Dataset Information

Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing.

ABSTRACT: CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co-transferred plasmid backbone and low edit rate in wheat, a large T0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium-delivered CRISPR/Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR/Cas9 system, we have developed 68 edit mutants for four grain-regulatory genes, TaCKX2-1, TaGLW7, TaGW2, and TaGW8, in T0 , T1 , and T2 generation plants at an average edit rate of 10% without detecting off-target mutations in the most Cas9-active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160-bp deletion in TaCKX2-D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium-delivered CRISPR/Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR/Cas9 remains active for novel mutations through generations.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC6662106 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing.

Zhang Zhengzhi Z Hua Lei L Gupta Ajay A Tricoli David D Edwards Keith J KJ Yang Bing B Li Wanlong W

Plant biotechnology journal 20190312 8

CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co-transferred plasmid backbone and low edit rate in wheat, a larg ...[more]

PMID: 30706614

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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.

Dataset Information

Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing.

Publications

Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing.

Similar Datasets

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