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.

Keywords: Leishmania; Lotmaria passim; Trypanosoma brucei; Trypanosoma cruzi; Kinetoplastids; trypanosomatids.

Fig 1.

Schematic representation of the two main strategies used for CRISPR/Cas9-mediated gene knockout in trypanosomatids. Both strategies involve the constitutive expression of Cas9 and provide a donor DNA template to induce DSB repair by HDR. A, The methodology originally described by Lander et al. (2015) in T. cruzi involves: i) co-transfection of an integrative vector for expression of Cas9 and a specific sgRNA, together with a donor DNA template containing a resistance marker (RM) flanked by homology regions; ii) co-expression of Cas9 and sgRNA, which will assemble in the nucleus of the cell to conform a ribonucleoprotein complex; and iii) Cas9 will be targeted by sgRNA to the gene of interest (GOI), producing a DNA cleavage that will be repair by homologous recombination, producing first allele replacement by the resistance marker. Constitutive expression of Cas9 and sgRNA allows sequential replacement of the second allele using the first replaced allele as template. B, Strategy developed by Beneke et al. [26] as a toolkit for genome editing in L. mexicana, L. major and T. brucei. i) The method involves the use of a parental cell line expressing Cas9 and T7 RNA polymerase (T7 RNA Pol) for nucleofection of three PCR products: two donor DNAs with different resistance markers (RM 1 and RM 2) and a DNA fragment holding the specific sgRNA sequence downstream the T7 promoter for in vivo transcription; ii) In the nucleus of the cell transiently expressed sgRNA assembles with Cas9; iii) Cas9 targets both alleles of the gene, which are simultaneously replaced by two different resistance markers.