Cas9 Expression Plasmid Construction

For expression of Cas9 in Leishmania, plasmid pRM006 was constructed: humanized S. pyogenes Cas9 from plasmid pX330 (Addgene #42230; [27 (link)]) was joined to Crithidia fasciculata PGKB 5′-UTR and GSPS 3′-UTR from pLENT V1 [12 (link)] and the backbone of pLENT V1, containing a phleomycin-resistance gene, which was subsequently replaced with a hygromycin B phosphotransferase gene. Finally, fragments from the 5′- and 3′-UTR of L. major β-tubulin from a constitutive expression plasmid [14 ] were inserted and the plasmid was linearized with Pac I before transfection. Insertion of the T7 RNAP cassette with calmodulin UTRs from pVY087 [29 (link)] into pRM006, downstream of Cas9 generated pTB007. For expression of Cas9 in T. brucei, hSpCas9 from pX330 was inserted into pPOTv4-Puro-mNG-Puro [12 (link)] between the aldolase and PFR2 intergenic sequences, replacing the mNG gene. The fragment containing the puromycin-resistance gene and Cas9 expression cassette was subsequently excised and inserted into a plasmid containing a blasticidin-resistance gene and targeting fragments for the T. brucei tubulin locus. The resulting plasmid pTB011 was linearized with Pac I before transfection. Plasmid sequences are in the electronic supplementary material, file S5.