Repositorium
MISSA 2.0: An updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems
257
Journal Article / 2017
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
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.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
603 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 ABI1 |
ABA and glucose insensitivity SDN1 |
Basic research Basic research |
604 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 ABI2 |
curled leafes SDN1 |
Basic research Basic research |
605 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 HAB1 |
curled leafes SDN1 |
Basic research Basic research |
606 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 TRY |
curled leafes SDN1 |
Basic research Basic research |
607 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 CPC |
curled leafes SDN1 |
Basic research Basic research |