Addressing the 2050 demand for terrestrial animal source food.

The high emissions intensity of terrestrial animal source food (TASF) and projected increasing demand in low- and middle-income countries (LMIC) have spurred interest in the development of animal-free alternatives and manufactured food items that aim to substitute for meat, milk, and eggs with the promise of reduced environmental impact of producing food. The developing world is the source of 75% of global emissions from ruminants and will house 86% of the world's human population by 2050. The adoption of cost-effective, genetic, feed and nutrition practices, and improving livestock health in LMIC are seen as the most promising interventions to reduce emissions resulting from projected increased TASF demand though 2050.

Global status of gene edited animals for agricultural applications.

Gene editing (GnEd) involves using a site-directed nuclease to introduce a double-strand break (DSB) at a targeted location in the genome. A literature search was performed on the use of GnEd in animals for agricultural applications. Data was extracted from 212 peer-reviewed articles that described the production of at least one living animal employing GnEd technologies for agricultural purposes.

Germline ablation achieved via CRISPR/Cas9 targeting of in bovine zygotes.

is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live knockout (KO) cattle have not been reported, and the specific role of in male cattle, or bulls, remains unexplored. This study generated KO cattle cytoplasmic microinjection of the CRISPR/Cas9 system produced bovine zygotes and evaluated the effect of elimination on bovine germline development, from fetal development through reproductive age.

Field demonstration analyzing the implementation of individual animal electronic identification and genetic testing in western range sheep flocks.

Adoption of electronic identification ear tags (EID) and DNA testing by commercial range sheep producers in the Western United States has been low, despite the availability of these technologies for over a decade. Jointly, these technologies offer an approach to provide individual animal performance data to improve flock health, genetic and reproductive management. This project involved a collaboration with five California sheep producers representing a broad geographic range, varying levels of pre-project EID adoption, and diverse operational practices.

Review: Progress in producing chimeric ungulate livestock for agricultural applications.

The progress made in recent years in the derivation and culture of pluripotent stem cells from farm animals opens up the possibility of creating livestock chimeras. Chimeras producing gametes exclusively derived from elite donor stem cells could pass superior genetics on to the next generation and thereby reduce the genetic lag that typically exists between the elite breeding sector and the commercial production sector, especially for industries like beef and sheep where genetics is commonly disseminated through natural service mating. Chimeras carrying germ cells generated from genome-edited or genetically engineered pluripotent stem cells could further disseminate useful genomic alterations such as climate adaptation, animal welfare improvements, the repair of deleterious genetic conditions, and/or the elimination of undesired traits such as disease susceptibility to the next generation.

LincRNA#1 knockout alone does not affect polled phenotype in cattle heterozygous for the celtic POLLED allele.

A long intergenic non-coding RNA (lincRNA#1) is overexpressed in the horn bud region of polled (hornless) bovine fetuses, suggesting a potential role in horn bud suppression. Genome editing was used to test whether the absence of this sequence was associated with the horned phenotype. Two gRNAs with high mutation efficiencies targeting the 5' and the 3' regions flanking the lincRNA#1 sequence were co-injected with Cas9 as ribonucleoprotein complexes into bovine zygotes (n = 121) 6 h post insemination.

A deletion at the polled P locus alone is not sufficient to cause a polled phenotype in cattle.

Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (P allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence.

Etiology and risk factors for bovine respiratory disease in pre-weaned calves on California dairies and calf ranches.

Our study objective was to estimate the magnitude of association of BRD risk factors including failure of passive immunity transfer, sex, age, and the detection of suspected BRD etiological pathogens in pre-weaned dairy calves in California. A conditional logistic regression model and a mixed-effects logistic regression model were used to estimate the association of these potential risk factors with BRD from a matched and nested case-control studies, respectively. For each exposure covariate, the odds ratio (OR) is the ratio of odds of an exposure in a BRD calf (case) to that in a non-BRD calf (control).

Electroporation-Mediated Genome Editing of Livestock Zygotes.

The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes.

Functional annotations of three domestic animal genomes provide vital resources for comparative and agricultural research.

