Leishmania

Trypanosoma brucei procyclic cells of the strain SMOXP9 [32 (link)] were grown at 28°C in SDM-79 medium supplemented with 10% FCS. The cultures were maintained between 2 × 10⁵ and 2 × 10⁷ cells ml⁻¹.
Trypanosoma brucei BSF cells of the strain SMOXB4 [32 (link)] were grown at 37°C in HMI-9 medium supplemented with 15% FCS. The cultures were maintained between 1 × 10⁵ and 2 × 10⁶ cells ml⁻¹.
Leishmania mexicana promastigote form cells of the WHO strain MNYC/BZ/62/M379 were grown at 28°C in M199 supplemented with 10% FCS, haemin and HEPES. The cultures were maintained between 1 × 10⁵ and 2 × 10⁷ cells ml⁻¹.
Cell densities were determined using a CASY Cell Counter.
Cycling and reaction conditions for PCR to generate the linear fragments of DNA for analysis of recombination rate in L. mexicana were performed as described for long primer PCR (see electronic supplementary material).
For epifluorescence microscopy of live cells expressing fluorescent fusion proteins, 5 × 10⁶ cells were harvested from culture by centrifugation, washed three times with PBS containing 500 ng µl⁻¹ Hoechst 33342, resuspended in 100 µl of PBS containing 500 ng µl⁻¹ Hoechst, and 15 µl was settled on a glass slide with a coverslip laid on top prior to imaging.
Leishmania were transfected using the Amaxa Nucleofector II T-cell system. Sterile DNA was prepared by either ethanol precipitation of cut plasmid DNA or by purification from a PCR using the Qiagen PCR purification kit in a sterile environment. The number of drug-resistant transfectants were determined by transferring the entire transfected culture to 96-well plates in MM199 (for L. mexicana) at 200 μl per well with the appropriate selection drugs 8 h following transfection. For more details, see the electronic supplementary material. After two weeks, the number of wells with visible growth, m, was counted and the corresponding number of transfectants, n, was calculated assuming random distribution among the wells according to the Poisson distribution

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.

An enzyme-linked indirect immunosorbent assay (ELISA) was performed on sera samples for the detection of specific antibodies of L. infantum (INGEZIM® Leishmania; Ingenasa, Madrid, Spain), Ehrlichia canis (INGEZIM® Ehrlichia; Ingenasa) and Anaplasma phagocytophilum (Anaplasma-ELISA Dog; AFOSA GmbH, Blankenfelde-Mahlow, Germany). All assays were performed following the respective manufacturer’s instructions. All sera samples collected on all SDs were tested for all of the above pathogens.