Application of the CRISPR/Cas9-riboswitch-based method to downregulate the inositol polyphosphate kinase (IP6K) expression in Trypanosoma cruzi

Abstract

Inositol pyrophosphates (PP-IPs) - mainly IP7, and IP8 - are involved in a wide range of cellular processes in eukaryotes. However, the mechanism of action of PP-IPs is not fully understood. IP7 and IP8 are synthesized by pathways involving the participation of IP6K and PP-IP5K kinases, respectively. Trypanosomatids have an ortholog gene for IP6K, but apparently do not have orthologs for PP-IP5K, i.e., they probably do not synthesize IP8, which makes them excellent models for the study of IP7. Using the CRISPR/Cas9 system and two rounds of sgRNA transfection, we successfully depleted a single IP6K allele in Trypanosoma cruzi (the causative agent of Chagas Disease), generating a single-null (IP6K-/+) lineage. Notably, we were also able to generate a double-null lineage (IP6K-/-). However, we were unable to select clones of this lineage as it was unable to proliferate, i.e., most T. cruzi IP6K-/- cells died a few days after transfection, leaving the doubt about the essentiality of IP6K in this organism. Removal of a single copy of IP6K causes several morphological effects, such as rounding and wrinkling of the cell body, increased number of glycosomes, and mitochondrial enlargement. Moreover, IP6K-/+ lineage showed a slight cell cycle arrest at G0/G1 phase, but no DNA damage was detected. Then, we developed a pioneering assay to measure quiescent (dormant) cells and figured out that the partial depletion of IP6K drives part of the T. cruzi population to dormancy. However, we still do not know whether IP6K is an essential gene and whether the effects observed in the IP6K-/- lineage were indeed due to the absence of IP6K. Thus, we intend to apply the CRISPR-Cas9-Riboswitch-based method in T. cruzi (CL Brener) in collaboration with Dr. Noelia Lander to downregulate the expression of IP6K. This approach consists of tagging the IP6K with the active (glmS) or inactive (M9) ribozyme and then inducing IP6K silencing with glucosamine. Next, we will evaluate the growth rate, cell cycle phases length, percentage of quiescent cells, morphological features, and possible metabolic alterations in the generated lineage. This approach will benefit both groups as it will make it possible to expand its applicability to other trypanosomatids. Furthermore, the expected results will clarify the effects of IP6K loss in the human pathogen T. cruzi, which will contribute significantly to a better understanding of the pyrophosphorylation performed by IP7, a non-enzymatic post-translational modification still little understood. (AU)