Authors

C Hughes¹; J Carnielli²; V Geoghegan¹; J Faria³; J Mottram¹;  ¹ University of York, UK;  ² York Biomedical Research Institute, UK;  ³ York Biomedical Research Institute, Department of Biology, University of York, UK

Discussion

Genetic modification has become a staple tool in Leishmania discovery research. The CRISPR-Cas9 system has enabled this process to be more flexible and efficient, as it can be used to make large-scale changes such as gene deletion, or small-scale changes, such as mutating individual codons of a protein-encoding gene, known as precision editing. Precision editing allows investigation into the role of specific amino acids in a protein in the parasite e.g. catalytic residues, ATP-binding site gatekeeper residues, and phosphorylation sites of protein kinases. To make precision editing more accessible, we have developed a method for Leishmania mexicana that is flexible, affordable, efficient, and selection-free. We use an oligonucleotide-based strategy to produce repair templates designed for synonymous and nonsynonymous mutations in both essential and nonessential genes;  a python script aids the design process. We applied this precision editing approach to phosphorylation sites on kinetochore proteins or gatekeeper residues in the ATP-binding sites of protein kinases involved in regulating differentiation. Thirty-five individual precision edits were attempted in L. mexicana T7Cas9 promastigotes. Up to 24 clones isolated from each of the 35 transfections were assessed by PCR for integration of the repair template, with at least 1 clone for each confirmed by Sanger sequencing to carry the expected homozygous mutation. The efficiency of integration varied for each precision edit. For all integration events (homozygous, heterozygous and “complex”), efficiency varied in each transfection from 8% to 83% (mean of 31%). Of all clones screened, homozygous mutants predominated (24%), with 4.2% heterozygotes and 2.0% “complex”. The high efficiency of this precision editing approach allows the dissection of protein kinase signalling events in Leishmania.