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October 06, 2012

What is the cost and speed of mass editing of DNA to convert human DNA into Neanderthal DNA ?

Geneticists have long been able to edit individual genes, but using a technique called MAGE/CAGE mass rewriting DNA is possible, turning the entire genome of a bacterium into an “editable and evolvable template”. MAGE and CAGE were described in Discover Magazine and is described in the George Church and Ed Regis book Regenesis

Their success was possible because the same genetic code underlies all life. The code is written in the four letters (nucleotides) that chain together to form DNA: A, C, G and T. Every set of three letters (or ‘codon’) corresponds to a different amino acid, the building blocks of proteins. For example, GCA codes for alanine; TGT means cysteine. The chain of letters is translated into a chain of amino acids until you get to a ‘stop codon’. These special triplets act as full stops that indicate when a protein is finished.

This code is virtually the same in every gene on the planet. In every human, tree and bacterium, the same codons correspond to the same amino acids, with only minor variations. The code also includes a lot of redundancy. Four DNA letters can be arranged into 64 possible triplets, which are assigned to only 20 amino acids and one stop codon. So for example, GCT, GCA, GCC and GCG all code for alanine. And these surplus codons provide enough wiggle room for geneticists to play around with.

Farren Isaacs, Peter Carr and Harris Wang have started to replace every instance of TAG with TAA in the genome of the common gut bacterium Escherichia coli. Both are stop codons, so there’s no noticeable difference to the bacterium – it’s like replacing every word in a document with a synonym. But to the team, the genome-wide swap will eventually free up one of the 64 triplets in the genetic code. And that opens up many possible applications.

MAGE stands for multiplex automated genome engineering. It makes it possible to introduce 10 million genetic modifications into a genome in a reasonable time.

CAGE stands for “conjugative assembly genome engineering”). The technique relies on the bacterial equivalent of sex – a process called conjugation where two cells sidle up, form a physical link between one another, and swap DNA.

MAGE can be used to substitute TAA for TAG in separate pieces of bacterial DNA, and CAGE, which knits the pieces together into a whole genome.



Modifying a genome with MAGE

MAGE uses the smallest bits of DNA that are unique to the genome to be changed. The smallest bits are in theory 12-mers for 5 million base pair bacterial genomes but in practice it is 90-mers. 2500-volt electric pulses get the 90-mers into cells. The 90-mers are protected from enzyme damage with a protein coating. Various tricks boost the mutations to several per hour.

Modifying human DNA to Neanderthal

1. Break human DNA into 10 thousand pieces of 300,000 base pairs
2. Replicate them in e. Coli (BAC - bacterial artificial chromosomes)or in yeast (YAC - Yeast artificial chromosomes)
3. Re-edit each piece with MAGE for about 1000 changes each
4. Using chip syntheses, 10 million oligos can be printed at about $5000 and double that to allow for error correction.
5. The human chromosomes can be reconstructed in parallel (about 500 steps)
6. Then combine them and put them into a stem cell



LA Times - recently mouse stem cells were turned into viable eggs. This could be developed for human cells or other methods can be used where the genetic material is transferred.

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