17. October 2020

CRISPR/Cas9: the beginning of a new era in life sciences


A 5-minute read

Since the term “genetic engineering” was introduced in the 1970s, the obstacles hindering its headway towards labs and industry were mostly related to the complex process of genome editing. In the last decade, a tongue-breaking abbreviation CRISPR-Cas9 has emerged as a fast and precise tool to modify genetic material, and has set molecular life science to a fast track to plant engineering, drug development and treatment of genetic diseases. As Jennifer Doudna and Emmanuelle Charpentier receive this year’s Nobel prize for chemistry, the CRISPR-Cas9 officially becomes a beginning of a new era in life sciences and a grand finale of a story about bacteria. So, what are CRISPR and CRISPR-Cas and CRISPR-Cas9, and what do dairy producers, structural biology and X-rays have to do with it?

Side observations, yogurt and immune system

As quite often in science, the phenomenon was first noticed as a side observation in an experiment with a different scope. In 1980s, as a group of scientists cloned bacteria, they saw a peculiar pattern in their genetic material: short sequences of DNA were repeated in the helix, and separated by unrelated sequences [1]. This brief note started drawing attention fifteen years later, when similar motifs were found in the DNAs of a number of bacteria and archea [2]. Is this just a coincidence? Investigations of bacterial DNA were picking up, and as it was found that the pattern always occurs in proximity of proteins [3, 4], the abbreviation CRISPR-Cas was born. Unusual pattern of bacterial DNA (short repetitions of DNA segments, separated by unrelated sequences) was baptized as “Clustered Regularly Interspaced Short Palindromic Repeats” (CRISPR), and the “CRISPR-associated” proteins Cas.

Although the function of Cas proteins nor the unusual DNA pattern had not been figured out yet, it was suspected that the CRISPR-Cas might have to do with bacterial “immune system”. Confirmation came from an unexpected source: dairy industry. Researchers at Danisco exposed a bacterium used for production of cheese and yogurt to a phage, a virus that infects bacteria, showed that this triggers an adaptable and hereditary defense against the invader [5]. As a closer look into bacterial CRISPR region revealed that the “separation sequences” (spacers) are elements from invading organisms, molecular life science was on a brink of revolution. Bacteria can cut invading DNA and insert it into own CRISPR regions, but how? Do bacterial Cas proteins cut the virus DNA? If so, how is the foreign DNA recognized?

CRISPR-Cas mechanisms: simplicity is the ultimate sophistication

Shedding light on CRISPR-Cas systems involved a range of interdisciplinary fields, including structural biology. Modern X-ray crystallography and cryo electron microscopy techniques yielded crystal structures of many Cas proteins and Cas complexes that occur during bacterial defense against invading DNA [6]. Although these structures helped to reveal a number of diverse CRISPR-Cas mechanisms, the one that has drawn the most attention was the simplest one – the Type II. 

Similar to other CRISPR-Cas mechanisms, Type II also relies on three basic steps to defend bacteria from the invading DNA: (1) recognition of particular fragments of invading DNA and incorporation of the fragment in the host CRISPR sequence (2) cleaving the CRISPR sequence into a specific RNA and (3) assembling the new RNA with Cas proteins, whereas the RNA guides the Cas to recognize and cleave the invading DNA in order to prevent the propagation of the foreign genetic material. However, while other mechanism types employ elaborate number of Cas proteins and their complexes to carry out this task, the Type II relies only one key protein: Cas9. This protein was found to participate in the processing of RNA and in destruction of the target DNA.

The revolution: single guide RNA opens up many gene-editing options

The Cas9 protein, however simple and powerful it is, still needed two RNAs to guide it in its task. In 2012, Jennifer Doudna and Emmanuelle Charpentier fused the two RNAs and re-engineered Cas9 to use this “single guide RNA” to locate the sequence where the DNA would be cut [7]. But how could this be used for gene editing? If a tailor-made single guide RNA is introduced into the system, the Cas9 is prompted to recognize and cleave a DNA at a site predetermined by the tailor-made single guide RNA. With this, CRISPR-Cas9 soon found its use for editing genes of many cells and organisms, and the research has continued its fast pace. One focus was to understand the structural basis of CRISPR-Cas9 mechanism, and the crystal structure of Cas9 was revealed from X-ray diffraction data, collected at the Swiss Light Source using the PILATUS detector [8] and at the beamlines of the Advanced Light Source. The other focus was to understand other Cas9 and Cas proteins. To date, three variants of the Cas9 have been reported, and many research groups are working on characterization of other Cas proteins that can be used for genome editing [9]. One of them is Cpf1, whose structure was solved in 2016 as the first structure obtained from the EIGER X 16M data [10].   

A fast track to future

The potential of the CRISPR-Cas genome scissors to efficiently cut, edit and mend any DNA seems to be unlimited. The method is powerful and precise, and the admission costs low: scientists can design their specific guide RNAs and make own CRISPR systems. Libraries of tens of thousands of guide RNA have been established, capable of targeting 90% of human genes.

While molecular life science is heading towards enormous progress in genome editing and its applications in prevention and treatments of diseases, some ethical issues still remain unresolved. But, that is another topic.


References

[1] Ishino, Y. et al. (1987) J. Bacteriol. 169(12), 5429-5433.

[2] Mojica, F.J.M. et al. (2002) Mol. Biol. 36(1)

[3] Tang, H-K. et al. (2002) PNAS 99(11), 7536-7541.

[4] Jansen, R. et al. (2002) Mol. Biol. 43(6), 1565-1575.

[5] Barrangou, R. et al. (2007) Science 315, 1709–1712.

[6] Jiang, F. & Doudna, J. (2015) Curr. Opin. Struct. Biol. 30, 100-111.

[7] Jinek, M. et al. (2012) Science 337, 816-821.

[8] Jinek, M. et al. (2014) Science 343, 1247997

[9] Jiang, F. & Doudna, J.A. (2017) Annu. Rev. Biophys. 46, 505-529.

[10] Yamano, T. et al. (2016) Cell 165, 949–962