Genome Editing of <italic toggle="yes">Babesia bovis</italic> 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 bread...

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Main Authors: Hassan Hakimi, Takahiro Ishizaki, Yuto Kegawa, Osamu Kaneko, Shin-ichiro Kawazu, Masahito Asada
Format: Article
Language:English
Published: American Society for Microbiology 2019-06-01
Series:mSphere
Subjects:
Online Access:https://journals.asm.org/doi/10.1128/mSphere.00109-19
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author Hassan Hakimi
Takahiro Ishizaki
Yuto Kegawa
Osamu Kaneko
Shin-ichiro Kawazu
Masahito Asada
author_facet Hassan Hakimi
Takahiro Ishizaki
Yuto Kegawa
Osamu Kaneko
Shin-ichiro Kawazu
Masahito Asada
author_sort Hassan Hakimi
collection DOAJ
description 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.
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spelling doaj.art-237770be9d024798a3040e3b75f300f22022-12-21T22:57:06ZengAmerican Society for MicrobiologymSphere2379-50422019-06-014310.1128/mSphere.00109-19Genome Editing of <italic toggle="yes">Babesia bovis</italic> Using the CRISPR/Cas9 SystemHassan Hakimi0Takahiro Ishizaki1Yuto Kegawa2Osamu Kaneko3Shin-ichiro Kawazu4Masahito Asada5Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, JapanDepartment of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, JapanDepartment of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, JapanDepartment of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, JapanNational Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, JapanDepartment of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, JapanABSTRACT 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.https://journals.asm.org/doi/10.1128/mSphere.00109-19Babesia bovisCRISPR/Cas9genome editingthioredoxin peroxidase
spellingShingle Hassan Hakimi
Takahiro Ishizaki
Yuto Kegawa
Osamu Kaneko
Shin-ichiro Kawazu
Masahito Asada
Genome Editing of <italic toggle="yes">Babesia bovis</italic> Using the CRISPR/Cas9 System
mSphere
Babesia bovis
CRISPR/Cas9
genome editing
thioredoxin peroxidase
title Genome Editing of <italic toggle="yes">Babesia bovis</italic> Using the CRISPR/Cas9 System
title_full Genome Editing of <italic toggle="yes">Babesia bovis</italic> Using the CRISPR/Cas9 System
title_fullStr Genome Editing of <italic toggle="yes">Babesia bovis</italic> Using the CRISPR/Cas9 System
title_full_unstemmed Genome Editing of <italic toggle="yes">Babesia bovis</italic> Using the CRISPR/Cas9 System
title_short Genome Editing of <italic toggle="yes">Babesia bovis</italic> Using the CRISPR/Cas9 System
title_sort genome editing of italic toggle yes babesia bovis italic using the crispr cas9 system
topic Babesia bovis
CRISPR/Cas9
genome editing
thioredoxin peroxidase
url https://journals.asm.org/doi/10.1128/mSphere.00109-19
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