Pea early-browning virus -mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis
Name:
Pea early-browning-Manuscript.pdf
Size:
619.7Kb
Format:
PDF
Description:
Accepted Manuscript
Type
Article
KAUST Department
Biological and Environmental Sciences and Engineering (BESE) DivisionBioscience Program
Desert Agriculture Initiative
Plant Science Program
Laboratory for Genome Engineering, Division of Environmental and Biological Sciences and Engineering, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Saudi Arabia
Date
2017-10-17Metadata
Show full item recordAbstract
The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system has enabled efficient genome engineering in diverse plant species. However, delivery of genome engineering reagents, such as the single guide RNA (sgRNA), into plant cells remains challenging. Here, we report the engineering of Tobacco rattle virus (TRV) and Pea early browning virus (PEBV) to deliver one or multiple sgRNAs into Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants that overexpress a nuclear localization signal containing Cas9. Our data showed that TRV and PEBV can deliver sgRNAs into inoculated and systemic leaves, and this resulted in mutagenesis of the targeted genomic loci. Moreover, in N. benthamiana, PEBV-based sgRNA delivery resulted in more targeted mutations than TRV-based delivery. Our data indicate that TRV and PEBV can facilitate plant genome engineering and can be used to produce targeted mutations for functional analysis and other biotechnological applications across diverse plant species.Key message: Delivery of genome engineering reagents into plant cells is challenging and inefficient and this limit the applications of this technology in many plant species. RNA viruses such as TRV and PEBV provide an efficient tool to systemically deliver sgRNAs for targeted genome modification.Citation
Ali Z, Eid A, Ali S, Mahfouz MM (2017) Pea early-browning virus -mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis. Virus Research. Available: http://dx.doi.org/10.1016/j.virusres.2017.10.009.Sponsors
We would like to thank member of the laboratory for genome engineering for continuous discussions. We would like to thank Professor Elisabeth Johansen, Danish Institute for food and veterinary research, for providing PEBV-containing binary constructs. This study is supported by King Abdullah University of Science and Technology (KAUST).Publisher
Elsevier BVJournal
Virus ResearchISSN
0168-1702PubMed ID
29051052Handle URI
http://hdl.handle.net/10754/626046Additional Links
http://www.sciencedirect.com/science/article/pii/S0168170217305543Scopus Count
Related items
Showing items related by title, author, creator and subject.
-
Antiviral lead compounds from marine sponges(MDPI AG, 2010-10-11)Marine sponges are currently one of the richest sources of pharmacologically active compounds found in the marine environment. These bioactive molecules are often secondary metabolites, whose main function is to enable and/or modulate cellular communication and defense. They are usually produced by functional enzyme clusters in sponges and/or their associated symbiotic microorganisms. Natural product lead compounds from sponges have often been found to be promising pharmaceutical agents. Several of them have successfully been approved as antiviral agents for clinical use or have been advanced to the late stages of clinical trials. Most of these drugs are used for the treatment of human immunodeficiency virus (HIV) and herpes simplex virus (HSV). The most important antiviral lead of marine origin reported thus far is nucleoside Ara-A (vidarabine) isolated from sponge Tethya crypta. It inhibits viral DNA polymerase and DNA synthesis of herpes, vaccinica and varicella zoster viruses. However due to the discovery of new types of viruses and emergence of drug resistant strains, it is necessary to develop new antiviral lead compounds continuously. Several sponge derived antiviral lead compounds which are hopedto be developed as future drugs are discussed in this review. Supply problems are usually the major bottleneck to the development of these compounds as drugs during clinical trials. However advances in the field of metagenomics and high throughput microbial cultivation has raised the possibility that these techniques could lead to the cost-effective large scale production of such compounds. Perspectives on biotechnological methods with respect to marine drug development are also discussed. 2010 by the authors; licensee MDPI.
-
Viral metagenomics: Analysis of begomoviruses by illumina high-throughput sequencing(MDPI AG, 2014-03-12)Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes) (genus, Begomovirus; family, Geminiviridae) were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA). Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS). CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions. 2014 by the authors; licensee MDPI, Basel, Switzerland.
-
CRISPR/Cas9-mediated viral interference in plants(Springer Nature, 2015-11-11)Background The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.
