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. 2020 Nov 24;10:581504. doi: 10.3389/fcimb.2020.581504

Figure 1.

Figure 1

Overview of steps in the construction of CRISPR/Cas9 and donor plasmids. (A) The 20 nucleotides viral genome target (highlighted in teal) is followed by the PAM recognition site (highlighted in purple), 5′-NGG. (B) Schematic on the construction and assembly of the Cas9/gRNA expression plasmid. Digestion of the plasmid containing Cas9 and gRNA scaffold with BbsI allows the insertion of the annealed gRNA oligos (teal) through replacing the type II restriction site (outlined in red). (C) Schematic on the construction of the donor plasmid harbouring the antigen and selectable marker. The T overhangs on the carrier plasmid is complementary to the overhangs in the expression cassette to facilitate ligation.