I've read that CRISPR/Cas9 is currently being implemented and tested for its ability to edit genomes in live cells, and that it is supplanting other genome editing tools in labs, such as TALENs and Zinc finger nucleases.

I understand that there may be a few metrics used for analyzing any genome editing system. One is the efficiency, which could be measured perhaps in edits per time or edits per molecule. Another metric might be error-rate. What is the probability that a genome is altered in something other than the intended way? Another metric might be the length of the genome that may be edited at once.

I've also read that CRISPR/Cas9 evolved in bacteria. When did CRISPR/Cas9 evolve in the history of life on earth?

If CRISPR/Cas9 evolved in bacteria, and before other evolutionary advances, including some of those that may have enabled certain multiceullar forms of life, I have to wonder if other systems like CRISPR/Cas9 but superior to it in some metric evolved as well.

How does recombination in sexual reproduction work? Might it involve biomolecular machinery like that of CRISPR/Cas9?

How does the adaptive immune system work? How does VDJ recombination work? Does it involve an advanced (relative to CRISPR/Cas9) genome editing system?

How might one estimate the probability that a system superior to CRISPR/Cas9 exists already?

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    $\begingroup$ could you further expand on the question and set out some parameters or perhaps share your thoughts or work on the matter so that any input is constructive. $\endgroup$ Commented Aug 21, 2014 at 18:53

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Lots of interesting questions! Let me try to address a few of them as I don't think I am qualified to answer them all but hopefully I can get this thread started. I am a graduate student in the biophysical chemistry field and have been following a little bit of the Crispr Cas9 craze in the last couple of years. So I am not an expert on Cas9 by any means but I do find it interesting.

Many speculate that as long as their have been cellular life, viruses or some obligate parasites have existed as well. This stems from the idea that viruses perhaps are not "non-self" but rather are parts of the host that happen to become inanimate particles that escape the cell and then find another suitable host. Some viruses have co-evolved with the host, perhaps to prevent other viruses from invading its "home/birth mother". Again, much of this paragraph is just speculation, but it is likely that CRISPR systems, or an adaptive immune systems like it have existed as long as viruses/obligate parasites have.

Error rates, you can look at some of the papers that have come out recently but the idea is that there are a lot of error rates, especially if the RNA is constitutively transcribed from a plasmid. Some people have suggested delivering, not the plasmids for the guide RNAs and the Cas9 protein, but instead, the Cas9 protein complexed to the RNA itself. Other ways to control the amount you deliver to a cell include delivering the RNA for the Cas9 protein so it is eventually degraded. This avoids the complication of delivering DNA which can be transcribed multiple times potentially flooding your cell with cas9 protein or guide RNAs, increasing the likelihood of off target effects. Or you can have the cas9 protein and the guide RNA plasmids under inducible promoters, so you only get expression say if you introduce a small molecule to your cultured organism and limit the amount you deliver of it.

As to if there are other systems "superior" to it, and by superior you mean more efficient, and has less off targeting effects? Sure those things may very well exist, since we haven't sequenced the entire planet yet! All joking aside, maybe start looking around at research groups that do adaptive immunity in prokaryotes, you might find some interesting ideas...

VDJ is a very cool system that I know only a little about but this review might be worth checking out:

"Mechanism and Control of V(D)J Recombination versus ClassSwitch Recombination: Similarities and Differences", which can be found here.

Also recombination in meiotic versus mitotic replication is covered in this review: "Meiotic versus Mitotic Recombination: Two Different Routes for Double-Strand Break Repair", which is available here.

So to answer your question, I will have to defer to someone else!

My question to you is what is "advanced"? Life as we understand it has been evolving for about 3-4 billion years. "Advanced" suggests that one organism has evolved better or faster. I don't know if I would call anything more advanced per se, but maybe more complicated and more capable of rapidly adapting to new environmental pressures? Bacteria have us trumped if there were rapid changes to the environment, global warming, asteroids careening into the planet etc.

Estimating this probability requires knowing how large the entire sequence space of the Earth is, some have estimated 5-50 million eukaryotic species but depends on what metric you would use to define a new species. It is unknown for prokaryotes. My guess is that you have 100's of millions of different organisms that are capable of having some type of adaptive immune system that may be inherently different from CRISPR/Cas9 system but still use some similar elements, like nucleic acid complementation, as a mode of defending against obligate parasites. Then the questions are how many of those organisms have we sequenced and how many proteins do we have any idea how they function based on sequence similarities to other proteins? Or if we have directly characterized their 3D structure based on Cryogenic Transmission Electron Microscopy (Cryo EM), NMR or X-ray crystallography. So, I imagine we have just hit the tip of the iceberg in finding natural enzymes that can function as gene editing and gene locating tools. Plus, we may start designing our own enzymes like what Professor David Baker at UW works on. Huge ocean to go exploring, bring a molecular fishing pole :-)

  • $\begingroup$ Thank you for getting the thread started with this interesting response. To respond to your question about what I mean by "advanced," I suppose I've conflated two separate meanings in the term. On one hand I am interested in whether a substantially different genome altering system evolved (I suppose that is true with VDJ?) after CRISPR/Cas9, and on the other I am interested in whether any such system provides a measurably "superior" genome editing system. So I conflated "advanced" in evolutionary time with some practical and measurable superiority for intentional genome editing by humans. $\endgroup$ Commented Oct 3, 2014 at 13:21
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    $\begingroup$ Well you could look at systems like the RISC complex in eukaryotes (RNA interference), or the other Cas9 based systems that function using different PAM recognition motifs or the Cas6 Endonuclease complex which supposedly cuts foreign RNA. Not sure what else may be available, obviously the TALENs systems and Zinc finger nucleases may still be useful tools as they become more high throughput in design. I think at the moment the type II cas9 system from Streptococcus pyogenes is pretty robust and decently well characterized. So far its a good platform to modify and optimize for exogenous hosts. $\endgroup$ Commented Oct 5, 2014 at 0:01

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