CRISPR-Cas in pictures

Just about complicated: CRISPR/Cas

Olga Volkova, "Biomolecule"

CRISPR/Cas is a system of adaptive immunity of bacteria and archaea, which was also useful for eukaryotes. We tried to reflect this mechanism very clearly, which caused an explosion in the biological community and probably greatly changed the future of science and humanity. From this infographic you will learn a brief history of the study, the mechanism and possible applications of the CRISPR/Cas system.

CRISPR/Cas, or CRISPR-Cas. This rather euphonious abbreviation by biological standards ennobles the term clustered regularly interspaced short palindromic repeats/CRISPR-associated genes (proteins). In the Russian substring: grouped regularly alternating short palindromic repeats and associated genes (proteins). Wikipedia offers such a first part: "short palindromic repetitions, regularly arranged in groups," and a post–science light version: "short palindromic cluster repeats".

Strikingly, in less than three decades, the CRISPR-Cas system has transformed from a "strange sequence of unknown biological purpose" into a promising genomic editing tool [8, 9]. Let's try together with the authors of a wonderful infographic to tell about this complex tool simply.

If you want to know how it was mastered, welcome to the page "CRISPR–epic and its heroes" with a brief summary of the amazing story told by a direct participant in the events, Eric Lander. The article "From words to deeds: CRISPR-Cas technology was used for the first time for the treatment of oncological diseases", provided with expert commentary, reports on the latest victories of CRISPR-Cas9 technology. – Ed.

How does the immune system of prokaryotes work?

CRISPR-Cas systems have been found in almost all known archaea and half of bacteria. More often they are on the chromosome, less often they are in the composition of phages (bacterial viruses) and other mobile genetic elements. These systems consist of two main blocks: a CRISPR cassette and an adjacent cluster of cas genes. A cassette is a block of direct almost palindromic ("mirror", mutually complementary sequences capable of folding into hairpins) repeats with a size of 24-48 nucleotide pairs. These repetitions are interspersed with spacers – unique inserts of approximately the same length. Spacers are identical to various sites of phages and other mobile elements that have ever penetrated this cell or its ancestors. The number of repetitions in different systems varies from units to hundreds.

Thus, CRISPR can be considered a collection of repeat-separated "photos" of violators of cellular boundaries. This collection is compiled by simply borrowing their pieces, and in order to resist the new invasion of the same molecular agents, the collection must be regularly "viewed" and updated. This function requires a leader sequence preceding a series of repetitions. It is rich in "fusible" AT-pairs and contains a promoter that controls the transcription of the CRISPR cassette ("viewing the collection").

cas genes encode proteins that take on the brunt of the work of embedding spacers and destroying agents with identical sequences (protospacers) and help process the CRISPR transcript: divide the photo garland into separate portraits. The destruction function is performed by Cas proteins called effector proteins. Depending on the type of effectors, all CRISPR systems are divided into two classes: in Class I, the target is destroyed by a multi–protein complex, and in class II - by a single large protein. Further, these classes are divided into six types. Most effectors attack DNA, only one – exclusively RNA [10], rare – both molecules. One organism can contain several different systems, and spacers differ in different cells even in the same population.

You can find out what this leads to from a competitive article about bacteriophages and the eternal arms race in the phage and bacterial worlds: "Bacterial eaters: murderers in the role of saviors" [11]. By the way, there are a lot of interesting author's electronic images of phages

For solving engineering problems, a type II system belonging to Class II is most suitable – it is the simplest. It is its effector protein called Cas9 – the very designation that appears in modern genome editing systems.

How is CRISPR-mediated immunity formed?

If a virus penetrates into a bacterium or archaea equipped with a CRISPR system, the adaptive functional module of the system is activated: specific Cas proteins - in all systems it is at least Cas1 and Cas2 - cut out the fragments they like from the alien. In some cases, an effector protein also helps to choose a protospacer. Proteins choose sites next to a special sequence of PAM (proto-spacer adjacent motif) – only a few nucleotides, but not the same for different CRISPR systems. Then the same adaptive proteins embed the fragment into the CRISPR cassette, always on the one hand - in the leader sequence. So a new spacer is formed, and along with it – a new repeat. This whole process is called adaptation, or acquisition, and in fact it is the memorization of the enemy. Information about all remembered enemies is received by all the progeny of the cell during divisions.

