Abstract
Babesia bovis, the most virulent causative agent of bovine babesiosis, is prevalent in tropical and subtropical regions of the world. Although the whole-genome sequence was released more than a decade ago, functional analysis of the genomics of this parasite is hampered by the limited breadth of genetic engineering tools. In this study, we implemented the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system for B. bovis and demonstrated its potential for genome editing. Cas9 and human dihydrofolate reductase (hDHFR) were simultaneously expressed by the B. boviselongation factor-1α bidirectional promoter, and a single guide RNA was expressed via the B. bovisU6 spliceosomal RNA promoter. Using a single plasmid construct, we were able to add an epitope tag to spherical body protein 3 (SBP3), introduce a point mutation into thioredoxin peroxidase 1 (tpx-1) to impair the function of the product, and replace the tpx-1 open reading frame with the other protein. Epitope tagging of SBP3 was efficient using this system, with a negligible number of remaining wild-type parasites and a pure transgenic population produced by allelic replacement of tpx-1 This advancement in genetic engineering tools for B. bovis will aid functional analysis of the genome and underpin characterization of candidate drug and vaccine targets.IMPORTANCEBabesia bovis is the most virulent cause of bovine babesiosis worldwide. The disease consequences are death, abortion, and economical loss due to reduced milk and meat production. Available vaccines are not effective, treatment options are limited, and emergence of drug and acaricide resistance has been reported from different regions. There is an urgent need to identify new drug and vaccine targets. Greater than half of the genes in B. bovis genome, including several expanded gene families which are unique for Babesia spp., have no predicted function. The available genetic engineering tools are based on conventional homologous recombination, which is time-consuming and inefficient. In this study, we adapted the CRISPR/Cas9 system as a robust genetic engineering tool for B. bovis This advancement will aid future functional studies of uncharacterized genes.
Highlights
Introduction of a point mutation intoB. bovis tpx-1 that impairs its function
The B. bovis U6 spliceosomal RNA promoter drove the expression of the single guide RNA (sgRNA), which contained a 20-nucleotide guide RNA that targets a region at the 3= end of B. bovis sbp3
Genome modification using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system does not require the insertion of a selection marker into the genome, allowing the possibility of targeting an unlimited number of genes in a single parasite
Summary
Introduction of a point mutation intoB. bovis tpx-1 that impairs its function. Tpx-1 is a cytoplasmic antioxidant enzyme peroxiredoxin with an essential peroxidatic Cys at its catalytic site functioning to reduce the peroxide substrate [21,22,23]. To evaluate the CRISPR/Cas system for making a point mutation in a target gene, without integrating a selectable marker, we made the BbU6-Cas9-hDHFR-tpx-1-mutant plasmid with donor DNA from a tpx-1 orf containing a mutation to change the peroxidatic Cys (Cys49) to Ser (Fig. 2A). The catalytic active site of B. bovis tpx-1, the peroxidatic Cys, was changed to Ser by transfecting parasites with the BbU6-Cas9-hDHFR-tpx-1-mutant plasmid. To confirm the expression of Cas, blood smears were immunostained with mouse anti-FLAG monoclonal antibody (M20008; Abmart, Shanghai, China) at 1:100 dilutions in PBS with 0.05% Tween 20 (T-PBS) and incubated at 37°C for 60 min. After 3 washes with T-PBS, the blood smears were incubated with Alexa Fluor 488-conjugated secondary goat anti-mouse IgG antibody (Invitrogen) (1:500 dilutions) at 37°C for 30 min. After 3 washes with T-PBS, the smears were examined under a Nikon A1R confocal laser scanning microscope (Nikon, Tokyo, Japan)
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