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. 2019 Mar 30;11(4):313. doi: 10.3390/v11040313

Figure 3.

Figure 3

Specific cleavage of pTGEV-GFP BAC by the CRISPR/Cas9 system in vitro. (A) The specific sequence at residues 17-240 were deleted from the N-terminal domain of the TGEV Spike gene. The GenBank accession number of the PRCV S protein partial sequence is BAG83239.1. (B) Annealing PCR with the two specific primers was performed to transcribe the targeted sgRNA in vitro. (C) Electrophoresis detection of the purity of the transcription product from the annealing DNA fragments. The targeted sites a and b of pTGEV-GFP used for digestion are marked with red arrows. (D) Specific cleavage of pTGEV-GFP to delete the sequence containing S_NTD224. The pTGEV-GFP BAC digested by Cas9, guided by sgRNA a and sgRNA b, was detected by electrophoresis. (E) Electrophoresis identification of the pTGEV-GFP-ΔS_NTD by RT-PCR using a pair of primers (rec-672SF and rec-672SR).