Mgcl2

We independently amplified ITS1 and ITS2 in each sample. The ITS PCR reactions were performed in 25 μl with the following composition: 5 μL 5 × buffer containing MgCl2 at 1.5 mM (New England Biolabs), 0.1 mM each dNTP, 0.2 µM each primer, and 0.02 U Taq high fidelity DNA-polymerase (Q5 High-Fidelity DNA Polymerase, New England Biolabs). We used a set of long primers developed to have a 5' flanking sequence complementary to the Nextera XT DNA index to facilitate adapter ligation during library construction:
 > ITS1-Flabel (for ITS1 amplification)
TCG TCG GCA GCG TCA GAT GT GTA TAA GAG ACA GTC CGT AGG TGA ACC TGC GG
 > ITS1-Rlabel (for ITS1 amplification)
GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGC TGC GTT CTT CAT CGA TGC
 > ITS3-Flabel (for ITS2 amplification)
TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG GCA TCG ATG AAG AAC GCA GC
 > ITS4-Rlabel (for ITS2 amplification)
GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GTC CTC CGC TTA TTG ATA TGC
Reactions included 30 cycles with the following conditions: 94 °C 15 s, 60 °C 30 s, and 72 °C 30 s. Amplified fragments were purified using spin columns (GenElute TM PCR Clean-Up Kit, Sigma-Aldrich) and were checked on agarose gel electrophoresis. Finally, we quantified the starting DNA concentration using the Infinite M200 PRO NanoQuant spectrophotometer (TECAN, Männedorf, Switzerland).

Genomic DNA from the drug-resistant cells was isolated 14 days posttransfection using a DNeasy blood and tissue kit (Qiagen) and analyzed for the presence of the LeishIF4E1 gene, using specific primers derived from the open reading frame (ORF) of LeishIF4E1. The primers used were LeishIF4E1 forward (5′-GGATCCATGTCATCTCCATCTTCAG-3′) and LeishIF4E1 reverse (5′-TCTAGAAGACGCCTCGCCGTGCTT-3′). A parallel reaction was performed to look for the presence of the G418 resistance gene, with primers derived from its ORF: G418 F (5′-GCCCGGTTCTTTTTGTCAAGAC-3′) and G418 R (5′-GTCACGACGAGATCATCATCGCCG-3′). Genomic DNA from Cas9/T7 L. mexicana cells was used as a positive control for the presence of the LeishIF4E1 gene. The reaction mixture consisted of 2 μM each primer, genomic DNA (gDNA) (100 ng), dNTPs (0.2 mM), and HiFi polymerase (1 unit of Phusion; NEB) in GC buffer with MgCl₂ (NEB) in a total volume of 50 μl. PCR conditions included initial denaturation at 98°C for 4 min, which was followed by 35 cycles of 98°C for 30 s, annealing at 60°C for 30 s, and an extension at 72°C for 2 min 15 s. A final extension was done for 7 min at 72°C. PCR products were separated over 1% agarose gels.

Genomic DNA from the drug-resistant cells was isolated 14 days post transfection using DNeasy Blood & Tissue Kit (Qiagen) and analyzed for the presence of the LeishIF4E2 gene, using specific primers derived from the UTRs of LeishIF4E2. The primers used were LeishIF4E2 (5’UTR) forward (P1) and LeishIF4E2 (3’UTR) Reverse (P2). A parallel reaction was performed to detect the presence of the G418 resistance gene with primers derived from its ORF: G418 Forward (P3) and G418 Reverse G418 Reverse (P4). An additional reaction confirming the insertion of the G418 cassette at the target site was performed with primers G418 Forward P3, and LeishIF4E2 (3’UTR) Reverse P2. All primers are listed in S1 Table. Genomic DNA from Cas9/T7 L. mexicana cells was used to detect the presence of the LeishIF4E2 gene. The reaction mixture consisted of 2 μM of each primer, gDNA (100 ng), dNTPs (0.2 mM), HiFi Polymerase (1 U Phusion, NEB) in GC buffer with MgCl₂ (NEB) in a total volume of 50 μl. PCR conditions included initial denaturation at 98°C for 4 min followed by 35 cycles of 98°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 2 min 15 s. Final extension was done for 7 min at 72°C. PCR products were separated on 1% agarose gels.

A PCR assay was conducted to amplify the E. coli housekeeping uidA [β-D glucuronidase] gene as described by Omolajaiye et al. [19 ] using the following primers: UidA-F AAAACGGCAAGAAAAAGCAG and UidA-R ACGCGTGGTTACAGTCTTGCG, which produces a 147 bp amplicon size. The PCR consisted of 12.5 μL of AmpliTaq Gold® DNA Polymerase, 0.05 units/L Gold buffer, 930 mM Tris/HCl pH 8.05, 100 mM KCl, 0.4 mM of each dNTP, and 5 mM MgCl2] (New England Biolabs, USA), 8.5 μL of RNase-nuclease free PCR water, 1 μL of 10 μM each primer and 2 μL of template gDNA. The cycling consisted of an initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 40 s, 60 °C for 30 s and 72 °C for 40 s and a final extension of 72 °C for 7 min using the ProFlex PCR System (Applied Biosystems, USA). The negative control was a DNA-free template (nuclease-free water). To allow standardization, molecular weight markers of 1 kb and 100 bp DNA (PROMEGA, Madison, WI, USA) were used to determine the size of the PCR amplicons. For product size confirmation and yield estimation, 5 µL of the PCR products were loaded onto 1% agarose gels, subjected to electrophoresis for 45 min at 80 V and stained with ethidium bromide.

Microcystin-LR was purchased from Sigma (Saint-Quentin Fallavier, France). PP2A was obtained from New England Biolabs Inc. Na₂S₂O₃, MgCl₂, MnCl₂, HCl, Ca(ClO)₂, high-purity CO₂, p-Nitrophenyldisodium orthophorphate (p-NPP), tris(hydroxymethyl)aminomethane (Tris), bovine serum albumin (BSA), dithiothreitol (DTT), neoprene rubber, sodium nitrobenzene disodium, and ascorbic acid were purchased from Sinopharm (Shanghai, China). HCOOH, CH₃OH, CF₃COOH, and HPLC acetonitrile were purchased from Merck (Darmstadt, Germany).