Animal Molecular Breeding 2024, Vol.14, No.2, 165-177 http://animalscipublisher.com/index.php/amb 170 Figure 1 Dual single-guide RNA (sgRNA)-directed deletion of MC1R in zygotes (Adopted from Xiao et al., 2019) Image caption: (A) Schematic diagram of sgRNA targeting the rabbit MC1R gene loci. The yellow rectangle represents the transmembrane domain of MC1R. Two sgRNA sequences, sgRNA1 (Sg1#) and sgRNA2 (Sg2#), are highlighted in green. Protospacer adjacent motif (PAM) sequences are presented in red with underline. Primers F and R are used for mutation detection in embryos; (B) Cytoplasmic injection of zygotes using the CRISPR/Cas9 system; Seven blastocysts are collected. Mutation detection in blastocyst by PCR. M, marker; numbers 1–7 represent different blastocysts used in this study.; The number in red represents the positive embryos. Scale bar, 100 μm; (C) T-cloning and Sanger sequencing of the modified MC1R alleles in blastocysts. Wild-type sequence is shown at the top of the targeting sequence. Sequences of sgRNAs are marked in green, the PAM sequences are in red, insertions are highlighted in lowercase red letters, and deletions are designated by dashes. E: embryos; WT: ED allele; deletion: “−”; insertion: “+” (Adopted from Xiao et al., 2019)
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