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

鸡是世界范围内重要的蛋白质来源，也是低收入和中等收入国家农村家庭的支柱。家禽群容易受到大量致命微生物的威胁，威胁到它们的健康和福利，这不仅削弱了低收入和中等收入国家的养殖者和农民的农业经济地位，也削弱了高收入国家的养殖公司和农民的农业经济地位。最近，转基因鸡被生产出来，这已经被证明是鸟类研究界有用的研究工具。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。位点特异性核酸酶(ZFN、TALEN和CRISPR/Cas9)的发展使人们能够更有效和准确地在基因组的特定位置操纵鸟类基因组，而不会留下转基因足迹。我们已经建立了一套产生基因组编辑的鸡的工作流程，通过将基因组编辑的原始生殖细胞(PGCs)注射到需要两轮育种的代理胚胎中，大约需要两年的时间。这是因为内源性生殖细胞与编辑的生殖细胞之间存在着种系传递的竞争。为了消除这种相对的竞争并缩短时间，我们培育了两个无菌的鸡代宿主鸡品系，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，我们建立了一条雄性(Sire)和雌性(Dam)代用宿主(Sire)直接交配(Sire)和(Dam)替代寄主(Dam)代用宿主(SDS)的管道，从外源编辑的PGCs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，在一个世代中产生了两个纯合的鸡品种，用于控制两个羽毛性状的遗传编辑。现在，我们的目标是通过建立一个利用连续替代宿主交配的鸡多重基因组编辑平台，瞄准与重要经济家禽疾病的抗病表型和生产力指标相关的多个基因座，来推进和完善这一管道。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PGC中多重基因组编辑的参数。其次，我们将通过测序来鉴定多重编辑的PGC，以确保没有染色体重排。第三，我们将使用无菌代理宿主交配在单代中产生多路基因组编辑的鸡。第四，我们将采用最近开发的共用性腺PGCS方法，在保留遗传多样性的同时创造多重基因组编辑鸡，从而防止近亲繁殖衰退、立即丧失适合度、胚胎成活率和繁殖力。这种鸡的多重系列基因组编辑技术具有潜在的变革性，将对农业和生物技术领域产生影响。

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.