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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American Society for Microbiology
2019
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oai:doaj.org-article:237770be9d024798a3040e3b75f300f22021-11-15T15:22:20ZGenome Editing of <italic toggle="yes">Babesia bovis</italic> Using the CRISPR/Cas9 System10.1128/mSphere.00109-192379-5042https://doaj.org/article/237770be9d024798a3040e3b75f300f22019-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00109-19https://doaj.org/toc/2379-5042ABSTRACT 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.Hassan HakimiTakahiro IshizakiYuto KegawaOsamu KanekoShin-ichiro KawazuMasahito AsadaAmerican Society for MicrobiologyarticleBabesia bovisCRISPR/Cas9genome editingthioredoxin peroxidaseMicrobiologyQR1-502ENmSphere, Vol 4, Iss 3 (2019) |
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Babesia bovis CRISPR/Cas9 genome editing thioredoxin peroxidase Microbiology QR1-502 |
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Babesia bovis CRISPR/Cas9 genome editing thioredoxin peroxidase Microbiology QR1-502 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 |
| 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. |
| format |
article |
| 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 |
| title |
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_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_sort |
genome editing of <italic toggle="yes">babesia bovis</italic> using the crispr/cas9 system |
| publisher |
American Society for Microbiology |
| publishDate |
2019 |
| url |
https://doaj.org/article/237770be9d024798a3040e3b75f300f2 |
| work_keys_str_mv |
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| _version_ |
1718427998184538112 |