Characterisation of Casein Kinase 1.1 inLeishmania donovaniUsing the CRISPR Cas9 Toolkit

Abstract

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.

Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

“…Our results demonstrate that the system is efficient both at the genomic and protein levels for PF16 (Figure S3c,d). Parasite growth was not affected after PF16 depletion (Figure S3e), in agreement with published results in L. mexicana and Leishmania donovani (Beneke et al, ; Martel et al, ). Surprisingly however, parasites of the [ PF16‐HA Flox Puro‐Neo] clones were not motile even before induction.…”

“…When an essential gene is used for a proof of concept, some deleterious events may not be noticed. So to have more insights in our system, that is, to be able to detect any limitations or deleterious events during parasites recovery, we decided to edit a non‐essential gene, PF16, encoding a protein of the central pair of microtubules of the flagellar axoneme (Beneke et al, ; Martel, Beneke, Gluenz, Spath, & Rachidi, ). Conditional editing of non‐essential genes is also interesting, in particular, to follow the early phenotypes secondary to protein depletion and to avoid parasite adaptation (see below).…”

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

“…; Martel et al. ). Figure describes the two strategies that have been most widely used for genome editing in kinetoplastids (Beneke et al.…”

“…; Martel et al. ). This cloning‐free system has also been adapted for gene editing in T. brucei and T. cruzi (Beneke et al.…”

“…In general, there were few molecular tools available for the genetic manipulation of T. cruzi and Leishmania spp., compared to T. brucei, thus limiting the study of biological processes in the first two parasites. Fortunately, genome editing by CRISPR/Cas9 has been recently achieved in T. cruzi (Lander et al 2015;, L. major (Sollelis et al 2015), L. donovani (Zhang and Matlashewski 2015), L. mexicana , T. brucei Rico et al 2018;Vasquez et al 2018) and the honey bee trypanosomatid Lotmaria passim (Liu et al 2019) and the system has been successfully used to investigate the role of several proteins in trypanosomatids, but mainly in T. cruzi Bertolini et al 2019;Chiurillo et al 2017Chiurillo et al , 2019Cruz-Bustos et al 2018b;Lander et al 2015Lander et al , 2018Martel et al 2017;Takagi et al 2019), as shown in Table 1. In this review we described the main strategies that have been developed to perform genome editing by CRISPR/Cas9 in trypanosomatids, presenting examples of their usefulness for solving biological questions.…”