a thoughtful web.
Good ideas and conversation. No ads, no tracking.   Login or Take a Tour!
comment by bristolstreet
bristolstreet  ·  3334 days ago  ·  link  ·    ·  parent  ·  post: A Cell Therapy Untested in Humans Saves a Baby With Cancer

Better target specificity in the best case scenario, maybe, but given the improvements in simplicity I would've thought CRISPR/Cas9 (or, now, CRISPR/Cpf1) still has the bigger fanbase. The length of the recognition sequence creates insignificant nonspecificity, something on the order of 10^-7 chance of repeat events in the human genome, if my professor did the calculations right. They did get that Zuckerberg rockstar award too...





thundara  ·  3333 days ago  ·  link  ·  

Well, CRISPR is more flexible in targeting a new sequence, but the current crop of therapies already have screened TALEN / ZFN mutants that target their gene of interest, so that advantage is somewhat moot.

The non-specificity is I think a much bigger problem than you expect due to the fact that Cas9 can cut sites where it has at least a partial match of its guide RNA to the DNA. Even if there is only one exact match to the gRNA in a genome, there are still millions of PAMs where Cas9 will at least transiently bind, and a partial match in the early sequence can cause the enzyme to stick around long enough to cleave the DNA in a non-trivial fraction of the time.

Keep in mind that Cas9 was primarily evolved to target viral genomes, which are generally pretty small (well less than 1 Mb) in a background of bacteria genomes (~ 2 Mb, say, for

S. pyogenes). In contrast, the human genome is about 3,000 Mb in size, which means you need 3 orders of magnitude more specificity than the protein was evolved for. Not impossible, but it means you need a bit of tweaking to the system.

There are several tricks people have come up with to reduce this off-target cleavage, but it's still not a solved problem to the point that you would feel safe putting the system in a life human body. Add to that the on-going patent dispute over Cas9 and you have a pretty good incentive to wait it out while academic researchers iron out the bugs with the technology.