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. 2020 Nov 24;5:274. [Version 1] doi: 10.12688/wellcomeopenres.16405.1

Figure 2. The SpEDIT pLSB plasmid allows one-step insertion of sgRNAs via Golden Gate cloning.

Figure 2.

A. Map of pLSB plasmid .Full sequence is available scanning the QR code in Figure 1 or at allshirelab.com/spedit. Versions with natMX6 (cloNAT), kanMX6 (G418) or hphMX6 (hygromycin) S. pombe resistance markers are available. A Cas9 codon optimised for S. pombe (SpCas9) is expressed from the adh15 promoter ( Padh15). B. Diagram of sgRNA cassette and cloning procedure. sgRNA cassette expression is driven by a tRNA Ser Pol III promoter (purple block arrow). A self-cleaving hepatitis delta virus (HDV) ribozyme is located at the 5’ end of the sgRNA cassette ( Ryan et al., 2014) (red block arrow). A superfolder green fluorescent protein (sfGFP) is used as placeholder (green block arrow). BsaI sites flanking sfGFP allow one-step insertion of a sgRNA target (light blue block arrow) into the sgRNA scaffold (grey block arrow) via Golden Gate cloning. The Pol III terminator sequence from S. cerevisiae SUP4 (tRNA Tyr) is present at the 3’ end of the sgRNA cassette (black block). C. The sfGFP placeholder allows cultures carrying empty (green) pLSB plasmids to be distinguished from sgRNA-loaded (non-green) pLSB plasmids. sgRNA, single guide RNA.