• CRISPR/Cas9
• genomic engineering
• marchantia polymorpha
• plant synthetic biology
• synthetic biology

Plant synthetic biology is an emerging and promising field in plant biology. The aim of synthetic biology is to apply engineering principles to plants in order to design new devices or functions in living organism. One of the goals of synthetic biology is creating a standard library of parts that can be shared between different organisms and work properly, without needing any optimization process. To achieve this purpose, devices should be tested and characterized in “chassis” organism, i.e. a common model that is used in the whole community. To date Arabidopsis thaliana has been the most used model in plant molecular biology, but it’s still a quite complex organism: it is diploid, needs about 6-8 weeks to complete its life cycle and it has a 135 Mbp genome. Marhantia polymorpha, instead, is a liverwort, the basal-most lineage of land plants, it has a 3-weeks haplodiplontic life cycle with a haploid predominant phase, a low gene redundancy and lots of these genes share sequence homology with other plant models. More-over it is easy to propagate vegetatively and protocols for its efficient transformation already exist. All the above mentioned characteristics make M. polymorpha a suitable chassis for plant synthetic biology, avoiding all the heterozygosity and multiple gene copies-related issues that are present in A.thaliana. Nevertheless a deeper understanding of M.polymorpha molecular biology is needed.
The aim of this thesis is to develop a tool to perform large deletion in Marchantia polymorpha genome. Using two different gRNAs, targeting two sequences far apart one from each other and a plant codon optimized Cas9, we want to delete up to 200kb fragments in the genome. We designed 13 vectors to perform deletions of increasing size around the Nop1 gene locus to validate the tool. Mutants for NOPPERABO 1 is a E3-ubiquitin ligase involved in air chambers formation and its mutant, nop1, show a clear phenotype that is easy to screen on plate and this result can be confirmed with PCR. The vectors were built using Gibson assembly and all the transcriptional units were included in between 40bp sequences optimized for homology-based cloning and reliable sequencing.
The validation of this genome-engineering tool will allow to perform large scale deletion from genomes. It can be used to delete entire biosynthetic pathways and re-introduce new ones, study the function of large sequences that cannot be targeted with other methodologies. It can be also exploit to build minimal-chromosome sequences in plant. Indeed, the future application of this project would involve the use of this tool to delete as much DNA sequence as possible from Marchantia polymorpha Y chromosome: the smallest and better annotated one nowadays.