The labs of Julian Sale and Jason Chin at the Medical Research Council (MRC) Laboratory of Molecular Biology in Cambridge, U.K., have created two new tools critical for rapid genome construction at scale.

These tools, BASIS (bacterial artificial chromosome stepwise insertion synthesis) and CGS (continuous genome synthesis), solve two of the most important problems in creating genomes: 1) putting together megabases of DNA quickly from smaller pieces and 2) replacing the genomic DNA of organisms with synthetic DNA in a scalable way. BASIS and CGS made it possible to build multiple fully synthetic genomes at the same time. The authors used these genomes to put together “scarless” large parts of the human genome on episomes. This technological advancement can facilitate high-throughput experimentation at the genome level and the creation of genome libraries.

The study detailing these methods, “Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly” was published in Nature.

The development of genomes offers previously unheard-of opportunities to define the relationship between genome sequences and the functions they encode as well as to create organisms with novel and useful functions. E. coli is an attractive host for assembling large stretches of DNA into episomes as a starting point for building synthetic gigabase-scale genomes. But the current methods for putting DNA together in E. coli can only be done once, using very small pieces of DNA, using site-specific recombinases that leave scars or require DNA fragments to be linearized in vitro and electroporated.

This paper presents a solution for the rapid stitching of DNA at the mega-base scale with BASIS and a strategy for the continuous replacement of host DNA with foreign DNA with CGS.

The Sale and Chin labs used BASIS to assemble a 1.1 Mb section of human chromosome 21 from bacterial artificial chromosomes (BACs) at high fidelity. This human DNA included numerous exons, introns, repetitive sequences, G-quadruplexes, and long and short interspersed nuclear elements (LINEs and SINEs).

They then demonstrated that BASIS BACs containing large DNAs provide a facile platform for rapid modification in E. coli and expression in human cells. To do so, they corrected a synthetic cystic fibrosis transmembrane conductance regulator (CFTR) BAC, transfected it into human cells, and demonstrated that the CFTR gene from the BAC had driven the expression of a corrected CFTR transcript.

For CGS to work, the Sale and Chin labs identified a strain of E. coli that is less prone to crossovers between exogenous DNA and the host genome, which they used for continuous insertion of 100 kb stretches of synthetic DNA. The authors used this strain to incorporate a 0.5 Mb section of DNA from five episomes into the E. coli genome in just 10 days. They believe that combining CGS with rapid oligonucleotide synthesis and episome assembly, as well as rapid methods for putting together a single genome from strains with different synthetic genome sections, will allow them to create whole E. coli genomes from functional designs in less than two months.

The DNA created with BASIS and CGS can be used with methods for moving large amounts of episomal DNA into human, animal, or plant cells and for iterative recombination in these cells to insert synthetic sequences into artificial chromosomes or replace natural sequences in chromosomes with synthetic sequences.

Overall, the ability to rapidly assemble megabase-scale DNA and the advancement of CGS lay important groundwork for rapid and scalable genome synthesis. With new methods for moving large DNA constructs between cells, these technologies take E. coli one step closer to becoming the platform for constructing synthetic genomics at the gigabase scale.

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