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

体外组装>20 kb的DNA片段最近被确定为书写的关键技术挑战 合成基因组迎接这一挑战将提高基因组编写项目的效率， 在大型合成DNA上，这些DNA以逐步的方式反复重组以替换染色体区域 部分或完全重写细菌或酵母的染色体。该项目的长期目标是开发 一种有效的、低成本的策略来产生完全合成的大DNA，从>50 kb到高达1 Mbp， 在酵母或细菌中同源重组。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热稳定连接酶组装（HTLA），以产生 大的DNA完全在体外。这项工作的基本原理是，成功完成将提供证明- 异源双链克隆可用于以低成本、可商业化的方法快速产生大的DNA。 方式为了在预算范围内实现这些目标，PCR产生的λ噬菌体基因组片段 将被用作从头合成DNA的范例。两个HTLA策略，以产生大的DNA将 通过实现三个相关但独立的具体目标来系统地探索：1）确定 最佳的HTLA条件，以产生约2 kb的片段与连接准备粘性末端的组装大的 DNA构建体。连接1 kb λ基因组部分以产生~2 kb粘性的单循环HTLA反应条件 将系统地改变末端嵌段（SEB）以使产率最大化。DNA部分重叠长度也将被优化。 然后改变SEB上的粘性末端长度以实现用T4或9°N™ DNA连接酶的最佳连接。SEB将 以增加的数量连接以确定最终产物长度的实际限制。序列保真度 2）确定~1 kb异源双链DNA的最大数量 分子可以通过HTLA以一锅一步法以高序列保真度可重复地连接 和环状异源双链热稳定连接组装（CHTLA）。一个kb的λ基因组部分， 将使用HTLA将数字连接起来，以确定SEB生成的尺寸上限，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反应也将用增加数目的1 kb λ基因组部分进行探测 CHTLA可产生的最终产品长度的上限。将确定序列保真度; 3） 为了使用目的1和/或2中确定的优化的异源双链体克隆策略来无缝地产生完整的 λ噬菌体基因组，其可以包装成感染性颗粒。使用优化的条件， 目的1和2，将产生完全组装的λ基因组，包装并用于感染E。杆菌 成功完成第一阶段项目将证明HTLA和/或CHTLA的实用性， 体外功能性~50 kb病毒基因组。在第二阶段，我们将扩大异源双链克隆的效用， 长度为100 kb至>1 Mbp的DNA服务于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%).