BIONÆR-Bionæringsprogram

Some of the ambitions in this project are to develop the resources and competence that enable gene editing (GE) experimentation in multiple species. Gene editing is a rapidly evolving field and significant, new technologies and approaches are frequently announced. The GENEinnovate project seeks to develop the essential resources, knowledge and competences required to implement gene editing (GE) in several key production species. At this stage our ambitions in this project are to deploy GE under controlled experimental conditions to perform proof-of-principle experiments and address certain specific scientific questions. Development of resources (WP1) activity transects and is synonymous with all activities in WP2, 3, and 4 and is therefore reflected in the following sections. Our initial plans for the species pig (WP2) was to use GE to explore specific aspects related to boar taint; an undesirable off-flavor and smell in pork meat. However, this proved to be an intractable question to explore due to the difficulties in identifying specific candidate genes and mutations responsible for this complex trait and (more importantly) the fact that we had no good phenotyping system to use where we would be able to detect a positive or negative effect of GE on the trait. For this reason we shifted focus towards porcine diarrhea caused by E.coli bacteria; a major issue for producers causing stock loss and creating animal welfare problems. We adopted an approach (CRISPR genome-screening ) that would allow us to screen for genes associated with the problem without any a priori assumptions (ie gene candidates). Whole genome screens are a powerful tool for identifying novel genes that control phenotypes and can be applied to any in vitro testable trait. Current status is that a genome-wide CRISPR library for porcine targeting 21,280 annotated protein coding genes, 6,801 regions coding for long-noncoding RNA, Y-chromosome genes, vault-RNAs, and approximately 1000 non-targeting controls are developed, and library produced. Protocols are currently optimized to perform the final screening in porcine intestinal epithelial cells. Similar to redesigning our pig plans, we decided early in the project to shift our phenotype focus in cattle (WP3) from the originally planned phenotype to another. Initially we hoped to develop GE competence and resources and direct them towards the problem of eliminating horns in cattle. While several gene candidates suitable for editing exist, the practical difficulties related to measuring the phenotype (horns vs no-horns) in an in vitro setting were not fully appreciated. For this reason we shifted our focus in this WP to bovine viral diarrhea virus (BVDV), which although a disease in itself, causes a number of transient infections which are often the cause of animal health and economic problems. Development of a genome wide CRISPR library for bovine is complete and will enter production early 2022. This library targets 21855 annotated protein coding genes and 1480 long-noncoding RNAs. In contrast to porcine library, bovine library will carry only targeting guides and will rely on Cas9 produced by targeted lung cells. The work with genetically modified salmon (WP4) has in the reporting period focused mainly on phenotyping the three CRISPR / CAS9 knock out (KO) lines generated earlier in the project. Intestinal and liver samples from the bco1, bco1 like or abcg2 lines (200g fish) are now analyzed for changes in gene expression using RNA sequencing and microscopy. So far, the microscope analyzes show the same phenotype for abcg2 in 200 g salmon as in 16 g fish, with increased lipid uptake in the intestine and also increased lipid deposition in the liver. In the work of immortalizing liver cells for the establishment of a new cell line, we have now improved the cell medium and also knocked out three different p53 paralogue genes using CRISPR / CAS9. In contrast to untreated cells, the KO cells proliferate so far without stimulation with growth factors. This means that we can establish a new cell line from Atlantic salmon that can be used for studies of liver metabolism and pathogens. In the reporting period large improvement have been achieved for plants (WP5) due to development of a new direct method for gene transfer, making in vivo gene transfer rather than in vitro transfer possible. Protocols for genotype independence in four different potato varieties have been tested. Promising results are obtained in both Nansen and Desiree species. In 2021, the main activities in the work package on legal, ethical and societal aspects of gene editing (WP6) have been linked to making the results of the consumer survey (from 2020) visible. Focus has been both on national and international arenas, and as input to various stakeholders.

Norwegian livestock, fish and plant breeding organizations sell genetic material (eggs, semen and seeds/plants) in a competitive global market and are under constant pressure from producers, food manufacturers, and consumers to develop innovative products. Traditional breeding methods, optimized to give small changes in many genes rather than fast changes in few, deliver steady but slow progress, and are limited by genetic variation present in the breeding population. Gene-editing (GE) technology can be used to precisely modify genes, introduce new genetic variation, or boost/repress existing variation in a population that would otherwise be difficult to select for (e.g. low frequency variants). This project will use GE (CRISPR/Cas9) to validate potential functional genetic variants associated with important industry concerns, and explore the societal, ethical and legal aspects of GE usage in production biology. This will be done with a view towards establishing responsible, effective frameworks for the future production of GE'ed genetic material with high commercial potential. Specific R&D challenges from the industry partners include (i) reducing boar taint in pigs, (ii) generating salmon with superior fillet color and resistance to sea lice, (iii) promoting the polled (hornless) mutation and improved fertility in cattle, and (iv) enhanced resistance to late blight in Norwegian potatoes. Expected scientific outcomes are, at the least, building technical expertise with GE across a range of species and challenges, and evaluating the role of specific genetic variants in these problem traits. Additionally, results we produce can improve current marker-assisted selection approaches to breeding. The project includes activity to engage with stakeholders, gauge public opinion, and monitor legal progress relating to GE usage in the bioindustry, and to communicate project aims and results in a transparent way to build understanding and trust in this powerful new technology.