What’s the big deal with CRISPR/Cas9?


Cas9 (grey) in complex with yellow guide RNA and red target DNA. PDB structure 4oo8 manipulated in PyMOL by yours truly. Cas9, like competing genome editing technologies (TALENs and ZFNs), is a nucelase. Click to view animated GIF.

Summary: Eliminate hereditary diseases. Re-program pathological tissue. Design babies. Bring back the T. rex. The peril and promise of genetic engineering has been a long-time coming. Generally speaking, none of the wonders we began collectively imagining with the deduction of DNA structure in the 1950s have come to fruition. At the turn of the millenium with the completion of the human genome project(s), we expected personalized medicine to eradicate inefficacies and side effects in modern medicine. Current development based on bacterial immune systems promises to either revolutionise the treatment of genetic disease or fill the world with ten-foot tall babies shooting lasers out of their perfect blue eyes while playing professional basketball and winning Nobel Prizes.

My first foray into a wet lab consisted of a project straight out of the astounding futures your favourite sci-fis promised you- or warned you about: incorporating functional genetic elements from humans into fungal cells. After a summer spent pushing the limits of what is possible and blurring the lines of what it means to be human, I created a terrible organism neither man nor yeast. Unable to find acceptance among people and no longer satisfied by nature’s intentions, these fungal colonies, the bizarre offspring of one man’s twisted mind and leavening products found the cruel world to be too much and jumped into an autoclave while reciting Macbeth.

Despite the hyperbolic passage above, the monsters yet live. The strain ended up in a laboratory-grade freezer at negative eighty degrees (Celsius, of course, the lab being free of both astrologers and barbarians). The little yeasties are probably still chilling in the small cardboard box where I left them, covered in frost and enjoying a nice bath of glycerol cryo-protectant, traveling through time in suspended animation until the world is ready for them.

The human genes and their counterparts in baker’s yeast are similar enough that in this case one could substitute for the other (at least in one direction). The function of these metabolic keystones known as ATP synthases is an ancient one: churning the potential energy of an electron gradient to make the cellular energy storage molecule adenine triphosphate (ATP). They are primeval enough that the human version acts as a suitable stand-in for a strain of Saccharomyces cerevisiae otherwise incapable of aerobic respiration. I had precisely engineered a genetic vector that inserted directly into the location of the yeast’s genome where the native version had been removed. And by “precisely engineered” I mean that it was so easy, an undergrad could do it, as I did.

Recently a technique based on CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) and CRISPR-Associated Proteins (such as Cas9) has garnered a lot of attention in the press as well as the scientific community. The word-sequences up-regulating all the excitement highlight the ease and effectiveness of CRISPR/Cas9 over previous methods. The technique’s critical reception has run the full range from drooling anticipation to worried alarm to bad puns.

Since my early days in the lab playing as a god with design of human-yeast splices, I’ve continued down the rabbit-hole of biological scale to the point that I now work more often with the single molecule(s) of biomolecular machinery than with cells directly. So I’m certainly out of the loop and out of a practical grasp of the rational underlying CRISPR/Cas9 genome editing. After all, spider silk proteins have been produced in mammalian cells since before 2002, and are regularly produced in goat’s milk. Does CRISPR/Cas9 change the game to such a degree that warrants the flood of interest?


The interest surrounding CRISPR

I’ll skip over the high-level technical overviews that you’ve probably read before, but for those with the time and interest I can recommend Jennifer Doudna’s Breakthrough Prize lecture. Instead I’ll compare two protocols, the first based on CRISPR/Cas9 and the second based on an older technique using another type of engineered nuclease known as zinc-finger nucleases (ZFNs). I scraped both protocols from the same publication, so apparent differences due to style should be small. To get a sense of the complexity of each technique, here are the two protocols as wordle word-clouds, displaying the size of the 256 most frequently used words in each protocol according to their relative usage.


ZFN protocol: word frequency word cloud


CRISPR/Cas9 protocol: word frequency word cloud

The table below compare the complexity and length of either protocol. The reading complexity measures were generated with this tool, and in short the first measure decreases with increased complexity while the second two increase with added complexity.


At first glance we see that the CRISPR/Cas9 protocol is much longer and more complicated, but if we consider that the Zn-finger nuclease protocol only describes the process up to in vitro validation of the process, we can make a much more equivalent comparison by truncating the CRISPR/Cas9 protocol to the first 13 steps. The resulting comparison:


The associated Wordle even looks a bit friendlier.


So suffice it to say that it’s not easy to see the underpinnings of the excitement surrounding major developments such as CRISPR/Cas9. Essentially the advantages of the CRISPR-based approach stems from the level of difficulty of engineering guide RNAs versus engineering DNA-binding domains based on amino acid residues required for competing techniques ZFNs and TALENs (not compared here). In the brewer’s yeast I modified “back in the day,” targeting the desired genes to the desired location was as simple as including a sequence from the target location on the DNA to be inserted; there are sufficient double-stranded breaks in a flask of yeast culture to allow the gene to find its target a few times. With the specifically targetable nucleases such as Cas9, Zinc-finger nucleases and TALENs, one doesn’t have to count on such an easy model organism to precisely manipulate a small number of cells for a desired change to the genome.

The increased interest alone is sure to drum up funding, public intrigue, and private investment, driving the impact forward as a self fulfilling prophecy. The more interested and excited people are for CRISPR/Cas9, particularly those people with the deep pockets to fill out scientists’ salaries, the more the technique will be subjected to use and refinement. More people using the tool drives the potential for meaningful breakthroughs. On the other hand, we have been promised and warned of this same onrushing biopunk dystopia before, and as they say: if this is the future, where are my gene-driven superpowers?


Published protocols referenced in this post:
[1] Carroll, D., Morton, J. J., Beumer, K. J., & Segal, D. J. (2006). Design, construction and in vitro testing of zinc finger nucleases. Nature Protocols, 1(FEBRUARY 2006), 1329–1341. http://doi.org/10.1038/nprot.2006.231

[2] Ran, F. A., Hsu, P. P. D., Wright, J., Agarwala, V., Scott, D. a, & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8(11), 2281–308. http://doi.org/10.1038/nprot.2013.143

[2015/12/14 EDIT – copyediting]

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