Tuesday, March 24, 2009

Codon's Type IIS Meganuclease

When I joined Codon Devices, I swore I would not use this space to shamelessly tout any results from the company. It turned out my resolve was never tested. It's not that there weren't interesting results being generated in the company, but that in one way or another they never became public. Some results were never meant to be public, but were within collaborations, whereas some intended to be public got held up by one snag or another.

Perhaps the universe does like to play subtle jokes on us. Now that I'm out, so is the first publication from the company, describing the engineering of a Type IIS restriction enzyme with a very large recognition sequence.

TypeIIS restriction endonucleases are handy for many purposes, but particularly for gene construction techniques. Whereas most restriction enzymes recognize and cut at the same site, Type IIS enzymes recognize a specific site but then cut a precise distance away (or cut at perhaps two different offsets; note Fig 2 of this reference). This is handy because it allows one to design two pieces to come together (via the sticky overhangs generated by the enzyme) but without the recognition sequence in the final product. Hence, Type IIS enzymes can allow virtually any sequence to be built.

The catch, of course, is that it is challenging to build in this fashion a sequence which itself contains the Type IIS recognition sequence. Ideally, these sequences would be very long and hence unlikely to appear by chance. Unfortunately, the known Type IIS enzymes almost all have 5 or 6 basepair long recognition sequences, which are not terribly rare once you get in the multiple kilobase range, and are certainly not rare if you want to build chromosome-sized DNA.

So the goal of a number of efforts has been to build a Type IIS restriction enzyme which has a very long recognition sequence. Enzymes called homing endonucleases have huge recognition sequences, with effective lengths of 12 or more basepairs (the actual lengths are greater, but there is also some positions which are not fully fixed to a particular nucleotide -- hence the term effective length). The advance of Lippow et al is that a new level of precision was obtained in the cutting sites, a level of precision compatible with gene engineering.

In a sense, the problem is analogous to that of a K9 unit. The handler has a potentially vicious dog which she would like to apply precisely. Give the dog too short a leash and you can't deploy its teeth; give it too long a leash and the teeth may sink into places other than where you want them to.

So what Lippow et al did is build different protein linkers to tie the DNA recognition domain (handler) to the cleavage domain (dog) from the Type IIS enzyme FokI. By run-off Sanger sequencing, in which the polymerase is allowed to extend to the end of a DNA strand, they showed that cutting is precise, particularly for one of the specific enzymes generated. The dog, alas, is not under complete control; some random off-site cutting is observed. But it is a step forward.

One last hitch: to be particularly useful, one really needs at least two Type IIS meganucleases, and ideally many. Alas, this paper provides only one -- but it is a roadmap to building more, as there are a number of other homing endonucleases which could be potentially used for recognition modules. Alternatively, a number of papers have generated Sce-I variants with different recognition specificities, so by introducing these mutations into the CdnI enzyme reported here should allow a new set of Type IIS meganuclease specificities.


Noah Sachs said...

Cool, but is the link supposed to go to the Supp. Info on Science for sequencing by ligation? I was hoping to read a whole article, none the less I was definitely thinking that this would be good for the Polonator, because it uses Mme I.

Keith Robison said...

Ugh! I apparently pasted in the same URL twice; only the second link is supposed to be going to the Polony paper's supplemental methods, because it has the best data on MmeI's dual offset (which, BTW, I consider an "easter egg" -- that data could have easily been a short note in a journal such as NAR);

In any case, I should have left a complete citation
S. M. Lippow, P. M. Aha, M. H. Parker, W. J. Blake, B. M. Baynes, and D. Lipovsek
Creation of a type IIS restriction endonuclease with a long recognition sequence
Nucleic Acids Res., March 20, 2009; (2009)

thanks for catching this

Brett said...

Sorry this comment is coming so long after the original post. Would you happen to know, by any chance, what happened to the CdnI nuclease engineered by Codon Devices? Did they license that to anyone, or did it die along with the company? Just wondering, as it would be very useful for some of the projects I am working on.

Keith Robison said...

Intellectual property never dies, though it may be in a bit of suspended animation. Someone at Flagship Ventures may be able to help you out; I'll try to make some inquiries as well (but I'm just back from a vacation so a bit in dig-out mode)