ArticleScientific reports2017
MISSA 2.0: an updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems.
Article in Scientific reports, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed.
- The Tomato Transcription Factor SlNAC063 Is Required for Aluminum Tolerance by RegulatingFrontiers in plant science · 2022Article
- Efficient Gene Stacking in Rice Using the GAANTRY System.Rice (New York, N.Y.) · 2021Article
- Gene Assembly in Agrobacterium via Nucleic Acid Transfer Using Recombinase Technology (GAANTRY).Methods in molecular biology (Clifton, N.J.) · 2021Article
- Comparative Physiological and Transcriptomic Analyses Reveal Altered Fe-Deficiency Responses in Tomato EpimutantFrontiers in plant science · 2021Article
- A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR/Cas Delivery in Maize.Plant physiology · 2019Article
- Harnessing Genome Editing Techniques to Engineer Disease Resistance in Plants.Frontiers in plant science · 2019Review
- Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.Plant molecular biology · 2018Article
- Plant genome editing with TALEN and CRISPR.Cell & bioscience · 2017Review
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Authors and funding
10 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Efficient generation of plants carrying mutations in multiple genes remains a challenge. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations, but assembly of these systems requires a robust, high-capacity toolkit. Here, we describe MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), an extensively updated toolkit for assembly of two or more CRISPR/Cas systems. We developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. We validated the utility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, and by creating transgenic Arabidopsis thaliana that constitutively or inducibly overexpress multiple genes. We then demonstrated that the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors facilitated the assembly of two orthogonal CRISPR/Cas systems including SpCas9 and SaCas9, and thus facilitated the creation of transgenic lines harboring these systems. We anticipate that MISSA 2.0 will enable substantial advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems, as well as in plant synthetic biology.
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