Gene Editing in Trypanosomatids: Tips and Tricks in the CRISPR-Cas9 Era

Gene editing in trypanosomatids has long been proven difficult. The develop-ment of CRISPR-Cas9 has improved this issue, opening the way to a better understanding of biological processes and drug-resistance mechanisms, and screening of drug targets. Different strategies have now been developed: either PCR-or plasmid-based, differing mainly in the nature of the donor DNA and the single guide RNA transcription. Here we review the main genetic tools avail-able for Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei for gene tagging, single-base editing, and deletion of nonessential and essential genes. We discuss the main advantages and challenges of different strategies and how to choose 'the right cut' depending on the importance of untranslated regions. These considerations allow selection of the most accurate gene editing approach for a given functional analysis.