De novo custom DNA synthesis using prime editing

脱氧核糖核酸（DNA）是所有生物普遍使用的一种储存信息的线状分子。类似于计算机以比特序列编码信息，比特序列可以有两个值（0或1），DNA以碱基序列编码信息，碱基序列可以有四个值（a、G、T或C）。存储在DNA中的信息被用作制造蛋白质和酶的蓝图——这些复杂的分子机器指导细胞内的所有过程。合成生物学领域的目标是设计生物系统来执行有用的医疗或工业功能。这只能通过重新设计和重新排列DNA来实现，从而改变细胞产生的蛋白质的功能或调节。另一个领域是DNA信息存储，旨在利用信息密集、持久的DNA来存储数据。这两个领域都强烈依赖于廉价和高效的定制DNA序列合成。在过去的30年里，这是通过一种被称为酰胺磷合成的化学过程实现的。这种方法效率很高，但只能合成非常短的DNA序列（少于200个碱基），而且会产生化学废物。如果需要更长的DNA序列，它们必须由较短的DNA序列组装而成，这大大增加了成本，而且有时是不可行的。在过去的十年里，一种被称为末端脱氧核苷酸转移酶（TdT）的酶被认为是一种更环保、更有效的磷酰胺化学替代品。两家公司，Molecular Assemblies和DNA script，已经将TdT合成技术推向商业化。然而，他们的技术仍然局限于短DNA序列，这并不能解决磷酸酰胺合成的主要问题。目的和相关性我项目的中心目标是开发一种新的DNA合成方法，能够合成长DNA分子。我计划使用启动编辑来实现这一目标，这是一种强大的技术，可以在任何选择的DNA位置以高特异性去除，添加或交换一些DNA碱基。利用光遗传（光定向）控制，可以指示引物编辑以周期性的方式将哪些碱基添加到序列中。该过程的每次迭代都应该包含一个检查点，以确保已经添加了基础。在这一过程的最后，细菌可以被低成本地培养，在自身内部复制最终测序的DNA，从而获得高滴度。如果成功，这项新技术将大大加速合成生物学和其他相关领域的发展，这些领域受到当前DNA合成方法的成本和限制的阻碍。这使得我的项目属于EPSRC“合成生物学”研究领域的范围。由于它与DNA信息存储相关，这一领域完全依赖于廉价定制DNA的可用性，因此该项目也与“生物信息学”研究领域相关。

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.