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.
Highlights
Background strainP254D P254D BW20767 P254D BW20767CTPa — F′DOT-TE chromosome F′DOT-TE chromosomeEngineered strainb P254DP254D-TE BW20767 P254D-TE BW20767Donor vector pLC2-GOI et al.— — pLC2-GOI et al pLC2-GOI et al.Donor strainc — P254D-TE BW20767 TEC-pLCM2-GOI et al SC-pLCM2-GOI et al
DNA assembly occurs at different levels, from parts to TUs, TUs to pathways, and pathways to networks and beyond; the quick and reliable assembly of standardized DNA parts into pathways and beyond can facilitate advances in synthetic biology[14,15,16]
MISSA is very suitable for the assembly of higher-order genetic units (HOGUs), in that it leaves only minor, harmless scar sequences between HOGUs
Summary
Background strainP254D P254D BW20767 P254D BW20767CTPa — F′DOT-TE chromosome F′DOT-TE chromosomeEngineered strainb P254DP254D-TE BW20767 P254D-TE BW20767Donor vector pLC2-GOI et al.— — pLC2-GOI et al pLC2-GOI et al.Donor strainc — P254D-TE BW20767 TEC-pLCM2-GOI et al SC-pLCM2-GOI et al. — — pLC2-GOI et al pLC2-GOI et al. Donor strainc — P254D-TE BW20767 TEC-pLCM2-GOI et al SC-pLCM2-GOI et al. DH10B EPI300 DH10B EPI300 DH10B-RV DH10B-RV. SRPa chromosome pAH57-Cre-TF chromosome pAH57-Cre-TF — pAH57-Cre-TF. Engineered strainb DH10B-SRP EPI300/SRP DH10B-SRP EPI300/SRP DH10B-RV DH10B-RV/SRP. Simplifying DNA assembly rules as far as possible is a key part of standardization efforts for synthetic biology. MISSA has a simple, main rule for DNA assembly: site-specific recombination reactions must occur between two loxP sites, between attL1 and attR1 sites, or between attL2 and attR2 sites. The final constructs can be plotted once the assembly order is determined (Fig. 3). Removal of the antibiotic resistance genes from the final recipient vectors can be achieved by two-step MISSA reactions in the final round of assembly (Fig. S2)
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