Abstract

Efficient isolation of genetically modified cells that are phenotypically indistinguishable from the unmodified cells remains a major technical barrier for the broader utilization of CRISPR/Cas9. Here, we report a novel enrichment approach to select the genome engineered cells by co-targeting a genomically integrated GFP gene along with the endogenous gene of interest (GOI). Using this co-targeting approach, multiple genomic loci were successfully targeted in chicken (DF1) and quail (CEC-32) fibroblast cell lines by transient transfection of Cas9 and guide RNAs (gRNAs). Clonal isolation of co-targeted DF1 cells showed 75% of cell clones had deletion of GFP and biallelic deletion of the GOI. To assess the utility of this approach to generate genome modified animals, we tested it on chicken primordial germ cells (PGCs) expressing GFP by co-targeting with gRNAs against GFP and endogenous ovomucoid (OVM) gene. PGCs enriched for loss of GFP and confirmed for OVM deletion, derived by co-targeting, were injected into Hamburger and Hamilton stage 14–15 chicken embryos, and their ability to migrate to the genital ridge was confirmed. This simple, efficient enrichment approach could easily be applied to the creation of knock-out or edited cell lines or animals.

 抽象的

有效分离表型上与未修饰细胞无法区分的转基因细胞仍然是更广泛利用 CRISPR/Cas9 的主要技术障碍。在这里，我们报告了一种新的富集方法，通过共同靶向基因组整合的 GFP 基因和内源感兴趣基因 (GOI) 来选择基因组工程细胞。使用这种共靶向方法，通过瞬时转染 Cas9 和引导 RNA (gRNA)，成功靶向鸡 (DF1) 和鹌鹑 (CEC-32) 成纤维细胞系中的多个基因组位点。共靶向 DF1 细胞的克隆分离表明，75% 的细胞克隆具有 GFP 缺失和 GOI 双等位基因缺失。为了评估这种方法生成基因组修饰动物的效用，我们通过与针对 GFP 和内源性卵类粘蛋白 (OVM) 基因的 gRNA 共同靶向，在表达 GFP 的鸡原始生殖细胞 (PGC) 上进行了测试。通过共靶向衍生的富集 GFP 丢失并确认 OVM 缺失的 PGC，被注射到汉堡包和汉密尔顿 14-15 期鸡胚胎中，并证实了它们迁移到生殖嵴的能力。这种简单、有效的富集方法可以很容易地应用于创建敲除或编辑的细胞系或动物。