Don't Fear the CRISPR - Comments

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  • #27
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@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.
 
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A major focus of research on gene editing is increasing the fidelity of cleavage by the CRISPR-Cas9 system.
Sounds like we need an update Insight :wink::smile:
 
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@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. I covered a lot of them, plus potential developments for the future when I was carrying out a risk assessment of whether Cas9 is safe to use in human beings or for widescale release to the environment. When I covered it 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.

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.

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.
 
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  • #30
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On the topic of Cas9 fidelity, there were two recent news articles posted today:
http://www.nature.com/news/biologis...molecular-scissors-for-genome-editing-1.18932
http://www.theatlantic.com/science/archive/2015/12/who-edits-the-gene-editors/418209/

Which are about a Science paper from Feng Zhang's lab published today on the topic.

@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 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).

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.
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.

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.
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.

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.
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.
 
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Like with any new technology, I think I feel a reasonable amount of apprehensiveness, most of which likely comes from the fact that I have absolutely no background in cell biology or genetics and can't speak to the limitations of this technology one way or another, but I also know that many other technological revolutions have come and gone and the world is a better place for it despite some people at the time being absolutely terrified of them.

I'm hopeful, to say the least.
 
  • #33
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Greg, just a technical comment. When the graphics in this article is under the mouse it gets dim and a blue point with two arrows appears. Klicking on that arrow, the graphics gets larger. But as the background of the graphics is tansparent, it becomes barely visible.
 
  • #34
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What Happens If Someone Uses This DIY Gene Hacking Kit to Make Mutant Bacteria?
http://motherboard.vice.com/read/wh...-diy-gene-hacking-kit-to-make-mutant-bacteria
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.

Personally, I would be more worried about people being able to stockpile assault weapons and materials for pipe bombs at home.
 
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More news on from those trying to engineer Cas9 proteins with increased fidelity:
Kleinstiver et al. 2016 High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature. Published online 06 Jan 2016. http://dx.doi.org/10.1038/nature16526[/URL]

[quote]CRISPR–Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used [I]Streptococcus pyogenes[/I] Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.[/quote]

News articles:
Enzyme tweak boosts precision of CRISPR genome edits [URL]http://www.nature.com/news/enzyme-tweak-boosts-precision-of-crispr-genome-edits-1.19114[/URL]
Improved Version Of CRISPR Gene Editing Tool Eliminates Errors [URL]http://www.popsci.com/new-form-crispr-is-more-precise[/URL]

The work seems complementary to the work published by the Zhang lab (mentioned in [URL='https://www.physicsforums.com/threads/dont-fear-the-crispr-comments.811056/page-2#post-5305836']post #30[/URL]), so the different modifications can probably be combined to engineer an even more precise enzyme.
 
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UK scientists are allowed to test CRISPR-Cas9 with human embryos.
http://www.bbc.co.uk/news/health-35459054
http://bigstory.ap.org/article/fdda5bf9f0314b748c7438c9659da83a/britain-approves-controversial-gene-editing-technique [Broken]
 
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