Construction of the CRISPR/Cas9 expression plasmid

Obeying the NGG PAM sequence rule, two target sites, MT1 and MT2, were designed in exon1 and exon2 of chicken MSTN via a convenient online tool (http://crispr.mit.edu/), respectively (Fig 1A). An ET1 target site was chosen in the envelope gene of EAV-HP (Fig 1B). The three targets are shown in Table 1 and the corresponding primers for sgRNA construction are displayed in Table A in S1 Text.

(A) A schematic diagram of the target sites in the chicken MSTN. MT1 and MT2 were chosen as target sites in exons 1 and 2 of MSTN, respectively. The two sites span 2.6 kb in the genome. A 50-mer ssODN harboring an EcoRI site was designed to integrate the MT1 site. (B) A schematic illustration of the ET1 site in the chicken EAV-HP viral genome. One target site, ET1, was determined in the Env gene, and a relative donor DNA containing the EGFP expression cassette and two homology arms of Env was constructed for the exogenous gene knock-in of the chicken genome. HL and HR: left and right homologous arms; pCMV: CMV promoter; PA: polyA. (C) The efficiencies of mutagenesis in the MT1 and MT2 sites of the chicken genome via the T7 EI assay. (D) The efficiency of the targeted mutation in the ET1 site of the chicken genome using the T7 EI assay. DF-1 cells were co-transfected with the CRISPR/Cas9 expression plasmid and the corresponding reporter vector. The target sequence was inserted into two homology repeats to disrupt Puro^R and eGFP genes in the reporter vector. When Cas9 cut the target sequence in the reporter vector to generate a DSB, Puro^R was restored via SSA. The target sequences in both the reporter and chicken genomes were simultaneously cut via CRISPR/Cas9. Thus, puromycin was added to the cell culture to enrich the gene-editing positive cells. Targeted mutations in the chicken genome not in reporter vectors were detected using the T7 EI assay.

Note: Underlining indicates PAM sequences.

DNA fragments harboring chicken MSTN and EAV target sequences were generated via overlap PCR. For target MT1, two short DNA fragments were amplified via the primer pairs U6F/MT1R and MT1F/U6R (Table A in S1 Text), in which the two PCR products both had MT1 target sequences as overlap regions. Then, two short fragments were ligated together via overlap PCR and cloned into the parent plasmid pll3.7-U6-sgRNA-Cas9 between the XbaI and NotI sites, thereby resulting in the MT1 CRISPR/Cas9 expression vector designated pll3.7-U6-MT1sgRNA-Cas9. Moreover, the CRISPR/Cas9 vectors for MT2 and ET1 were obtained using the same strategy and designated pll3.7-U6-MT2sgRNA-Cas9 and pll3.7-U6-ET1sgRNA-Cas9. The sgRNA sequencing of the expression plasmids was performed using the U6F primer.

Another CRISPR/Cas9 nuclease expression vector was initially designed that expressed yeast Rad52 and Cas9 as a fusion protein. Yeast Rad52 was amplified via the primers yRadF and yRadR (Table A in S1 Text) from the yeast genome. The forward and reverse primers containing BsaI sites and the same sticky ends with NheI and BamHI were generated via the BsaI digestion of the PCR products. Then, the yRad52 fragments were cloned into plasmid pll3.7-U6-sgRNA-Cas9 between NheI and BamHI, thereby resulting in the yRad52-Cas9 fusion protein vector pll3.7-U6-sgRNA-yRad-Cas9. Subsequently, ET1 and MT1 target sequences were obtained via the double digestion of pll3.7-U6-ET1sgRNA-Cas9 and pll3.7-U6- MT1sgRNA-Cas9 and then subcloned into plasmid pll3.7-U6-sgRNA-yRad-Cas9 XbaI/NotI sites, thereby resulting in two plasmids, pll3.7-U6-ET1-yRad52-Cas9 and pll3.7-U6-MT1-yRad52-Cas9, respectively.