Genome Editing of Babesia bovis Using the CRISPR/Cas9 System

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. bovis elongation factor-1α bidirectional promoter, and a single guide RNA was expressed via the B. bovis U6 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. IMPORTANCE Babesia 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.

Novel Babesia bovis exported proteins that modify properties of infected red blood cells

“…The CRISPR/Cas9 system was employed to delete BBOV_III004280 or insert myc and glmS sequences into the 3' end of the original locus [56]. Briefly, homologous regions (HRs) were PCR-amplified from B. bovis genomic DNA and inserted into the BamHI site of the BbU6-Cas9-hDHFR plasmid.…”

Experimental Infection of Calves with Transfected Attenuated Babesia bovis Expressing the Rhipicephalus microplus Bm86 Antigen and eGFP Marker: Preliminary Studies towards a Dual Anti-Tick/Babesia Vaccine

“…As ticks and tick-borne parasitic diseases become emerging concerns for humans and domestic and wild animals, the development of integrated strategies to control simultaneously ticks and tick-borne parasites are urgently needed. Genetically modified parasites produced by gene manipulation methods have been used to develop novel next generation strategies aimed at controlling vector-borne diseases [ 33 , 34 , 35 ]. Furthermore, a recent study demonstrated stable incorporation of heterologous genes into the B. bovis genome and expression of foreign proteins on the surface of parasite merozoites [ 36 ].…”

Bovine Babesiosis in Turkey: Impact, Current Gaps, and Opportunities for Intervention

“…On the other hand, state-of-the-art technologies, such as next-generation and nanopore sequencing approaches, can help our understanding of the composition of local strains, population dynamics of the parasites, and help identify virulence factors that can be used for developing novel vaccines. These techniques combined with gene manipulation techniques already available for the transfection of Babesia parasites [ 156 , 159 ] may also lead to better defined and non-tick transmissible live vaccines incorporating DIVA markers to differentiate vaccinated from naturally-infected animals. These DIVA vaccines are much needed in Turkey, especially considering the importation of a large number of live cattle from countries that are endemic for bovine babesiosis.…”