The almighty CRISPR-Cas9 technology: How does it work?

CRISPR-Cas9 took the whole world of biology by storm. Selected Science’s 2015 Breakthrough of the Year, the CRISPR-Cas9 technology is revolutionizing science. Within five years of the official announcement (Jinek et al. 2012), it became the genome-editing technique of choice. The secret? It’s easy, cheap and precise.

Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA (Nishimasu et al. 2014)

Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA (Nishimasu et al. 2014)

Step by step, the CRISPR-Cas9 technology is infiltrating scientific fields. One of the fields where this technology might turn out to be a game-changer is conservation biology. But the future directions of CRISPR-Cas9 implementation to conservation practice have to be thoroughly considered and discussed. Only during the last twelve months, major scientific journals like Nature Review Genetics, Cell, Trends in Biotechnology, and PNAS published reviews on genome engineering for conservation purposes.

So, if phrases like gene drive, guide RNA, and protospacer make your head spin, this post is for you. If you didn’t know they existed, it’s for you too. And even for you who have no idea what the acronym CRISPR actually means. Like me until yesterday.

Prokaryotic vaccination

Clustered regularly interspaced short palindromic repeats-CRISPR associated system, i.e. CRISPR-Cas, is a bacterial and archaeal adaptive immune mechanism that works on the basis of cut-and-paste DNA editing. Approximately 50% of bacteria and 90% of archaea (Makarova et al. 2015) carry a record of previous infections incorporated into their DNA as spacers within the CRISPR system.

“In 2007, it was shown that a spacer matching a phage genome immunizes the host microbe against the corresponding phage and that infection by a novel phage leads to the expansion of the CRISPR array by addition of new spacers originating from the phage genome (Barrangou et al., 2007).” (Wright et al. 2016)

Whoa, spacers, arrays… Let’s slow down again.

The CRISPR locus, called ‘CRISPR array’, consists of an AT-rich ‘leader’ sequence and a series of ~20-50 bp repeats separated by unique spacers, which originate from the DNA of viral and plasmid invaders (Wright et al. 2016).

To put it boldly, it’s basically a prokaryotic way of vaccination.

A simplified scheme of CRISPR array. Source: WikimediaCommons/AnnaJune

A simplified scheme of CRISPR array. Source: WikimediaCommons/AnnaJune

Protospacer incorporation into the CRISPR locus is performed by the Cas1-Cas2 integrase complex. Wright et al. 2016, Fig. 2B.

Protospacer incorporation into the CRISPR locus is performed by the Cas1-Cas2 integrase complex. Wright et al. 2016, Fig. 2B.

The miraculous endonuclease system CRISPR-Cas9

There are different types of CRISPR-Cas systems, but probably the best known is Type II with its famous Cas9 endonuclease. CRISPR-Cas9 is an endonuclease system that, given a guide RNA is provided, accurately targets and modifies specific nucleotide sequences (Champer et al. 2016).

It’s those fancy attributes ‘accurate’ and ‘specific’ that make it so widely used in genome engineering. As Champer and colleagues noted in a review in Nature Reviews Genetics published early this year:

“Remarkably, this system can be designed to universally target virtually any genomic sequence, and has recently been adapted to modify the genomes of yeast, plants, worms, fruitflies, mosquitoes, zebrafish, mice, monkeys and human cells, among others.”

A naturally occurring phenomenon

But before going into how the CRISPR-Cas9 is used in biotechnology, let’s take a look at how it works in bacteria and archaea.

The mechanism needs “DNA to be identified, integrated into the CRISPR locus, transcribed, processed, and assembled into an interference complex that must then begin the search for appropriate targets” (Wright et al. 2016).

