Developing multiplex genome editing in the chicken using serial surrogate host mating

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The chicken represents an important source of protein worldwide and is the backbone of rural households in low and middle-income countries. Poultry flocks are susceptible to a plethora of deadly microorganisms threatening their health and welfare, which leads to weakening the agro-economic status of not only for breeders and farmers in low and middle-income countries, but also for breeding companies and farmers in high-income countries. Recently, genetically modified chicken was produced, which has proven a useful research tool for the avian research community. The initial steps to making the genetically modified chicken were inefficient and lacked precision and were based on transgenes. The development of site-specific nucleases (ZFN, TALEN, and CRISPR/Cas9) enables to manipulation of the avian genome more efficiently and precisely at the specific location of the genome without leaving a transgenic footprint. We have established a workflow for generating genome-edited chicken by injecting genome-edited primordial germ cells (PGCs) into the surrogate embryos that need two rounds of breeding taking around 2 years. It is because there is a competition between endogenous PGCs to edited PGCs for germline transmission. To eliminate this relative competition and reduce time, we have generated two sterile chicken surrogate host chicken lines that either does not have their own germ cells or can be ablated of germ cells using an inert chemical compound. As technological advancement in this field, we established a pipeline of direct mating of the male (Sire) and female (Dam) surrogate hosts (SDS) produced pure genome-edited offspring deriving from the exogenous edited PGCs in a single generation. Using this approach, we produced two breeds of chicken homozygous for genetic edits controlling two feather traits in a single generation in around six months.We now aim to advance and refine this pipeline by establishing a multiplex genome-editing platform in chicken using serial surrogate host mating, targeting multiple loci associated with the disease resistance phenotype of economically important poultry diseases and productivity traits.Based on our preliminary data, we propose four objectives. Firstly, we will optimize parameters for multiplex genome editing in chicken PGCs by both simultaneous and serially editing multiple genes of interest. Secondly, we will characterize the multiplex edited PGCs by sequencing to assure that there are no chromosomal rearrangements. Thirdly, we will generate multiplexed genome-edited chicken in a single generation using sterile surrogate host mating. Fourthly, we will create multiplex genome editing chicken while preserving genetic diversity by adopting the recently developed pooled gonadal PGCs approach thus preventing inbreeding depression, immediate loss of fitness, embryonic viability, and fertility.This multiplex serial genome editing technology in chicken has the potential to be transformative in its impact to agriculture and the biotechnology sectors.