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      Plant Virus Vectors 3.0: Transitioning into Synthetic Genomics

      1 , 2 , 1
      Annual Review of Phytopathology
      Annual Reviews

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          Abstract

          Plant viruses were first implemented as heterologous gene expression vectors more than three decades ago. Since then, the methodology for their use has varied, but we propose it was the merging of technologies with virology tools, which occurred in three defined steps discussed here, that has driven viral vector applications to date. The first was the advent of molecular biology and reverse genetics, which enabled the cloning and manipulation of viral genomes to express genes of interest (vectors 1.0). The second stems from the discovery of RNA silencing and the development of high-throughput sequencing technologies that allowed the convenient and widespread use of virus-induced gene silencing (vectors 2.0). Here, we briefly review the events that led to these applications, but this treatise mainly concentrates on the emerging versatility of gene-editing tools, which has enabled the emergence of virus-delivered genetic queries for functional genomics and virology (vectors 3.0).

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          Most cited references78

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          Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

          CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.
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            Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.

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              Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems.

              Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements.
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                Author and article information

                Journal
                Annual Review of Phytopathology
                Annu. Rev. Phytopathol.
                Annual Reviews
                0066-4286
                1545-2107
                August 25 2019
                August 25 2019
                : 57
                : 1
                : 211-230
                Affiliations
                [1 ]Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843, USA;
                [2 ]Shriram Center for Biological and Chemical Engineering, Stanford University, Stanford, California 94305, USA
                Article
                10.1146/annurev-phyto-082718-100301
                31185187
                3d948475-cfe1-4970-ab6d-c22b896a3497
                © 2019
                History

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