Taming Parasites by Tailoring Them

Abstract

The next-generation gene editing based on CRISPR (clustered regularly interspaced short palindromic repeats) has been successfully implemented in a wide range of organisms including some protozoan parasites. However, application of such a versatile game-changing technology in molecular parasitology remains fairly underexplored. Here, we briefly introduce state-of-the-art in human and mouse research and usher new directions to drive the parasitology research in the years to come. In precise, we outline contemporary ways to embolden existing apicomplexan and kinetoplastid parasite models by commissioning front-line gene-tailoring methods, and illustrate how we can break the enduring gridlock of gene manipulation in non-model parasitic protists to tackle intriguing questions that remain long unresolved otherwise. We show how a judicious solicitation of the CRISPR technology can eventually balance out the two facets of pathogen-host interplay.

Keywords: CRISPR; genetic engineering; genome editing; parasite manipulation; protozoan infections.

Figure 1

Abridged phylogenetic tree depicting main super-phyla of the kingdom protozoa, namely Alveolata and Excavata. Two of the all shown phyla, Apicomplexa, and Kinetoplastida, comprise a vast majority of human and animal pathogens. Only the selected genera representing each class are displayed. While most apicomplexans (except for gregarines) favor an intracellular lifestyle, kinetoplastids prefer an extracellular life (barring certain stages of T. cruzi and Leishmania). Besides, apicomplexan parasites exhibit well-defined asexual as well as sexual reproduction, whereas the latter phase is not yet known in most kinetoplastids. Individual genera or even species have evolved a notably distinct lifecycle in specific host organisms, which often involves a perpetual inter-host transmission in nature.

Figure 2

Current implementation of CRISPR or CRISPR-like tools in prototypical parasitic protists and their mammalian hosts. As noted, CRISPR/Cas9 and ensuing methods (dCas9, Cpf1, and NgAgo) have been successfully established in the mammalian cells, but remain widely marginalized in parasitology. Only the original CRISPR/Cas9 in designated parasites has been used so far.

Figure 3

Traffic light gradient depiction of genome engineering applications in parasites and mammalian cells. A comparative color-coding indicates the current progress in different organisms. The green and red colors display a “comprehensive” or “not-at-all” scenario, respectively. The yellow light reflects an incremental success achieved through customary methods. While the green and yellow colors in model parasites encourage for more innovative applications (inducible gene silencing, epigenetic/epigenomic studies, multi-site editing), the widespread red color, mostly in non-model pathogens, advocates for a systematic application of the CRISPR technology.

Figure 4

Major applications of Cas9, dCas9, and Cpf1 in genome editing. The three widely accepted CRISPR-dependent systems (A–C) broadly complement each other, even though the specified usage are not always restricted to individual proteins, i.e., Cas9 (A) and Cpf1 (C) can substitute each other to perform at least some of the mentioned tasks. Both enzymes require unique protospacer adjacent motif (PAM) to locate the gene of interest, making them complementary when no proper PAM is available for any of them. RNA-guided double-strand break is vital to their functioning. Whereas, Cas9 incision generates blunt ends, Cpf1 produces cohesive ends. The dCas9 (B) method can recompense for Cas9, when the target genomic loci happen to be essential. The inactivated isoform of Cas9 serves as a blocker, enabling a silencing of gene transcription instead of disrupting the gene locus. Its low toxicity also allows the assembly of stable progenitor lines for successive transgenic research. Not least, the feature that dCas9 can bind harmlessly to a locus of interest permits a fusion of dCas9 with reporter or effector proteins, further expanding its utility to epigenetic and epigenomic studies. Cas9, CRISPR associated protein 9; Cpf1, Centromere and Promoter Factor 1; dCas9, catalytically dead Cas9; cr-RNA, CRISPR-RNA; Indel, insert or deletion (of bases in the genome).