Generation of genome-wide CRISPR knockout libraries for pig and chicken

The benefits of selective breeding within livestock to maintain and improve beneficial traits have long been practiced in agriculture. Traits such as increased size, milk production and resistance to infection are, for the most part, determined by relatively small differences in the DNA code of livestock animals such as pigs, cows, sheep and chickens. Recent technological advances allow scientists to read the genetic code of animals in greater detail, faster and for less money. So much so that we are beginning to understand how specific changes in the genomes of animals result in specific beneficial and detrimental characteristics. The aim in the near future is to be able to predict and select for beneficial characteristics in offspring based entirely on the animals DNA code.

Although our ability to read the genetic code of animals has greatly improved our ability to translate the genetic code into physical attributes lags far behind. This is because we don't fully understand the function of most genes. A central approach to understanding the function of a particular gene involves deleting the gene and observing the effect this has on the animal or the animal's cells. Recent advances have allowed scientists to independently inhibit every individual gene within human cells, allowing them to ask how particular genes are involved in specific biological processes. These approaches allow us to link the genetic code of human cells to particular biological traits or processes. Such technologies have not been developed in livestock species due to the cost involved and comparative lack of detailed knowledge of livestock genomes compared to human genomes.

In the last few years a new technology has been developed that allows scientists to rapidly and specifically edit the genomes of animals. These approaches have been shown to be more cost effective and technically superior to previous strategies for inhibiting genes. Currently the technique is limited to only a few species such as human and mouse. In this application we propose developing this technology for chicken and pig two of the most important livestock animals. Achieving this requires the latest detailed knowledge of the genome sequence of these animals and the technical skills required to develop the technology. At the Roslin institute we have two of the leading researchers involved in mapping the pig and chicken genomes as well as researchers with extensive knowledge of the newly developed technology. In addition we will be working closely with the Broad institute in the US who originally developed the technology in question. Therefore the Roslin Institute is uniquely positioned to successfully develop and apply this technology.

Initially we will be asking a relatively straightforward question using the technology. Influenza virus is an important pathogen both in humans and in livestock animals. Spread of the virus in livestock such as birds and pigs contributes to outbreaks of the virus in humans, including potentially dangers pandemics. In many cases genes expressed by the host (such as human, chickens and pigs) are required by the virus and can restrict the replication of the virus. Significant amounts of work has been dedicated to understanding which genes are important following infection of human cells. Relatively little research has been performed to answer the same question in other species. We will use this technology to answer these questions. Although this will be an important aspect of the research, it is only one of many ways in which this new technology will be of benefit to scientists working on livestock animals. This technology has the potential to greatly improve our ability to understand the link between the genes encoded in a genome the functional roles they play. Such advances will have major impacts on the potential for improved breeding of livestock and will also contribute to our basic understanding of biology.

- Tools for genome wide functional screens do not exist for livestock species

- Genome wide functional screens in human cells using CRISPR technology have proven highly effective

- We propose developing genome wide CRISPR knockout libraries for pig and chicken

- The Roslin Institute is uniquely positioned to successfully develop and apply this technology

- sgRNAs and CRISPR libraries will be made freely available maximizing the potential beneficial impact on livestock research

CRISPR/Cas9 technology allows for specific manipulation of genomes based on the expression of modified Cas9 protein and simple small guide RNAs. Genome wide CRISPR libraries consist of pooled lentiviruses expressing multiple guide RNAs against every annotated transcript within a genome. With effective phenotypic screens these libraries provide the opportunity to interrogate the role of host genes in specific biological processes. Currently such libraries have only been developed for a small number of species. No such libraries exist for livestock animals. In this application We propose generating genome wide CRISPR libraries for pig and chicken and screening for host factors involved in influenza infection as a basis for library validation.

Long term these libraries will represent a valuable resource for diverse studies (e.g. Marek's disease virus, Bluetongue virus, African Swine Fever virus) in these economically and strategically important species.

The development of genome wide CRISPR libraries for chicken and pig will have a major impact on research involving livestock animals. The development of genome wide RNAi technology has provided major advances in human research and research involving model organisms, such as mouse and drosophila. The lack of such tools for livestock species has proved a major disadvantage for the development of functional screens in these species.

There are three major areas where the development of these tools will have major impact;

1. Enhancing translation of livestock genomic data into practical improvements in breeding programs and livestock generation
2. Contributing to our basic understanding of cell biology in livestock species
3. Establishing expertise in genome wide CRISPR library generation and implementation

1. Improving translation of livestock genomic data into practical improvements in breeding programs and livestock generation

Advances in genome sequencing of livestock has provided opportunities for increasingly sophisticated approaches to identifying links between gene sequence and desirable traits such as improved breeding and resistance to infection. The availability of CRISPR libraries in livestock species will allow for rapid and cost effective studies to determine the functional effects associated with genetic variants. This can be achieved in two ways. Genome wide association studies can be compared to data from genome wide functional studies to identify significant overlaps. Or a more focused approach involves the use of specific CRISPR lentiviruses to disrupt expression of particular genes identified from large-scale genomic data. Once sgRNAs have been designed, cloning and application of such constructs is relatively straight-forward. Another major avenue of impact is the specific engineering of livestock animals based on genome wide CRISPR studies.

2. Contributing to our basic understanding of cell biology in livestock species

Genome-wide functional studies have provided a wealth of information on basic cell biology in humans and other species where the technology exists. There are aspects of livestock research such as development, fertility and infectious diseases that can complement and in some cases advance our understanding over research conducted in humans and model organisms. Generation of functional CRISPR screens would be critical in realizing these possibilities.

3. Establishing expertise in genome wide CRISPR library generation and implementation

Although the short-term plan is to generate pig and chicken CRISPR libraries, an essential aspect of this application will be establishing the expertise for the generation and application of further libraries. This will involve the generation of libraries for additional livestock species such as cow and sheep, but also the development of CRISPR libraries that cause gene expression enhancement. Again, our short term goal is to investigate the role of host factors in influenza infection, but long term the aim will be to expand the approaches developed in this application to other important veterinary infectious diseases and to other aspects of livestock biology such as development and fertility. We predict that the availability of these tools will also encourage the development of new areas of research - genome wide functional studies are theoretically applicable to any situation where a suitable screening method can be devised.

As we plan to make both the libraries and the sgRNAs freely available the impact of these tools should go beyond the research at the Roslin Institute and have major impact at a national and international level.