Efficient CRISPR-Cas9-mediated genome editing for characterization of essential genes in .

This protocol outlines a new genetic complementation strategy to investigate gene function in , the parasite causing Chagas disease. We combine CRISPR-Cas9 technology with recombination of variants of the target gene containing the desired mutations that are resistant to Cas9-cleavage, which enables detailed investigation of protein function. This experimental strategy overcomes some of the limitations associated with gene knockouts in . For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).