Optimization Of Plant Synthesis Biology Toolbox And Its Applications In Genome Editing

Many important traits,complex regulatory networks and metabolic pathways in plants are often the result of multiple gene interactions,so assembly of multiple gene cassettes onto a binary vector and then introduction into plants is important for both basic theory and applied research.The establishment of efficient and standardized DNA assembly system has been one of the goals in the field of synthetic biology.Good DNA assembly systems must have highly standardized interfaces,very simple assembly rules,and excellent compatibility.Although CRISPR/Cas has been widely used,the efficient production of plant polygenic mutants remains a challenge.Theoretically,multiple sgRNAs can be used to obtain polygenic mutants,but too much sgRNAs dilute the Cas9 protein,which affects the efficiency of the target recognition and cleavage.Thus,the use of two or more orthogonal CRISPR/Cas systems provides a new strategy for addressing this challenge.The orthogonal CRISPR/Cas9 system refers to two or more CRISPR/Cas9 systems that do not interfere with each other in the same cell for gene editing.However,fast and efficient assembly of two or more CRISPR/Cas systems faces a new challenge.In this paper,the MISSA 2.0/3.0(Multiple-round in vivo site-specific assembly 2.0/3.0)system has been established to lay the foundation for solving the challenges of genome editing.MISSA 2.0 is an upgraded version of the MISSA system.The MISSA 2.0 donor system contains a new suicide donor vector system based on the plasmid RK2,which has a higher cloning capacity(>300 kb)than the original R6K plasmid system.The new donor vector system is compatible with two sets of transfer systems from the plasmids F and RK2.In MISSA 2.0,this study constructed a new,reproducible receptor vector that is compatible with Agrobacterium-mediated transformation.While also modifying the chromosomes of E.coli,making it a giant receptor vector.The new donor strains constructed in this study were used for the replication and conjugation of suicide donor vectors and the receptor strains for MISSA 2.0 recombinant reactions.The MISSA 2.0 system was validated by assembling multiple DNA fragments into the chromosomes of E.coli and by assembling multiple expression frames into the binary vector to obtain transgenic Arabidopsis that constitutively or induce multiple genes.The results show that the R6K vector can not be used to assemble two orthogonal CRISPR/Cas9 systems due to the limitation of capacity,and the RK2 donor vector can be successfully assembled with large capacity.In this study,SpCas9 and SaCas9 systems,which were driven by egg cell-specific promoters and which did not interfere with each other,and six sgRNAs were assembled onto the receptor vector by three rounds of MISSA 2.0.Subsequent mutations in transgenic plants were analyzed.The results show that the assembly of the carrier is correct.In order to further improve the efficiency of assembly,this study established MISSA 3.0.MISSA 3.0 technology can not only multiround assembly,and can be multisite assembly,each round can be assembled three fragments,greatly improving the assembly efficiency.The MISSA 3.0 was successfully assembled with four rounds of 12 fragments.Transgenic Arabidopsis analysis showed that the vector was correctly assembled.In summary,the MISSA 2.0 and 3.0 systems which established in this study contribute to the application of two or more orthogonal CRISPR/Cas9 systems in the field of multiple genome editing.