cebpd Knockout Generation via CRISPR/Cas9

The cebpd knockout allele was generated with a pair of guide RNAs using CRISPR/Cas9 technology. The gRNAs were designed using the CHOPCHOP website^{61 (link)}, and the target sequences were 5’- GCCCAGCTCATGTTGCATGGTGG-3’ and 5’CGAAGCCTCGTTTGGGTCGGCGG-3’, with PAM sequences underlined. gRNAs were generated through in vitro transcription using T7 RNA polymerase and co-injected with Cas9 protein (CP01-200, PNA Bio) into one-cell stage embryos. Animals with cebpd knockout alleles are screened by PCR with primers cebpdseq1: 5’-ACACTTTCCTTGGGACAGCC-3’ and cebpdseq2: 5’-CCATCATCGTCGTCTAACGTGTAAC-3’. The wild-type allele is identified by PCR with primers cebpdseq1: 5’-ACACTTTCCTTGGGACAGCC-3’ and cebpdseq3: 5’-CTCCATGGCCCAGCTCATG-3’. Deletions were confirmed by Sanger sequencing (Eton Bioscience Inc.). The allele designation for this line is pd354.