How is CRISPR-mediated immunity implemented?

To search for re-invading agents, the CRISPR cassette must be expressed. As a result of its transcription, a long RNA molecule is formed – pre-sgRNA. With the help of RNase III and, as a rule, Cas proteins, the transcript is cut into repeats into separate SRNA molecules containing one spacer and pieces of the repeats surrounding it (one of them is longer). In type II systems, this process, called maturation, requires another participant – tracrRNA (trans-activating CRISPR RNA), which is encoded next to the cas cluster [12].

Further, in Class I systems, sgRNA interacts with a complex of Cas proteins, and in class II systems, sgRNA or tracrRNA-sgRNA duplexes bind to a single effector protein, for example Cas9. This is how an interference functional module is formed – a working immune unit consisting of a guide RNA and an effector protein (or complex). The totality of such units "scans" the cell in search of interventionists.

When a complementary SRNA sequence is detected, that is, a protospacer, the module "sticks together" with it and determines whether it is marked as "its own", cellular. If not, and if the same PAM is adjacent to it, then the effector protein, which is an endonuclease, cuts both DNA chains in strictly defined places. The whole process is called interference. In a special case, in a type VI system, RNA interference occurs because the effector protein is a ribonuclease and destroys RNA. One way or another, the attacked phages or plasmids are disabled. Well, there is an extra opportunity to "steal" new spacers.

What problems can arise when implementing an immune response? It is possible that as we move away from the leader sequence, that is, from the CRISPR promoter, the chances of the spacer being transcribed and maturing decrease. In addition, it is believed that remote spacers may accumulate mutations over time that prevent effective interference with the target, or even be removed altogether. But since the adaptation of new spacers occurs near the promoter, the removed spacers are photos of agents that have not attacked this cell line for a long time, and the cell does not need constant combat readiness in relation to them. Even single nucleotide mutations of the target can become a real problem. In general, complementarity in this case is above all.

And should we tame someone else's immunity?

Having studied in detail the principles of operation of the CRISPR-Cas9 streptococcal system (type II), scientists thought: and why not try to use it to correct the genomes of other organisms? There were new hopes regarding the treatment of genetic (and not only) human diseases, because this method of editing in vivo could be more effective than the ZFN and TALEN nucleases already being tested at that time [13].

All that was required for the new technology was to place the Cas9 protein gene and a CRISPR cassette on the vectors, where the spacers could be made identical to the places of the genome that needed to be changed. By changing the number and type of spacers, several different sections of the genome can be modified at once. It was quickly realized that tracrRNA and sgRNA can be painlessly combined into one chimeric single-guide RNA molecule, and RNase III in eukaryotic cells is quietly replaced by other ribonucleases. Well, it was also necessary to optimize the system for eukaryotic cells: to correct the codon composition and add a nuclear "address" so that it clearly followed the place of work – chromosomes.

The result is a simple and, importantly, cheap two-component system: the cas9 gene and the CRISPR cassette are transcribed in the cell nucleus of the selected organism, the CRISPR transcript is cut into separate CDRNAS that combine with Cas9 proteins and search for a target. When the SRNA finds a complementary site in the genome of an organism, Cas9 cuts both DNA chains "tightly". That's it, the work of the CRISPR system is over. Now the baton is passed to the repair systems of the body itself. They decide how best to patch the incision: whether to simply stitch the pieces (this will be a non-homologous connection of the ends, NHEJ), or, if there is a suitable matrix with flanks complementary to DNA sections on both sides of the gap, put a "patch" (this will be homologous recombination). So, the first option is advantageous if you need to cut something, the second - if you need to insert something or replace a defective DNA section with a normal one, which is simply injected on a suitable vector. Sometimes homology with a paired chromosome is used if the desired locus is not defective on it.

