Heteroduplex thermostable ligation assembly: a new platform to rapidly generate large DNA molecules

DNA片段的体外组装最近被认为是写作的关键技术挑战 合成基因组。迎接这一挑战将提高基因组编写项目的效率，这些项目依赖于 在大的合成DNA上，这些DNA被迭代重组，以逐步取代染色体区域 部分或全部重写细菌或酵母染色体。这个项目的长期目标是发展 一种高效、低成本的策略来生成完全合成的大DNA，从50 kb到1 MBP，而不需要 酵母或细菌中的同源重组。这里提出的工作的目标是优化 这种新方法的参数我们称之为异源双链耐热连接酶组件(HTLA)来产生 大的DNA完全在体外。这项工作的基本原理是，成功完成将提供以下证据： 异源双链克隆可用于以低成本、可商业化的方式快速生成大DNA的概念 举止。为了在预算内实现这些目标，聚合酶链式反应产生了噬菌体lambda基因组片段 将被用作从头合成DNA的范例。生成大型DNA的两个HTLA战略将是 通过实现三个相关但独立的具体目标而系统地探讨：1)确定 生成具有连接就绪粘性末端的~2kb片段用于大分子组装的最佳HTLA条件 DNA构建。单循环HTLA反应条件加入1kb的Lambda基因组部分产生~2kb的粘性 将系统地改变端块(SEB)以最大限度地提高产量。DNA部分重叠长度也将得到优化。 然后改变SEBS上的粘端长度，以实现与T4或9°N™DNA连接酶的最佳连接。SEBS将 越来越多地被连接，以确定最终产物长度的实际极限。序列保真度 将确定组装的序列；2)确定~1kb异源双链DNA的最大数量 可通过HTLA以高序列保真度以一锅一步的方法重复连接的分子 和环状异双链耐热连接组件(CHTLA)。1kb的lambda基因组片段在增加 将使用HTLA将数字连接起来，以确定SEB生成的上限，其产量足以 后续步骤。将确定DNA部分重叠长度(100-500)对反应效率的影响。10- 循环CHTLA反应也将随着1kb的Lambda基因组部分的增加而进行 CHTLA可以生成的最终产品长度的上限。将确定序列的保真度；3) 使用AIMS 1和/或2中确定的优化异双链克隆策略无缝生成完整的 Lambda噬菌体基因组可以被包装成具有感染性的颗粒。使用中确定的优化条件 目的1和2，将产生一个完全组装的lambda基因组，包装并用于感染大肠杆菌。 成功完成此第一阶段项目将展示HTLA和/或CHTLA的效用，以构建 体外有功能的~50kb病毒基因组。在第2阶段，我们将扩展异源双工克隆的实用程序以生成 长度为100 kb至1 MBP的DNA，服务于价值16.2亿美元的基因组工程和编辑市场(CAGR，24.4%)。

In vitro assembly of DNA fragments >20 kb has recently identified as a critical technical challenge for writing synthetic genomes. Meeting this challenge will increase the efficiency of genome writing projects that depend on large synthetic DNAs, which are iteratively recombined to replace chromosome regions in a step-wise manner to partially or completely re-write bacterial or yeast chromosomes. The long-term goal of this project is to develop an efficient, low-cost strategy to generate completely synthetic large DNAs, from >50 kb up to 1 Mbp, without homologous recombination in yeast or bacteria. The objective of the work proposed here is to optimize the parameters of this novel approach we term Heteroduplex Thermostable Ligase Assembly (HTLA) to generate large DNAs completely in vitro. The rationale for this work is that successful completion will provide proof-of- concept that heteroduplex cloning can be used to rapidly generate large DNAs in a low-cost, commercializable manner. To accomplish these goals within the budget, PCR-generated bacteriophage lambda genome fragments will be used as a paradigm for de novo synthesized DNAs. Two HTLA strategies to generate large DNAs will be systematically explored by achieving the goals of three related but independent Specific Aims: 1) To determine optimal HTLA conditions to generate ~2 kb fragments with ligation-ready sticky ends for assembly of a large DNA construct. One-cycle HTLA reaction conditions to join 1 kb lambda genome parts to generate ~2 kb sticky end blocks (SEBs) will be systematically varied to maximize yield. DNA part overlap length will also be optimized. Sticky end length on SEBs will then be varied to achieve optimal ligation with T4 or 9°N™ DNA ligase. SEBs will be ligated in increasing numbers to determine the practical limit of final product length. Sequence fidelity of assembled sequences will be determined; 2) To determine the maximum number of ~1 kb heteroduplex DNA molecules that can be reproducibly joined in a one-pot, one-step method with high sequence fidelity by HTLA and Cyclic Heteroduplex Thermostable Ligation Assembly (CHTLA). One kb lambda genome parts in increasing numbers will be joined using HTLA to determine the upper size limit for SEB generation with a yield sufficient for subsequent steps. The effect of DNA part overlap length (100-500) on reaction efficiency will be determined. 10- cycle CHTLA reactions will also be performed with increasing numbers of 1 kb lambda genome parts to probe the upper limit on final product length that can be generated by CHTLA. Sequence fidelity will be determined; 3) To use optimized heteroduplex cloning strategies identified in Aims 1 and/or 2 to seamlessly generate a complete lambda phage genome that can be packaged into infectious particles. Using optimized conditions determined in Aim 1 and 2, a completely assembled lambda genome will be generated, packaged, and used to infect E. coli. Successful completion of this Phase 1 project will demonstrate the utility of HTLA and/or CHTLA to build a functional ~50 kb viral genome in vitro. In Phase 2, we will extend the utility of heteroduplex cloning to generate DNAs 100 kb to >1 Mbp in length to service the $1.62B Genome Engineering and Editing Market (CAGR, 24.4%).