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Development of simple and readily adoptable methods to mediate germline engineering of the chicken genome will have many applications in research, agriculture and industrial biotechnology. We report germline targeting of the endogenous chicken Interferon Alpha and Beta Receptor Subunit 1 (IFNAR1) gene by in vivo transgenic expression of the high-fidelity Cas9 (Cas9-HF1) and guide RNAs (gRNAs) in chickens. First, we developed a Tol2 transposon vector carrying Cas9-HF1, IFNAR1-gRNAs (IF-gRNAs) and green fluorescent protein (GFP) transgenes (pTgRCG) and validated in chicken fibroblast DF1 cells. Next, the pTgRCG plasmid was directly injected into the dorsal aorta of embryonic day (ED) 2.5 chicken embryos targeting the circulating primordial germ cells (PGCs). The resulting chimera roosters generated a fully transgenic generation 1 (G1) hen with constitutive expression of Cas9-HF1 and IF-gRNAs (G1_Tol2-Cas9/IF-gRNA). We detected a spectrum of indels at gRNA-targeted loci in the G1_Tol2-Cas9/IF-gRNA hen and the indels were stably inherited by the G2 progeny. Breeding of the G1_Tol2-Cas9/IF-gRNA hen resulted in up to 10% transgene-free heterozygote IFNAR1 mutants, following null-segregation of the Tol2 insert. The method described here will provide new opportunities for genome editing in chicken and other avian species that lack PGC culture.

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.

Exogenous RNA polymerase III (pol III) promoters are commonly used to express short hairpin RNA (shRNA). Previous studies have indicated that expression of shRNAs using standard pol III promoters can cause toxicity in vivo due to saturation of the native miRNA pathway. A potential way of mitigating shRNA-associated toxicity is by utilising native miRNA processing enzymes to attain tolerable shRNA expression levels. Here, we examined parallel processing of exogenous shRNAs by harnessing the natural miRNA processing enzymes and positioning a shRNA adjacent to microRNA107 (miR107), located in the intron 5 of the Pantothenate Kinase 1 (PANK1) gene. We developed a vector encoding the PANK1 intron containing miR107 and examined the expression of a single shRNA or multiple shRNAs. Using qRT-PCR analysis and luciferase assay-based knockdown assay, we confirmed that miR30-structured shRNAs have resulted in the highest expression and subsequent transcript knockdown. Next, we injected Hamburger and Hamilton stage 14-15 chicken embryos with a vector encoding multiple shRNAs and confirmed that the parallel processing was not toxic. Taken together, this data provides a novel strategy to harness the native miRNA processing pathways for shRNA expression. This enables new opportunities for RNAi based applications in animal species such as chickens.