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. 2021 Jan 8;11:625862. doi: 10.3389/fmicb.2020.625862

FIGURE 1.

FIGURE 1

Construction of the Cas9i system and sgRNA array. (A) Sketch of pUF1-Cas9 and pL6cs-sgRNA. pUF1-Cas9 expresses the Cas9 protein with the BSD drug-selectable marker. A flag tag and two NLSs are fused to Cas9 protein. pL6cs-sgRNA carries the sgRNA cassette, the donor DNA including the homologous regions (HR1 and HR2) and modifications, and the hDHFR marker. (B) Diagram illustrating the Cas9e and Cas9i system. The Cas9e system employs two episomal plasmids in the nucleus while the Cas9i system employs an episomal pL6cs plasmid and an integrated pUF1-DHODH-Cas9i plasmid which was integrated into the endogenous P230p locus through a single crossover in advance. pUF1-DHODH-Cas9i carries the Cas9 expression cassette (the same as pUF1-Cas9), yDHODH selection marker, and the C-terminal of P230p coding sequence. Primers indicated as P1–P3 were used for verification of integration. (C) Sketch of pL6cs bearing the dual-sgRNA array. Multiple sgRNAs are constructed in a single array, consisting of the 20- or 30-bp target sequences following by the 78-bp sgRNA scaffold. Donor DNAs were constructed adjacent to each other. (D) Diagram illustrating drug off/on selection cycle. Episomal mutant is cultured for 3 weeks without selection drug (DSM1 in this case), and for another 2 weeks with the drug. This process can be repeated for several cycles.