Gene regulatory elements are central drivers of phenotypic variation and thus of critical importance towards understanding the genetics of complex traits. The Functional Annotation of Animal Genomes consortium was formed to collaboratively annotate the functional elements in animal genomes, starting with domesticated animals. Here we present an expansive collection of datasets from eight diverse tissues in three important agricultural species: chicken (Gallus gallus), pig (Sus scrofa), and cattle (Bos taurus).

Comparison of Gene Editing Versus Conventional Breeding to Introgress the Allele Into the Tropically Adapted Australian Beef Cattle Population.

Dehorning is the process of physically removing horns to protect animals and humans from injury, but the process is costly, unpleasant, and faces increasing public scrutiny. Genetic selection for polled (hornless), which is genetically dominant to horned, is a long-term solution to eliminate the need for dehorning. However, due to the limited number of polled Australian Brahman bulls, the northern Australian beef cattle population remains predominantly horned.

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes.

Background: The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells.

Results: By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.

Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos.

The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing.

Genetic Engineering of Livestock: The Opportunity Cost of Regulatory Delay.

Genetically engineered (GE) livestock were first reported in 1985, and yet only a single GE food animal, the fast-growing AquAdvantage salmon, has been commercialized. There are myriad interconnected reasons for the slow progress in this once-promising field, including technical issues, the structure of livestock industries, lack of public research funding and investment, regulatory obstacles, and concern about public opinion. This review focuses on GE livestock that have been produced and documents the difficulties that researchers and developers have encountered en route.

Efficient One-Step Knockout by Electroporation of Ribonucleoproteins Into Zona-Intact Bovine Embryos.

Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knock-out. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development.

A comparative analysis of chromatin accessibility in cattle, pig, and mouse tissues.

Background: Although considerable progress has been made towards annotating the noncoding portion of the human and mouse genomes, regulatory elements in other species, such as livestock, remain poorly characterized. This lack of functional annotation poses a substantial roadblock to agricultural research and diminishes the value of these species as model organisms. As active regulatory elements are typically characterized by chromatin accessibility, we implemented the Assay for Transposase Accessible Chromatin (ATAC-seq) to annotate and characterize regulatory elements in pigs and cattle, given a set of eight adult tissues.

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos.

Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR).

Genome editing approaches to augment livestock breeding programs.

The prospect of genome editing offers a number of promising opportunities for livestock breeders. Firstly, these tools can be used in functional genomics to elucidate gene function, and identify causal variants underlying monogenic traits. Secondly, they can be used to precisely introduce useful genetic variation into structured livestock breeding programs.

Author Correction: Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.

Genome editing followed by reproductive cloning was previously used to produce two hornless dairy bulls. We crossed one genome-edited dairy bull, homozygous for the dominant P Celtic POLLED allele, with horned cows (pp) and obtained six heterozygous (Pp) polled calves. The calves had no horns and were otherwise healthy and phenotypically unremarkable.

Management of lethal recessive alleles in beef cattle through the use of mate selection software.

Background: Recessive loss-of-function (LOF) alleles at genes which are essential for life, can result in early embryonic mortality. Cattle producers can use the LOF carrier status of individual animals to make selection and mate allocation decisions.

Methods: Two beef cattle breeding strategies i.

Application of genome editing in farm animals: cattle.

Milk and meat from cattle and buffaloes contribute 45% of the global animal protein supply, followed by chickens (31%), and pigs (20%). In 2016, the global cattle population of 1.0 billion head produced 6.

Proposed U.S. regulation of gene-edited food animals is not fit for purpose.

Dietary DNA is generally regarded as safe to consume, and is a routine ingredient of food obtained from any living organism. Millions of naturally-occurring DNA variations are observed when comparing the genomic sequence of any two healthy individuals of a given species. Breeders routinely select desired traits resulting from this DNA variation to develop new cultivars and varieties of food plants and animals.

Genome to Phenome: Improving Animal Health, Production, and Well-Being - A New USDA Blueprint for Animal Genome Research 2018-2027.

In 2008, a consortium led by the Agricultural Research Service (ARS) and the National Institute for Food and Agriculture (NIFA) published the "Blueprint for USDA Efforts in Agricultural Animal Genomics 2008-2017," which served as a guiding document for research and funding in animal genomics. In the decade that followed, many of the goals set forth in the blueprint were accomplished. However, several other goals require further research.