MISSA 2.0: an updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems

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...

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Bibliographic Details
Published in:Scientific reports 2017-02, Vol.7 (1), p.41993, Article 41993
Main Authors: Zhang, Hai-Yan, Wang, Xing-Hui, Dong, Li, Wang, Zhi-Ping, Liu, Bing, Lv, Jie, Xing, Hui-Li, Han, Chun-Yan, Wang, Xue-Chen, Chen, Qi-Jun
Format: Article
Language:English
Subjects:
38/61
42/41
42/70
631/449/447/2311
631/61/447
Arabidopsis - genetics
Cloning
Cloning vectors
CRISPR
CRISPR-Cas Systems
Deoxyribonucleic acid
DNA
E coli
Escherichia coli
Genetic Engineering - methods
Genetic Vectors - genetics
Genome editing
Genomes
Humanities and Social Sciences
multidisciplinary
Mutation
Plants, Genetically Modified - genetics
Science
Science (multidisciplinary)
Suicide
Synthetic biology
Synthetic Biology - methods
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep41993