InViDA: Exploiting Cas12a for scarless conjugation-based in vivo DNA assembly technology
Lead Research Organisation:
Rothamsted Research
Department Name: Plant Sciences and the Bioeconomy
Abstract
This proposal aims to revolutionise the technology of DNA assembly, the process joining multiple fragments of DNA together. Construction of large artificial DNA using standard biological parts as "Lego- blocks" is one of the main challenges in synthetic biology, metabolic engineering and engineering of synthetic genomes. Approaches need to be quick, easy, cheaper, and more environmentally friendly. Current state-of-the-art technologies, such as multilevel golden gate assembly (GGA), require frequent switching between manipulations with DNA (in vitro) and the microbial host (in vivo). However, every transition in each direction, i.e., DNA extraction (in-vivo to in-vitro) or DNA transformation (in-vitro to in-vivo), requires an extra experimental day. Such transitions constitute a considerable part of typical projects, which inevitably delays progress.
Here, we propose transformative research to develop the most efficient DNA assembly technology, based solely on in vivo procedures. Standard biological parts can be distributed and stored as a collection of microbial strains. Assemblies themselves will be based on DNA transfer between bacterial strains, which should be accompanied by recombination inside the cells. Then, cell-to-cell transfers can deliver assembled constructs into a final host, including plants.
So far, only two techniques enable multiple step assemblies without DNA isolations. Multiple-round In vivo Site-Specific Assembly is based on site-specific recombination. However, it can affect gene functions. Moreover, it cannot be used in a convergent pairwise assembly scheme, which is the only efficient way to bring together high numbers of fragments by cell-to-cell transfer. On the contrary, Conjugative Assembly Genome Engineering is based on homologous recombination and requires long arms between joined parts; thus, it can be used for assemblies in strictly pre-designed order and therefore cannot be used for standard biological parts.
To overcome drawbacks of current systems we propose a new recombination mechanism. Similar to GGA, it will be based on the simultaneous digestions and ligations, but will operate in vivo and utilize the RNA-guided endonuclease Cas12a. To prove the concept, we will build a bacterial strain, able in a programmable manner, to remove a particular DNA part from a tester plasmid. Such a strain will carry out digestions and ligations in a programmable and precise manner, while its genetic background will suppress degradation of linear DNA. Using this strain, we will demonstrate assembly of DNA parts that will be brought together by transfer. Therefore, we will functionally check all the key elements required to revolutionise DNA assembly technology in the near future.
Here, we propose transformative research to develop the most efficient DNA assembly technology, based solely on in vivo procedures. Standard biological parts can be distributed and stored as a collection of microbial strains. Assemblies themselves will be based on DNA transfer between bacterial strains, which should be accompanied by recombination inside the cells. Then, cell-to-cell transfers can deliver assembled constructs into a final host, including plants.
So far, only two techniques enable multiple step assemblies without DNA isolations. Multiple-round In vivo Site-Specific Assembly is based on site-specific recombination. However, it can affect gene functions. Moreover, it cannot be used in a convergent pairwise assembly scheme, which is the only efficient way to bring together high numbers of fragments by cell-to-cell transfer. On the contrary, Conjugative Assembly Genome Engineering is based on homologous recombination and requires long arms between joined parts; thus, it can be used for assemblies in strictly pre-designed order and therefore cannot be used for standard biological parts.
To overcome drawbacks of current systems we propose a new recombination mechanism. Similar to GGA, it will be based on the simultaneous digestions and ligations, but will operate in vivo and utilize the RNA-guided endonuclease Cas12a. To prove the concept, we will build a bacterial strain, able in a programmable manner, to remove a particular DNA part from a tester plasmid. Such a strain will carry out digestions and ligations in a programmable and precise manner, while its genetic background will suppress degradation of linear DNA. Using this strain, we will demonstrate assembly of DNA parts that will be brought together by transfer. Therefore, we will functionally check all the key elements required to revolutionise DNA assembly technology in the near future.
