mfb said:
@Ygggdrasil: Can you comment on
this news? It looks very interesting.
A major focus of research on gene editing is increasing the fidelity of cleavage by the CRISPR-Cas9 system. The Cas9 enzyme can tolerate a number of mismatches in its target site, so it is prone to cutting at sites that resemble the target site. These off-target cleavages can be problematic as they can lead to unwanted edits to the genome that could be problematic, especially in clinical applications (in the worst case scenario, an off target mutation could lead to a serious disease like cancer). The news article (which references
http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.3624.html from the journal
Nature Methods) discusses a new method that combines elements from two different gene editing approaches to increase the potential for off target effects of gene editing.
Prior to the discovery of the CRISPR-Cas9 system, researchers had been working with two different technologies, zinc-finger (ZFs) and tal effector (TALEs), as ways to perform gene editing. These are proteins which can be engineered to recognize and cut specific sequences in the human genome to perform gene editing. The advantage they have over CRISPR-Cas9 is that they are generally better at distinguishing the correct sequence from incorrect sequences and are less likely to introduce unwanted edits at other sites in the genome. However, it is relatively difficult to engineer new ZFs or TALEs to recognize new sequences. In contrast, it is trivial to program the CRISPR-Cas9 to target new sequences, but as mentioned above, suffers from problems with off target cleavage.
To increase the fidelity of the CRISPR-Cas9 system, the authors add either a ZF or a TALE as a DNA-binding domain (DBD) to a weakened version of the Cas9 enzyme. The weakened Cas9 is not capable of binding to its target site on its own, but instead relies on the DBD for recruitment. Once tethered near its target site by the DBD, the Cas9 can then read its target sequence and cleave the DNA for gene editing purposes. Because cleavage depends on recognition of both the binding site for the DBD and the guide RNA of the CRISPR system, the engineered protein shows increased fidelity. This strategy also helps expand the types of targets that CRISPR can recognize.
The main drawback here is that the technique relies on ZFs and TALEs, and as mentioned previously, it is difficult to reprogram ZFs or TALEs to recognize new sequences. The authors of this study targeted a site in the genome that they have studied extensively for which ZFs and TALEs had previously been designed (and these ZFs and TALEs have been well validated in the literature). Designing a new construct to target a new site would involve much more work to redesign and test new ZFs or TALEs. In contrast, other methods exist for increasing the fidelity of the CRISPR-Cas9 system, which does not sacrifice the ease of reprogramming the nucleases to recognize new sequences (for example, see
http://www.nature.com/nbt/journal/v32/n6/full/nbt.2908.html or
http://www.cell.com/abstract/S0092-8674(13)01015-5). The authors of the
Nature Methods study claim, ~100-fold reduction of off target cleavage, whereas the double-nicking strategy in the
Cell paper claims a 50-1000 fold reduction in off-target cleavage, so it's not clear that the method is better than other existing methods. There may be some contexts, however, where the DBD-fusion strategy will work where some of the other strategies might not work.
While the ease of programming CRISPR-Cas9 recognition will be useful in basic research for screening efforts, it's likely that the fidelity of the CRISPR-Cas9 system will not be sufficient for clinical applications. Clinical applications will probably still require testing many different approaches (CRISPR-based, ZF-based, TALE-based, and various combinations) in order to find the approach that leads to the least off target effects. Whether one approach is superior in all cases or whether the best approach depends on the particular target and application likely remains to be seen.
... I'll kick it off with, you bet! Not too much in the article convinces one (if fearful) that all the crazy things imagined in the movies couldn't or won't happen. The breakthrough seem to declare with seamless authority, it is now truly, more realistically possible.
