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

In vitro assembly of DNA fragments andgt;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 andgt;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 andgt;1 Mbp in length to service the $1.62B Genome Engineering and Editing Market (CAGR, 24.4%).The proposed research is relevant to public health because it will provide a new technology to generate large DNA molecules for synthetic biology applications. This project is relevant to NIH’s mission because new therapeutics and diagnostics for a wide array of diseases are currently being developed in industry and academia using synthetic biology approaches.