Primordial germ cells (PGCs), the embryonic precursors of the sperm and egg, are used for the introduction of genetic modifications into avian genome. Introduction of small defined sequences using genome editing has not been demonstrated in bird species. Here, we compared oligonucleotide-mediated HDR using wild type SpCas9 (SpCas9-WT) and high fidelity SpCas9-HF1 in PGCs and show that many loci in chicken PGCs can be precise edited using donors containing CRISPR/Cas9-blocking mutations positioned in the protospacer adjacent motif (PAM). However, targeting was more efficient using SpCas9-HF1 when mutations were introduced only into the gRNA target sequence. We subsequently employed an eGFP-to-BFP conversion assay, to directly compare HDR mediated by SpCas9-WT and SpCas9-HF1 and discovered that SpCas9-HF1 increases HDR while reducing INDEL formation. Furthermore, SpCas9-HF1 increases the frequency of single allele editing in comparison to SpCas9-WT. We used SpCas9-HF1 to demonstrate the introduction of monoallelic and biallelic point mutations into the FGF20 gene and generate clonal populations of edited PGCs with defined homozygous and heterozygous genotypes. Our results demonstrate the use of oligonucleotide donors and high fidelity CRISPR/Cas9 variants to perform precise genome editing with high efficiency in PGCs.

中文翻译：

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高保真CRISPR / Cas9增加了原始生殖细胞中精确的单等位基因和双等位基因编辑事件。

原始生殖细胞（PGC）是精子和卵的胚胎前体，用于将遗传修饰引入禽类基因组。尚未在鸟类中证明使用基因组编辑引入小的定义序列。在这里，我们比较了使用野生型SpCas9（SpCas9-WT）和高保真度SpCas9-HF1在PGC中的寡核苷酸介导的HDR，并显示可以使用包含位于原间隔子中的CRISPR / Cas9阻断突变的供体来精确编辑鸡PGC中的许多基因座。相邻主题（PAM）。但是，当仅将突变引入gRNA靶序列时，使用SpCas9-HF1进行靶向更有效。随后，我们采用了eGFP到BFP的转化试验，直接比较了SpCas9-WT和SpCas9-HF1介导的HDR，发现SpCas9-HF1增加了HDR，同时减少了INDEL的形成。此外，与SpCas9-WT相比，SpCas9-HF1增加了单等位基因编辑的频率。我们使用SpCas9-HF1来证明将单等位基因和双等位基因点突变引入FGF20基因，并产生具有定义的纯合和杂合基因型的已编辑PGC的克隆种群。我们的结果证明了使用寡核苷酸供体和高保真度的CRISPR / Cas9变体在PGC中高效执行精确的基因组编辑。