In the late 1980s and mid-1990s, genomes of diverse lineages of bacteria and archaea (the latter representing a domain of single-celled prokaryotic microorganisms) revealed clusters of regularly interspaced short palindromic repeats, known today under the abbreviation CRISPR. Later it was found that these repeat sequences (previously considered disparate) share a common set of features.
In 2005, a link between CRISPR arrays and protection of the host against invading genetic elements has been established, which boosted a myriad of genetic and biochemical studies exploring the intricate details of this complex genetic barrier. This in turn led to the discovery of CRISPR-associated (Cas) proteins, which are (alongside repeats, spacers and partially conserved leader regions) essential functional components of this astonishing type of adaptive immune system.
Today, CRISPR/Cas9 is successfully adapted for genome editing of various organisms, offering a revolutionary technique for researchers around the world. It offers a number of advantages over other genome editing approaches (such as transcription activator-like effectors and zinc fingers).
Steps of CRISPR-Cas action
As it is the case with other types of immune systems, CRISPR-Cas systems function on the principle of the “self-nonself” discrimination. In short, the goal is to incorporate snippets of foreign DNA (also known as spacers) into a CRISPR locus between a series of short repeats, followed by the transcription of the loci and the processing of those transcripts to generate small RNAs (crRNAs), which subsequently guide endonucleases that target invading DNA.
Therefore the action of CRISPR-Cas system is usually divided into three steps. An initial step is the acquisition phase where alien DNA is integrated in into the bacterial genome, i.e. the adaptation of spacer incorporation takes place. Cas1 and Cas2 are proteins that form a quasi-autonomous and conserved complex pivotal for this step.
Second step is crRNA biogenesis, where CRISPR loci are transcribed and processed into those small RNAs. It takes place either by employing an RNA endonuclease complex or through an alternative mechanism with bacterial RNase II and an auxiliary RNA species. Mature crRNAs can be bound by one Cas protein (this is where Cas9 comes to play), but also by several other Cas proteins, which is the basis of classification of different CRISPR systems.
Third and final step is DNA or RNA interference, where recognition and destruction of target nucleic acid takes place by joint activity of crRNA and Cas proteins. These three steps form an effective stand-alone system that can take place in an individual cell, which is a prerequisite for organisms with unicellular behavior.
Classification of CRISPR systems and the role of Cas9
As there are a plethora of diverse Cas proteins, CRISPR loci, as well as potential for horizontal transfer events, the classification of CRISPR-Cas systems is quite a cumbersome task. The most commonly used classification distinguishes three types of CRISPR-Cas system, each with several subgroups. A recent proposition of a fourth type is still controversial in the scientific community.
Type 1 system is determined by the occurrence of the hallmark protein Cas3, which contains both DNAase and helicase domains used to degrade the target. Six subtypes of this system have been identified, and besides cas1, cas2 and cas3 genes, all of them encode a Cascade-like complex that binds crRNA and pinpoints the target.
Type 2 system is somewhat unique in comparison to the other CRISPR-Cas systems, since only one Cas protein is actually needed for gene silencing – this is the Cas9 protein. It is involved in the processing of crRNAs, but is also in charge of the target DNA destruction. The simplicity of this system makes it complaisant to conversion for genome editing, which is a rapidly progressing field that excites biomedical researchers around the world.
Type 3 system is determined by the occurrence of the hallmark protein Cas10, whose functions are still not completely clear. The specificity of this CRISPR-Cas system has the possibility to target both DNA and RNA (whereas type 1 and 2 systems target only DNA). Alongside type 1 system, it can be found in bacteria and archaea alike (in comparison to type 2 systems that are found exclusively in bacteria).
All the aforementioned CRISPR-Cas systems are considered to be mobile genetic elements that exhibit frequent horizontal transfers, which explains their high prevalence in the prokaryotic world. The degree of their variability can be used as a marker of the diversity and evolution of certain species, and it can also be seen as a genetic record of “vaccination” events (reflecting the changes in environmental conditions over time).