De novo custom DNA synthesis using prime editing

背景脱氧核糖核酸（DNA）是一种被所有生物普遍使用的储存信息的线状分子。类似于计算机如何将信息编码成比特序列，它可以取两个值（0或1），DNA将信息编码成碱基序列，它可以取四个值（A，G，T或C）。储存在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。合成生物学领域旨在设计生物系统以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，DNA信息存储，旨在使用信息密集，持久的DNA来存储数据。这两个领域都强烈依赖于廉价而有效的定制DNA序列合成。在过去的30年里，这是通过一种称为亚磷酰胺合成的化学过程实现的。这种方法效率很高，但只用于合成非常短的DNA序列（少于200个碱基），并产生化学废物。如果需要更长的DNA序列，它们必须从较短的DNA序列组装，这大大增加了成本，有时是不可行的。在过去的十年中，一种称为末端脱氧核苷酸转移酶（TdT）的酶被提出作为亚磷酰胺化学的更绿色，更有效的替代品。Molecular Assemblies和DNA script这两家公司的TdT合成技术已经达到了商业化的程度，但是他们的技术仍然局限于短DNA序列，这并没有解决亚磷酰胺合成的主要问题。目的和相关性我的项目的中心目标是开发一种能够合成长DNA分子的新的DNA合成方法。我计划使用prime editing来实现这一目标，prime editing是一种强大的技术，可以在任何选择的DNA位置上删除，添加或交换一些DNA碱基，具有高度特异性。使用光遗传学（光定向）控制，prime editing可以指示哪些碱基以循环方式添加到序列中。这个过程的每次迭代都应该包括一个检查点，以确保已经添加了基础。在这一过程的最后，细菌可以廉价地生长，在自己体内复制最终测序的DNA，从而获得高滴度。如果成功，这项新技术将大大加快合成生物学和其他相关领域的发展，这些领域受到当前DNA合成方法的成本和局限性的阻碍。这使得我的项目属于EPSRC“合成生物学”研究领域的范围。由于其在DNA信息存储领域的相关性，这一领域完全依赖于廉价的定制DNA的可用性，该项目也与“生物信息学”研究领域有关。

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.