Friday, May 2, 2025
5/2/2025
Multiplexed In Vivo DNA Assembly - PROJECT SUMMARY/ABSTRACT High-throughput and low-cost synthesis of long DNA fragments could open up new frontiers in genomics and synthetic biology by facilitating the production of genes, genetic pathways, variant libraries, or entire synthetic genomes. Microarray printing technologies provide for high-throughput and low-cost synthesis of short oligonucleotides (currently up to 300 bases long), while new technologies, such as enzymatic synthesis, have the potential to synthesize longer sequences. Libraries of open reading frames or regulatory elements have also been constructed. However, assembly of the above or other DNA elements into longer fragments remains a challenge. Widely-used in vitro DNA assembly methods, such as Gibson assembly and polymerase chain assembly, require considerable hands-on time, are difficult to multiplex and scale, and have size constraints on the length of the assembled product. In addition, accurate long assemblies from de novo synthesized DNA are difficult to achieve without a high-throughput error correction step. To overcome these hurdles, we have developed a novel, multiplexed method to parse, sequence verify, and stitch together DNA of various sizes in vivo. Using this method, complex pools of array-synthesized oligonucleotides or other DNA elements can be turned into positionally-ordered cellular arrays, with each position containing a sequence-verified input DNA. Input DNA in these arrays can then be stitched together recursively by bacterial mating. A key feature of our technology is early error detection, whereby individual DNA inputs are sequence-verified prior to stitching. That is, errors that occur during short DNA synthesis are detected and removed, rather than propagated into longer assemblies. At maximal throughput, we estimate that our technology has the potential to produce thousands of DNA assemblies in parallel at a cost that is at least an order of magnitude cheaper than commercially available gene synthesis technologies. Because DNA stitching can occur within bacterial artificial chromosomes, in vivo DNA parsing and stitching can perform multiplexed assembly of DNA constructs exceeding 100kb, at least an order of magnitude longer than current in vitro assembly techniques. In vivo assembly uses a stepwise stitching process that can be branched, allowing the construction of a wide variety of variants at low incremental cost. Here, we propose to continue development of this technology i) by building many genes to learn the error modalities of the process, ii) by assembling a ~100kb product, and iii) by developing a multi-step assembly procedure to reduce assembly time and increase product accuracy. Together, this integrated set of aims will result in a new high-throughput DNA assembly platform capable of synthesizing long and complex DNA constructs and variant libraries at low cost.
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