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.
Yes, I was probably unclear here. I meant that the fidelity of a single CRISPR-Cas9 in the standard gene editing protocols is probably insufficient for many clinical applications. Modified versions (such as the engineered Cas9 version described above or the dimeric versions) may end up being useful. Of course, the amount of off target activity that is tolerable will depend a lot on the particular application (e.g. off target activity will be more acceptable when treating serious diseases whereas it will be much less acceptable when editing genes in healthy individuals).@Yggdrasil I think you're wrong there. There are various methods developed in very recent times which have been shown to increase the fidelity of cleavage.
Yes, I linked to this study in my post #27. One problem with this approach is that DNA binding by dCas9 is much more promiscuous than DNA cleavage by catalytically competent Cas9 (see for example http://www.nature.com/nbt/journal/v32/n7/full/nbt.2889.html showing widespread off-target binding of dCas9 compared to Cas9 cutting with the same gRNA and http://www.nature.com/nature/journal/v527/n7576/full/nature15544.html providing a mechanistic basis for the additional proofreading that occurs between DNA binding and DNA cleavage). A more promising approach is the other paper I cited from Feng Zhang's lab that uses a double nicking approach that likely preserves the fidelity of Cas9 compared to strategies using dCas9.a Cas9-FokI nuclease combination was shown to reduce off-target effects by requiring dimerization and two gRNA sequences to achieve a DSB. My understanding was that the gRNA still served as the means of targeting the DNA, and the attached FokI domains were used as a nuclease, to replace the nuclease function of dCas9 (de-activated Cas9). FokI simply requires dimerization to act as a nuclease (see "Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing" http://www.nature.com/nbt/journal/v32/n6/full/nbt.2908.html). Within that paper it's referred to as wild-type FokI nuclease that was attached to dCas9, used on multiple sites.
No, the main drawback of ZFNs and TALENs is the difficulty in engineering them to target new sequences. See Table 1 in http://www.nature.com/nm/journal/v21/n2/full/nm.3793.html for a comparision of various genome editing technologies.My understanding is that the Zinc-finger part of the ZFN is the DNA-binding domain, FokI is simply a nuclease attached to the Zinc-finger protein (https://en.wikipedia.org/wiki/Zinc_finger_nuclease), same with TALENs, TALE is the DNA-binding domain and FokI is simply an attached nuclease. The main problem I thought was the efficacy of cutting and maybe that's where protein engineering might be needed.
At some point, with Cas9 you will begin having problems finding suitable target sites due to the invariant PAM recognition site of Cas9. At some targets you will not have a suitable number of PAM sites positioned correctly to use dimeric or tetrameric Cas9 strategies, so ZFNs or TALENs may be required for these targets. There have been some successes in reengineering Cas9 to recognize other PAM sequences, however, so these advances could remove some of these limitations.Given the availability of the ZF nickase technique (where a pare of FokI dimers is converted to a nickase rather than a nuclease), if you can find further efficiency gains (as I recall efficacy is quite poor even in a Zinc finger-nickase), you could use with Cas9 to the point it would require dimerization to make a single nick - thus 4 gRNA sequences would be required to make a single DSB. You've also got the achievement of enhanced specificities through use of tru-gRNA (truncated gRNAs - reduces undesired mutagenesis some 5,000 fold http://www.nature.com/nbt/journal/v32/n3/full/nbt.2808.html), so my conclusion is that if you get these two working in concert with improvements in predictive software, you're gonna end up with Cas9 of quite a high targeting specificity.
You don't really need CRISPR to make precision edits to bacteria, and the technology to do this has been around since the late 80s (https://en.wikipedia.org/wiki/Recombineering). The Indiegogo page for the kit in question describes the types of experiments that might be performed in freshman biology courses. These are not dangerous experiments. Yes, in theory, someone could use CRISPR to engineer something potentially illegal (for example, yeast that allow you to brew illegal drugs), but doing so would take much more expertise than the general public would have.What Happens If Someone Uses This DIY Gene Hacking Kit to Make Mutant Bacteria?