome edited zygotes

Transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) genome editing technology enables site directed engineering of the genome. Here we demonstrate for the first time that both TALEN and ZFN injected directly into pig zygotes can produce live genome edited pigs. Monoallelic as well as heterozygous and homozygous biallelic events were identified, significantly broadening the use of genome editor technology in livestock by enabling gene knockout in zygotes from any chosen mating.

Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus

VOLUME 34 NUMBER 1 JANUARY 2016 NATURE BIOTECHNOLOGY To the Editor: Porcine reproductive and respiratory syndrome (PRRS) is the most economically important disease of swine in North America, Europe and Asia, costing producers in North America more than

A form of albinism in cattle is caused by a tyrosinase frameshift mutation

600 million annually1. The disease syndrome was first recognized in the United States in 1987 and described in 1989 (ref. 2). The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), was subsequently isolated and characterized in Europe in 1991 (ref. 3). Vaccines have been unable to control the disease. It has been suggested that CD163 is the receptor for entry of PRRSV into cells4. Thus, we hypothesized that pigs with defective CD163 would be immune to PRRSV. Previously we used CRISPRCas9 to generate pigs lacking functional CD163 (ref. 5). Here we demonstrate that these animals are resistant to the PRRSV isolate NVSL 97-7895, a well-characterized, relatively virulent viral isolate that is commonly used in experimental PRRSV infection trials. After infection, they showed no clinical signs (fever or respiratory signs), lung pathology, viremia or antibody response and remained healthy for the 35 d after infection measured in this study. Because CD163 was edited using CRISPR-Cas9, the pigs challenged in this study do not contain any transgenes5. PRRSV is a member of the mammalian arterivirus group, which also includes murine lactate dehydrogenase-elevating virus, simian hemorrhagic fever virus and equine arteritis virus. The arteriviruses share important pathogenesis properties, including macrophage tropism and the capacity to cause both severe disease and persistent infection. In young pigs, infection with PRRSV results in respiratory disease, including cough and fever and reduced growth performance. In pregnant sows, PRRSV infection can result in reproductive failure, as well as persistently infected and low birth weight piglets.The virus is associated with polymicrobial disease syndromes, including porcine respiratory Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus

A gene-based SNP linkage map for Pacific white shrimp, Litopenaeus vannamei.

We used PCR amplification of cDNA prepared from skin biopsies to determine the full-length protein-coding sequence of tyrosinase (TYR) in cattle of several coat colors. An insertion of a cytosine was detected in an albino Braunvieh calf, which resulted in a frameshift which caused a premature stop codon at residue 316. This insertion was found in the homozygous state in this calf and the genomic DNA of two related albino calves. All six parents of these calves were heterozygous for this insertion. However, an albino Holstein calf did not have this insertion, nor was any other mutation detected in the partial TYR sequence obtained from the genomic DNA available. Diagnostic genotyping tests were developed to detect this mutation in Braunvieh cattle.

A mapping and evolutionary study of porcine sex chromosome genes

Pacific white shrimp (Litopenaeus vannamei) are of particular economic importance to the global shrimp aquaculture industry. However, limited genomics information is available for the penaeid species. We utilized the limited public information available, mainly single nucleotide polymorphisms (SNPs) and expressed sequence tags, to discover markers for the construction of the first SNP genetic map for Pacific white shrimp. In total, 1344 putative SNPs were discovered, and out of 825 SNPs genotyped, 418 SNP markers from 347 contigs were mapped onto 45 sex-averaged linkage groups, with approximate coverage of 2071 and 2130 cm for the female and male maps, respectively. The average-squared correlation coefficient (r(2)), a measure of linkage disequilibrium, for markers located more than 50 cm apart on the same linkage group, was 0.15. Levels of r(2) increased with decreasing inter-marker distance from approximately 80 cm, and increased more rapidly from approximately 30 cm. A QTL for shrimp gender was mapped on linkage group 13. Comparative mapping to model organisms, Daphnia pulex and Drosophila melanogaster, revealed extensive rearrangement of genome architecture for L. vannamei, and that L. vannamei was more related to Daphnia pulex. This SNP genetic map lays the foundation for future shrimp genomics studies, especially the identification of genetic markers or regions for economically important traits.

Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function

A combination of FISH and RH mapping was used to study the evolution of sex chromosome genes in the pig. In total, 19 genes were identified, including 3 PAR genes (STS, KAL, PRK). The gene order of the porcine X Chromosome (Chr) closely resembled the human X Chr (PRK/STS/KAL–AMELX–EIF2s3X/ZFX–USP9X–DBX–SMCX), suggesting that the porcine X has undergone very little rearrangement during evolution. For the porcine Y Chr, two linkage groups of 10 NRY genes were found, and the following order was established: Ypter–(AMELY–EIF2S3Y/ZFY–USP9Y–DBY/UTY)–(TSPY–SMCY–UBE1Y–SRY)–CEN. This gene order showed greater conservation with the murine Y than with the human Y Chr. In addition, all porcine Y Chr genes mapped to Yp, which is similar to the mouse and included EIF2s3Y and UBE1Y, which are not present in humans. Interestingly, complete conservation of X/Y homologous gene order was found between the pig X and Y Chrs, indicating that the porcine Y Chr has not undergone extensive reorganisation with respect to the X. This suggests that the order of the X/Y homologous genes of the porcine X and Y Chrs may closely resemble the ancestral gene order of the eutherian sex chromosomes.

A major SNP resource for dissection of phenotypic and genetic variation in Pacific white shrimp (Litopenaeus vannamei)

Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests differently in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV). PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 has been described as a fusion receptor for PRRSV, whereby the scavenger receptor cysteine-rich domain 5 (SRCR5) region was shown to be an interaction site for the virus in vitro. CD163 is expressed at high levels on the surface of macrophages, particularly in the respiratory system. Here we describe the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of Exon 7 of the CD163 gene, encoding SRCR5. Deletion of SRCR5 showed no adverse effects in pigs maintained under standard husbandry conditions with normal growth rates and complete blood counts observed. Pulmonary alveolar macrophages (PAMs) and peripheral blood monocytes (PBMCs) were isolated from the animals and assessed in vitro. Both PAMs and macrophages obtained from PBMCs by CSF1 stimulation (PMMs) show the characteristic differentiation and cell surface marker expression of macrophages of the respective origin. Expression and correct folding of the SRCR5 deletion CD163 on the surface of macrophages and biological activity of the protein as hemoglobin-haptoglobin scavenger was confirmed. Challenge of both PAMs and PMMs with PRRSV genotype 1, subtypes 1, 2, and 3 and PMMs with PRRSV genotype 2 showed complete resistance to viral infections assessed by replication. Confocal microscopy revealed the absence of replication structures in the SRCR5 CD163 deletion macrophages, indicating an inhibition of infection prior to gene expression, i.e. at entry/fusion or unpacking stages.

Highly efficient targeted chromosome deletions using CRISPR/Cas9 †

Bioinformatics and re-sequencing approaches were used for the discovery of sequence polymorphisms in Litopenaeus vannamei. A total of 1221 putative single nucleotide polymorphisms (SNPs) were identified in a pool of individuals from various commercial populations. A set of 211 SNPs were selected for further molecular validation and 88% showed variation in 637 samples representing three commercial breeding lines. An association analysis was performed between these markers and several traits of economic importance for shrimp producers including resistance to three major viral diseases. A small number of SNPs showed associations with test weekly gain, grow-out survival and resistance to Taura Syndrome Virus. Very low levels of linkage disequilibrium were revealed between most SNP pairs, with only 11% of SNPs showing an r(2)-value above 0.10 with at least one other SNP. Comparison of allele frequencies showed small changes over three generations of the breeding programme in one of the commercial breeding populations. This unique SNP resource has the potential to catalyse future studies of genetic dissection of complex traits, tracing relationships in breeding programmes, and monitoring genetic diversity in commercial and wild populations of L. vannamei.