Authors
T Beneke1; R Madden1; J Valli1; L Makin1; E Gluenz1; 1 University of Oxford, Sir William Dunn School of Pathology Discussion
The CRISPR-Cas9 system offers a powerful method for precise genome editing, which promises to revolutionise genetic manipulation of Leishmania spp., offering for the first time a simple method to generate loss-of-function mutants. We have generated a cloning-independent, PCR-only toolkit for rapid CRISPR-Cas9 genome editing with the following key components: (1) L. mexicana cell lines that constitutively express Cas9 and T7 RNA polymerase, (2) a set of plasmids with several resistance genes and tags to generate donor DNA, in a one-step PCR, for homology directed repair of Cas9-induced double strand breaks using 30nt homology arms, and (3) a new protocol for delivery of single-guide RNAs using PCR-generated DNA templates, which are transcribed by T7 RNA polymerase in vivo. Using simultaneous selection with different drugs, two or more alleles can be targeted at once; here we show examples of gene tagging and of knockout phenotypes produced in a single round of transfection. This method can allow for targeting of multi-copy gene families and can circumvent the technical challenges posed by the plasticity of the Leishmania genome. Our toolkit is rapid, facile and scalable to a 96-well plate format, allowing targeting of large cohorts of genes for localisation and mutant phenotype screening. 