Transcription of the CRISPR locus creates CRISPR RNAs (crRNAs), each containing a  ‘protospacer’ sequence, and its flanking repeat sequence. Each crRNA hybridizes with a transactivating CRISPR RNA (tracrRNA) complementary to the crRNA’s repeat sequence and encoded by a gene located within or nearby the CRISPR array. The crRNA-tracrRNA complex then teams up with a Cas9 endonuclease, also encoded by a gene flanking the CRISPR array.

For those of you who work better with visual stuff, just stare at the pictures below as long as you need.

The three stages of CRISPR immunity. Wright et al. 2016, Fig. 1A.

The three stages of CRISPR immunity. Wright et al. 2016, Fig. 1A.

Naturally occurring CRISPR-Cas system. Sander & Joung 2014, Fig. 2a.

Keep your friends close, but enemies closer

Once the crRNA-tracrRNA-Cas9 ‘effector complex’ is ready, Cas9 looks for a specific ‘protospacer adjacent motif’ (PAM) in the phage/plasmid sequence. PAMs work as a way to recognize new protospacer, as well as to identify invaders upon reinfection. In phage and plasmid DNA, PAM is located close to the protospacer, which is not the case in the CRISPR array because the protospacer sequence is incorporated without the PAM. This way CRISPR-Cas9 avoids cleaving itself (Sander & Joung 2014, Wright et al. 2016).

A (near-)perfect match between the spacer and target (= invader’s) DNA means that Cas9 uses it’s endonuclease superpower to cleave both DNA strands of the invader. Checkmate.

To be continued…

In this post, my intention was to finally grasp this CRISPR thing and then reflect on it’s implementation to biotechnology and conservation practice. However, this turned out to be quite an overwhelming task, so I’ve decided to split it into two separate posts.

In the second part I’ll hope to finally get to the way that CRISPR-Cas9 is used in biotechnology for engineering gene drives and what are the potential applications in conservation. I’ll also try to touch upon the question whether CRISPR is the savior or the threat for conservation. So stay tuned.

And btw, if you are looking for some really cool figures of CRISPR-Cas9 structure, check out the paper by Nishimasu and colleagues.

References

Champer, J., Buchman, A. & Akbari, O.S. (2016). Cheating evolution: engineering gene drives to manipulate the fate of wild populations. Nat. Rev. Genet. 17, 146–159. DOI: 10.1038/nrg.2015.34

Corlett, R.T. (2016). A Bigger Toolbox: Biotechnology in Biodiversity Conservation. Trends Biotechnol. In press. DOI: http://dx.doi.org/10.1016/j.tibtech.2016.06.009

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A. & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821. DOI: 10.1126/science.1225829

Makarova, K.S., Wolf, Y.I., Alkhnbashi, O.S., Costa, F., Shah, S.A., Saunders, S.J., Barrangou, R., Brouns, S.J.J., Charpentier, E., Haft, D.H., et al. (2015). An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13, 722–736. DOI: 10.1038/nrmicro3569

Nishimasu, H., Ran, F.A., Hsu, P.D., Konermann, S., Shehata, S.I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. (2014). Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156(5):935–49. DOI: 10.1016/j.cell.2014.02.001

Sander, J.D. & Joung, J.K. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32(4):347–355. DOI: 10.1038/nbt.2842

Webber, B.L., Raghu, S. & Edwards, O.R. (2015). Opinion: is CRISPR-based gene drive a biocontrol silver bullet or global conservation threat? Proc. Natl Acad. Sci. USA 112, 10565–10567. DOI: 10.1073/pnas.1514258112

Wright, A.V., Nu~nez, J.K. & Doudna, J.A. (2016). Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 164, 29–44. DOI: http://dx.doi.org/10.1016/j.cell.2015.12.035

Travis, J. (2015). Making the cut. Science 350, 1456–1457.

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About Patrícia Pečnerová

I am a PhD candidate at Stockholm University. I study paleogenomics of the last population of the woolly mammoth before its extinction and I am mainly interested in combining ancient DNA, population and conservation genomics to trace pre-extinction changes in genetic diversity.
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