Cutting and pasting with the human genome

Highlights in Chemical Biology ^ | 16 September 2009 | Philippa Ross

[neverdem]

A DNA cutting tool that can manipulate human genomic DNA could ultimately find applications in gene therapy, say Japanese scientists.

Makoto Komiyama, Narumi Shigi and colleagues at the University of Tokyo recently made the DNA cutter - ARCUT - and used it to cut bacterial DNA at one target site. Now they have shown that it can be tuned to cut human genomic DNA selectively and also to repair it.

"ARCUT's selectivity meant that the Tokyo team was able to use the cutter to target one site in human genomic DNA"

ARCUT consists of a cerium(IV) complex which cuts the DNA and a recognition system of peptide nucleic acids, which resemble natural nucleic acids but with a peptide rather than a sugar backbone. The cutter's target site is pre-determined by base pairing between the DNA being cut and the bases in the cutter, which can be easily designed to bind a specific scission site. This is currently not possible with naturally occurring DNA cutting enzymes, which cut the genome repeatedly every 4⁴ or 4⁶ base pairs (approximately 700000 times in human DNA). ARCUT's selectivity meant that the Tokyo team was able to use the cutter to target one site in human genomic DNA - a system 1000 times larger than the Escherichia coli DNA they tested previously.

Site-specific cleavage of double-stranded DNA by ARCUT

The researchers have also shown that ARCUT can promote homologous recombination, a process in which nucleic acid fragments are exchanged between lengths of DNA. Whilst several technologies already exist that do this by introducing double strand breaks in DNA, their design and preparation is often difficult and slow. This is because they use protein based nucleic acid cleaving enzymes which recognise the DNA target site through complex protein-DNA interactions. 'We have been working on chemistry based artificial cutting enzymes believing that they could be the solution to these problems,' says Komiyama. 

"The team has already used ARCUT to repair damaged DNA in human cells"

Peter Nielsen works with peptide nucleic acids at the University of Copenhagen, Denmark, and is an expert in DNA recognition and gene targeting. He comments that, although further work is needed to test ARCUT in vivo, it is an exciting breakthrough. 'The DNA cleavage system is very interesting, being a truly chemical system with good efficiency,' he says. 'It is also interesting in connection with regimes for gene repair.' 

Indeed, the team has already used ARCUT to repair damaged DNA in human cells. The next step for the group is to apply the system in vivo so that it can be used for gene therapy and preliminary experiments have been encouraging. Komiyama's team has demonstrated that blue fluorescent protein incorporated into the human genome can be converted to its green analogue using ARCUT-promoted homologous recombination. 
 

 

Link to journal article

Site-selective scission of human genome by artificial restriction DNA cutter
Kenichiro Ito, Hitoshi Katada, Narumi Shigi and Makoto Komiyama, Chem. Commun., 2009
DOI: 10.1039/b911208a

Homologous recombination in human cells using artificial restriction DNA cutter
Hitoshi Katada, Hsuan Jung Chen, Narumi Shigi and Makoto Komiyama, Chem. Commun., 2009
DOI: 10.1039/b912030k

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[Myrddin]

Just looking at the book titles tells me what I learned in college is ancient history. What we had in 1974 is like a kid's toy toolbox in comparison to the present capability.

[Myrddin]

The big deal when I was in school was the EcoR1 mutant. It was E. coli with the restriction and modification enzymes removed. That left the organism defenseless against insertion of foreign DNA. You could insert a sequence for something useful like human insulin and get the organism to replicate massive amounts of the protein.

[neverdem]

Converting this knowledge into useful gene therapy would be awesome, but the barriers seem huge.

I wouldn’t envy being most kids today, except for being kids talented in the sciences.

[Myrddin]

Being able to target the exact spot to cut is outstanding. The problem from a gene therapy position is delivering the "fix" to the right population of cells. The differentiation process results in sections of DNA that are active and others that are purposely "protected" to elicit the proper functioning of the cell. A snip that is beneficial to the desired target population may result in dysfunction in a different cell type.