Fig. 5: RECKLEEN 3 platform for simultaneous multi-target genome editing.
A Design of the RECKLEEN 3 plasmid containing the main transcription units of the lambda Red components, Cas9, ACRIIA4, as described for RECKLEEN 1 and a sfgfp dropout part where two to five different sgRNA cassettes can be introduced using eight sgRNA helper plasmids (RECKLEEN 3plus_helper sgRNA plasmids). Customizing of the RECKLEEN 3 plasmid for simultaneous editing of different targets is done by two Golden Gate Cloning steps as detailed in the method section. B A scheme of the sgRNA helper plasmids showing the different plasmids which can be combined to assemble plasmids with sgRNA modules for targeting 2, 3, 4, and 5 different targets simultaneously. Fusion sites (5′→3′ direction) are introduced based on predicted assembly efficiency36 and are indicated in the inlay box. Evaluation of the RECKLEEN 3 platform for simultaneous deletion of two genes (C) and three genes (D) using a single editing plasmid.
