Researchers Develop MISSA 2.0 for Assembly of Orthogonal CRISPR/Cas Systems

Efficient generation of plants carrying mutations in multiple genes remains a challenge to this day. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations. However, assembly of these systems requires a robust, high-capacity toolkit. Researchers from the China Agricultural University developed MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), a toolkit for assembly of two or more CRISPR/Cas systems.

The team developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. They then tested the feasibility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, as well as by developing a transgenic Arabidopsis thaliana that overexpress the assembled multiple genes.

The study also demonstrated the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors helped assemble two orthogonal CRISPR/Cas systems, aiding the development of transgenic lines harboring these systems.

MISSA 2.0 is expected to enable advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems.

For more information on this promising technology, read the article in Nature.