Fig 1: Production of OVAL-null chickens. (A) Schematic illustration of OVAL targeting donor plasmid containing OVAL intron #1 and exon 2, human IgG construct and CMV PuroR expression cassette. The donor plasmid will be inserted into gRNA target site mediated by non-homologous end joining (NHEJ) DNA repair process. PAM sequence is indicated as blue bar and gRNA recognition site is indicated as red bar. PCR primers specific for 5′ and 3′ junctions are indicated as arrows. (B) Validation of targeted insertion of donor plasmid into target site in PGCs by genomic DNA PCR of 5′ and 3′ junctions. Wild type PGC was used as negative control. (C) Images of donor-PGC derived progeny (I/i) and OVAL targeted progeny after testcross of germline chimera with Korean Ogye (KO) hens. (D) Genomic DNA sequencing analysis of G1 OVAL targeted progenies at 5′ and 3′ junctions amplified by each junction specific primers. PAM sequence is indicated as blue bar and gRNA recognition site is indicated as red bar. Endogenous OVAL gene and donor plasmid sequences are described (E) Pedigree of OVAL targeted chickens. G0 germline chimeric rooster was mated with wild type hen and produced G1 OVAL±chickens. G2 OVAL+/+, OVAL±and OVAL-/- chickens were produced by mating between G1 OVAL±individuals. (F) Genotyping of G2 progenies by genomic DNA PCR using specific primers for wild type OVAL allele and modified allele. Primers used for genotyping are indicated as arrows.