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Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system.

ABSTRACT: Precise genetic modifications in model animals are essential for biomedical research. Here, we report a programmable "base editing" system to induce precise base conversion with high efficiency in zebrafish. Using cytidine deaminase fused to Cas9 nickase, up to 28% of site-specific single-base mutations are achieved in multiple gene loci. In addition, an engineered Cas9-VQR variant with 5'-NGA PAM specificities is used to induce base conversion in zebrafish. This shows that Cas9 variants can be used to expand the utility of this technology. Collectively, the targeted base editing system represents a strategy for precise and effective genome editing in zebrafish.The use of base editing enables precise genetic modifications in model animals. Here the authors show high efficient single-base editing in zebrafish using modified Cas9 and its VQR variant with an altered PAM specificity.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC5524635 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system.

Zhang Yihan Y Qin Wei W Lu Xiaochan X Xu Jason J Huang Haigen H Bai Haipeng H Li Song S Lin Shuo S

Nature communications 20170725 1

Precise genetic modifications in model animals are essential for biomedical research. Here, we report a programmable "base editing" system to induce precise base conversion with high efficiency in zebrafish. Using cytidine deaminase fused to Cas9 nickase, up to 28% of site-specific single-base mutations are achieved in multiple gene loci. In addition, an engineered Cas9-VQR variant with 5'-NGA PAM specificities is used to induce base conversion in zebrafish. This shows that Cas9 variants can be ...[more]

PMID: 28740134

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Project description: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.

Project description:Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites. Importance: Malaria, caused by infection of Plasmodium parasites, remains a world-wide public health burden. Although the genomes of many malaria parasites have been sequenced, we still do not know the functions of approximately half of the genes in the genomes. Studying gene function has become the focus of many studies; however, editing genes in malaria parasite genomes is still inefficient. Here we designed several efficient approaches, based on the CRISPR/Cas9 system, to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. Using this system, we achieved high efficiencies in gene deletion, reporter tagging, and allelic replacement in multiple parasite genes. This technique for editing the malaria parasite genome will greatly facilitate our ability to elucidate gene function.

Dataset Information

Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system.

Publications

Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system.

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