Fig. 7.

GI screens using multiplexed CRISPR/Cas gene editing. a Schematic of the DNA molecules and manipulations resulting in the generation of a double mutant using multiplexed CRISPR/Cas to generated two gene deletions replaced with two different selectable markers. b High-throughput workflow to generate multiplex edits. All CRISPR/Cas manipulations are performed in 96-well plates. In this example, all wells carry competent cells of the same strain (wild-type or mutant), but two different sgRNA-Donor plasmids are combined in each well. All wells also receive the same Cas9 expression plasmid. After selection of triply transformed cells, Cas9 expression is induced for multiple generations. Counterselection of the Cas9 plasmid shuts off gene editing, and doubly edited cells are selected (each edit has a different selectable marker). The red text indicates how many days are required for each step in the workflow. kanMX: G418-resistance marker, natMX: nourseothricin-resistance marker; colored wells indicate that the contents of each well are different