De novo custom DNA synthesis using prime editing

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BackgroundDeoxyribonucleic acid (DNA) is a string-like molecule used universally by all living beings to store information. Analogous to how a computer encodes information in a sequence of bits, which can take two values (0 or 1), DNA encodes information in a sequenc8e of bases, which can take four values (A, G, T, or C). The information stored in DNA is used as a blueprint to create proteins and enzymes - complex molecular machines which direct all processes inside a cell.The field of synthetic biology aims to engineer biological systems to perform useful medical or industrial functions. This can only be achieved by re-designing and re-arranging DNA, thus altering the function or regulation of the proteins created by a cell. Another field, DNA information storage, aims to use the information-dense, long-lasting DNA to store data. Both areas are strongly dependent on cheap and efficient synthesis of custom DNA sequences.For the last 30 years, this was achieved through a chemical process known as phosphoramidite synthesis. This method is highly efficient but only for the synthesis of very short DNA sequences (less than 200 bases) and produces chemical waste. If longer DNA sequences are desired, they must be assembled from the shorter ones, which greatly increases costs and is sometimes unfeasible.In the last decade, an enzyme called Terminal deoxynucleotidyl transferase, or TdT, has been proposed as a greener, more efficient alternative to phosphoramidite chemistry. Two companies, Molecular Assemblies and DNA script, have advanced TdT synthesis enough to commercialise it. However, their technology is still limited to short DNA sequences, which does not solve the main caveat of phosphoramidite synthesis.Aims and relevanceThe central goal of my project is to develop a new DNA synthesis method capable of synthesising long DNA molecules. I plan to achieve this using prime editing, a powerful technology which allows to remove, add, or exchange a few DNA bases at any chosen DNA location with high specificity.Using optogenetic (light directed) control, the prime editor could be instructed which bases to add to the sequence in a cyclical manner. Each iteration of this process should include a checkpoint to ensure that the base has been added. At the end of the process, the bacteria can be inexpensively grown to replicate inside themselves the finalised DNA sequenced, allowing to obtain high titres.If successful, this new technology would significantly accelerate the development of synthetic biology and other related fields hampered by the costs and limitations of current DNA synthesis methods. This makes my project fall within the scope of the EPSRC "Synthetic Biology" research area. Due to its relevance in DNA information storage, a field entirely dependent on the availability of cheap custom DNA, this project is also related to the "Biological Informatics" research area.