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The recent adaptation of CRISPR Cas9 genome editing to  Leishmania  spp. has opened a new era in deciphering  Leishmania  biology. The method was recently improved using a PCR-based CRISPR Cas9 approach, which eliminated the need for cloning. This new approach, which allows high-throughput gene deletion, was successfully validated in  L. mexicana  and  L. major . In this study, we validated the toolkit in  Leishmania donovani  targeting the flagellar protein PF16, confirming that the tagged protein localizes to the flagellum and that null mutants lose their motility. We then used the technique to characterise CK1.1, a member of the casein kinase 1 family, which is involved in the regulation of many cellular processes. We showed that CK1.1 is a low-abundance protein present in promastigotes and in amastigotes. We demonstrated that CK1.1 is not essential for promastigote and axenic amastigote survival or for axenic amastigote differentiation, although it may have a role during stationary phase. Altogether, our data validate the use of PCR-based CRISPR Cas9 toolkit in  L. donovani , which will be crucial for genetic modification of hamster-derived, disease-relevant parasites.

Characterisation of Casein Kinase 1.1 inLeishmania donovaniUsing the CRISPR Cas9 Toolkit