DNA-seq of F0 Oryctolagus cuniculus: GGTA1 gene edtion by Crispr
The 8th Exon of the rabbit GGTA1 gene (gene ID: LOC100348435) with full length of 694 bp was selected as a rabbit GGTA1 domain. To target the rabbit GGTA1, single guide RNAs were designed using online tools. Rabbit embryos at the pronuclear stage were collected and transferred into oocyte manipulation medium. A mixture of Cas9 and sgRNA mRNA was microinjected into the embryo cytoplasm to edit the GGTA1 gene. The injected embryos then were transferred into the oviduct of recipient rabbits. Genomic DNA from the ear tissue of wild type (WT) and GGTA1 edited rabbit pups was isolated and amplified. The PCR products of the 8th Exon of the GGTA1 gene were purified and cloned into the pGM-T vector. The colonies were picked, and sequenced and analyzed by Sanger sequencing to confirm exact gene mutations.