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

Abstract

CRISPR/Cas9 technology has revolutionized biology. This prokaryotic defense system against foreign DNA has been repurposed for genome editing in a broad range of cell tissues and organisms. Trypanosomatids are flagellated protozoa belonging to the order Kinetoplastida. Some of its most representative members cause important human diseases affecting millions of people worldwide, such as Chagas disease, sleeping sickness and different forms of leishmaniases. Trypanosomatid infections represent an enormous burden for public health and there are no effective treatments for most of the diseases they cause. Since the emergence of the CRISPR/Cas9 technology, the genetic manipulation of these parasites has notably improved. As a consequence, genome editing is now playing a key role in the functional study of proteins, in the characterization of metabolic pathways, in the validation of alternative targets for antiparasitic interventions, and in the study of parasite biology and pathogenesis. In this work we review the different strategies that have been used to adapt the CRISPR/Cas9 system to Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., as well as the research progress achieved using these approaches. Thereby, we will present the state‐of‐the‐art molecular tools available for genome editing in trypanosomatids to finally point out the future perspectives in the field.

Disruption of Intracellular Calcium Homeostasis as a Therapeutic Target Against Trypanosoma cruzi

“…The advanced application of the CRISPR/Cas9 system to investigation of T. cruzi biology (Lander and Chiurillo, 2019) has enormously facilitated the finding and characterization of many proteins in these parasites. The recent identification in T. cruzi of a mitochondrial pyruvate dehydrogenase phosphatase (TcPDP) that is sensitive to physiological Ca 2+ concentration and is able to stimulate the activity of a mitochondrial pyruvate dehydrogenase, stimulating energy metabolism through the (1) Sphingosine-activated Ca 2+ channel, responsible for Ca 2+ entry.…”

A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in Trypanosoma cruzi

“…Few genetic tools were available to work with T. cruzi (Docampo, 2011;Burle-Caldas Gde et al, 2015) until the recent introduction of the CRISPR/Cas9 technique for gene knockout (Peng et al, 2014;Lander et al, 2015;Costa et al, 2018;Romagnoli et al, 2018;Takagi et al, 2019) endogenous gene tagging (Lander et al, 2016b(Lander et al, , 2017Soares Medeiros et al, 2017;Costa et al, 2018), gene complementation (Chiurillo et al, 2017), and gene knock-in (Chiurillo et al, 2019). These studies have been recently reviewed in Lander and Chiurillo (2019).…”

Signaling pathways involved in environmental sensing in Trypanosoma cruzi

“…Further work is also needed to investigate signaling events acting at other levels, such as transcription, translation, cell cycle, and DNA damage response. The development of gene‐editing tools for the study of T. cruzi (Lander & Chiurillo, 2019; Lander et al, 2019) will have an important role in the elucidation of these pathways.…”