The Multiplex Automated Genome Engineering (MAGE) technique developed in George Church's lab at Harvard Medical School provides a promising alternative to whole-genome synthesis. It's certainly going to work out a lot cheaper than writing a million base-pair sequence from scratch and can cope with situations where the changes needed to something such as E coli involve edits throughout the chromosome.
In their paper for Nature that was published online over the weekend, Harris Wang, Farren Isaacs and their co-authors describe how they used MAGE to essentially perform rapid prototyping on a genome and increase the amount of the chemical lycopene that E coli could produce. It takes advantage of an aspect of MAGE that lets you tune how much variation out of the edits the system makes to a genome.
Talking about MAGE last year at the Royal Society, Isaacs explained how it was being used to perform hundreds of edits on the E coli genome in a bid to rework the genetic code itself to build a wider range of proteins than natural genetic systems can today. The first step was to remove one of the stop codons used by E coli to terminate translation. All of the 300 or so instances of the sequence TAG would be replaced by the more common TAA variant of the code. This allows the three-base TAG codon to be redeployed as the code for a non-natural amino acid. Isaacs said initial results from this project are nearing completion.
Full-scale codon replacement is the kind of thing you can do if you push MAGE through many cycles. The process starts off with a single, isogenic genome. As MAGE proceeds, the replacement pieces of DNA do not supplant the original sections all at once. So, if you have a lot of simultaneous replacements to make, there will be a lot of variation between the genomes of individual cells. But, as you push MAGE further, ultimately all the sections get replaced and you wind up with a new isogenic genome. To get many different mutants, you simply stop the MAGE process in the middle and analyse what comes out. In the latest experiment, the process came up with more than 4 billion genomic variants each day. They then isolated variants that showed a significant increase in lycopene production - finding one that had a five-fold increase over the wild type in just three days.
The core of the MAGE techique is a protein used by the λ-Red virus to introduce its own changes into a bacterial genome with a little help from genetic engineering. The β protein binds to oligos that are intended to replace sections of DNA in the actual genome. The protein helps the sections displace the Okazaki fragments that the cell's own machinery uses to build complementary DNA strands on the lagging strand during DNA replication. Each end of the section provides a match to the original DNA to let it stick, with the new 30 base pairs or so of DNA lying somewhere in the middle. Normally, the cell's mismatch repair proteins would spot this alien DNA - because it does not marry up with the original complementary sequence - but one of the key genes for the repair mechanism has already been knocked out.
When the DNA replicates again, the new DNA is copied and becomes fully part of the genome. Some of the replacement fragments don't make it or, in the case of this experiment, are replaced by other near matches, which gives rise to the huge genetic variation at the edit points.
The edit points are not picked at random. Isaacs explained that some of them are simple knock-outs, putting stop codons in the middle of genes that might divert feedstock chemicals away from the pathway that produces lycopene. The other target was the gene that codes for the protein that makes lycopene itself. One of the directions of Isaacs' research is on the efficiency of translation as mRNA is used to produce the final proteins.
The Shine-Dalgarno sequence (TAAGGAGGT) in the section of messenger RNA where the ribosome first attaches generally boosts translation efficiency but it does not work in all cases. The structure of the messenger RNA seems to have an effect. Rather than try to design the most-efficient sequence a priori, Wang and Isaacs decided to let evolution have a go. They developed a variety of oligos that contained subtly different variants of the Shine-Dalgarno sequence and added them to the MAGE pool. They could then pull out the one that worked the best at the selection stage. The first selection of variants were made by looking for colonies that produced an intense red pigmentation on Luria-Bertani agar plates with the best performers screened from the ten thousand or so that process identified.
Isaacs thinks the technique can go much further and use evolution to pick out winners from more extensive reworkings of the genome, acting on promoters as well as ribosome binding sites. It will, in a sense, press the fast-forward button on evolution by letting biology explore genetic states that might be inaccessible because single changes on the way to them would kill off the candidate cells. Experiments such as the one performed by Mark Isalan and Luis Serrano and the team at CRG in Barcelona have shown how swapping bits of gene promoters can alter the fitness of bacteria. And these techniques could show how regulatory networks of genes affect cell behaviour.
"We want to think of general strategies that we can use with this method to introduce diversity with a universal oligo pool, and allow the cells to explore new genetic landscapes that will confer new properties. And then come up with screens and selections that let us pull out cells with specific behaviours," said Isaacs.
The aim is to target all 4000 genes of E coli with on the order of 15 000 oligos. "We could target every known coding and regulatory locus. You are seeing just the beginning of what we are trying to do," said Isaacs.
The core MAGE technique could work in other cell types, such as yeast, Isaacs claimed: "The mechanism that we use is something that is conserved across biology."
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