Abstract
The sexually-transmitted parasite Trichomonas vaginalis infects ~1/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying T. vaginalis pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single T. vaginalis cell. We then created DNA constructs required to express several components essential to drive CRISPR/Cas9-mediated DNA modification: guide RNA (gRNA), the Cas9 endonuclease, short oligonucleotides and large, linearized DNA templates. Using these technical advances, we have established CRISPR/Cas9-mediated repair of mutations in genes contained on circular DNA plasmids harbored by the parasite. We also engineered CRISPR/Cas9 directed homologous recombination to delete (i.e. knock out) two non-essential genes within the T. vaginalis genome. This first report of the use of the CRISPR/Cas9 system in T. vaginalis greatly expands the ability to manipulate the genome of this pathogen and sets the stage for testing of the role of specific genes in many biological processes.
Highlights
Trichomonas vaginalis is an obligate extracellular, unicellular flagellated protozoan parasite and the causative agent of trichomoniasis, an infection that afflicts ~1⁄4 billion people worldwide[1]
The double stranded breaks are repaired by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ) or homology-directed repair (HDR) pathways[36,38]
In order to improve the ability to modify T. vaginalis genes, we have developed the use of the CRISPR/Cas[9] system as a means to directly modify genes in this parasite
Summary
Trichomonas vaginalis is an obligate extracellular, unicellular flagellated protozoan parasite and the causative agent of trichomoniasis, an infection that afflicts ~1⁄4 billion people worldwide[1]. Despite being a very common sexually transmitted infection, the basic mechanisms underlying pathogenesis and drug resistance are poorly understood This is in part due to the slow adoption of molecular tools to study the genes and proteins involved in these processes[8,9]. Cas9-gRNA expression induces repair of the double stranded break by the NHEJ and MMEJ pathways, which can result in insertion (NHEJ) or deletions (NHEJ and MMEJ) of nucleotides that may cause gene disruption and loss-of-function mutations. A more directed approach utilizes Cas9-gRNA in combination with a provided DNA template containing homology to the repair site to direct specific user-defined gene modifications. This approach requires a small region of donor DNA homology around the site of modification. This report lays the foundation for further development and utilization of this CRISPR/Cas[9] to rigorously test gene function in this understudied parasite
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