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

鸡是世界范围内蛋白质的重要来源，是中低收入国家农村家庭的支柱。家禽群容易受到威胁其健康和福利的过量致命微生物的影响，这不仅削弱了低收入和中等收入国家的育种者和农民的农业经济地位，而且也削弱了高收入国家的育种公司和农民的农业经济地位。最近，转基因鸡被生产出来，这已被证明是鸟类研究界的一个有用的研究工具。制造转基因鸡的最初步骤效率低下，缺乏精确度，而且是基于转基因的。位点特异性核酸酶（ZFN， TALEN和CRISPR/Cas9）的发展使得在基因组的特定位置更有效和精确地操纵鸟类基因组而不会留下转基因足迹。我们已经建立了基因组编辑鸡的工作流程，通过将基因组编辑的原始生殖细胞（PGCs）注射到代孕胚胎中，需要大约2年的时间进行两轮繁殖。这是因为内源性PGCs与编辑过的PGCs在种系传播方面存在竞争。为了消除这种相对竞争并减少时间，我们已经产生了两种无菌的鸡代宿主鸡系，它们要么没有自己的生殖细胞，要么可以使用惰性化合物去除生殖细胞。随着这一领域的技术进步，我们建立了一个雄性（Sire）和雌性（Dam）代宿主（SDS）直接交配的管道，通过外源编辑的PGCs在一代内产生纯基因组编辑的后代。利用这种方法，我们在大约6个月的时间里生产了两种纯合子鸡，用于基因编辑，以控制单代鸡的两种羽毛特征。我们现在的目标是通过在鸡中建立一个多重基因组编辑平台来推进和完善这一管道，该平台使用串行代宿主交配，针对与经济上重要的家禽疾病和生产力性状的抗病表型相关的多个位点。根据我们的初步数据，我们提出了四个目标。首先，我们将通过同时和连续编辑多个感兴趣的基因来优化鸡PGCs的多重基因组编辑参数。其次，我们将通过测序来表征多重编辑的PGCs，以确保没有染色体重排。第三，我们将通过不育代宿主交配，在一代中产生多重基因组编辑鸡。第四，我们将创建多重基因组编辑鸡，同时通过采用最近开发的汇集性腺PGCs方法保持遗传多样性，从而防止近交抑制、适应性、胚胎活力和生育能力的立即丧失。这种鸡的多重序列基因组编辑技术有可能对农业和生物技术部门产生变革性的影响。

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.