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.