Of course, the technology is not without drawbacks yet. Cas9, for example, may exhibit inappropriate activity, "turning a blind eye" to minor inconsistencies between the SRNA and the target. According to K. Severinov, the main problem is the bioinformatic prediction of targets, since, in addition to the presence of a PAM site, it is necessary to take into account a lot of factors, including the state of chromatin. In addition, the scenario according to which the incision will be repaired does not always correspond to what is desired, so now they are actively looking for factors influencing the choice of this scenario by the cell. In addition to optimizing CRISPR-Cas9 and its delivery mechanisms to the desired cells, other types of CRISPR systems are being tested [14].

The range of applications of CRISPR-Cas9 and its modifications

The application points of CRISPR technology can be conditionally grouped into three large groups: "CRISPR - for research", "CRISPR – for biotechnology" and "CRISPR – for therapy".

• to improve the properties of farm animals and plants. We have already created and tested CRISPR systems for rice, wheat, corn, sorghum and many other crops. This kitchen is analyzed in detail in the review [17]. In addition to improving nutritional qualities, such tools can easily endow crops with resistance to pests and chemicals, and animals can be rid of unwanted genes. For example, recently endogenous retroviruses were inactivated in pig cells (not for the sake of pig health, but keeping in mind plans for transplantation of its organs to humans) [18];
• to control the spread of infections carried by animals. For example, they are already seriously considering the "introduction" of plasmodium resistance genes or population control genes into natural populations of malaria mosquitoes [19]. This has become fundamentally possible thanks to the "gene drive" technology (something like "gene promotion"), based on a change in classical inheritance. With its help, the gene embedded in one individual quickly spreads throughout the population. The principle of the technology is illustrated by the sensational mutagenic chain reaction in fruit flies [20];
• for the construction of new metabolic pathways and the implementation of directed evolution of biomolecules. New or optimized enzyme systems of bacteria and fungi, obtained so easily and cheaply, are the ultimate dreams of technologists from a number of industries. But even a simple embedding of CRISPR-Cas systems with desired properties into industrially important bacterial strains can protect them from bacteriophages and unwanted plasmids.

The advantages of genome correction in the germ line (as a set of any generative cells linking generations of organisms with each other) and stem cells are obvious, but even changes made to somatic cells of already developed organs have an effect. Especially when it comes to fighting liver and muscle diseases. A recent review tells about the results of the therapeutic use of CRISPR-Cas9 in different cell types [21].

A separate promising area is the fight against chronic viral diseases such as hepatitis and HIV infection. If the pathogen persists in the body in the form of a provirus (viral DNA embedded in the cellular genome), then it can simply be cut out. This is exactly what a team of biologists from the USA did, ridding human lymphocytes of HIV (this was reported by two "biomolecular" articles at once: "The Battle of the Century: CRISPR VS HIV" [22] and "CRISPR/Cas9 as an assistant in the fight against HIV" [23]). True, the HIV object is extremely changeable, and you will still have to break spears with it.

One can dream that variants of the recently described type VI CRISPR system will be used in tumor therapy - the one that destroys only RNA, and, as it turned out, any cellular RNA indiscriminately: launching such a system into a cancer cell is like sending a curse on it [14].

CRISPR-Cas is not just about immunity

It turns out that this system means much more to bacteria and their evolution.

Non-canonical activities of CRISPR systems or their individual components appeared as by-products of their immune function or as independently selected signs. Most likely, CRISPR cassettes and Cas proteins once worked separately, and the initial task of the latter was to regulate gene expression and DNA repair [7]. Modern CRISPR-Cas components are noticed:

• in the regulation of gene activity. These systems can interfere with the communication of bacteria by the quorum sensing type [24] and thus regulate group behavior: the formation of fruit bodies and spores in myxococci and biofilms in Pseudomonas aeruginosa. Cas9 proteins (type II systems) regulate the virulence of Legionella pneumophila, Francisella novicida, Campylobacter jejuni and possibly Neisseria meningitidis pathogens;
• in DNA repair. The ability to cut CRISPR cassettes for embedding new spacers is most likely a secondary functional acquisition of Cas1. Initially, he cut typical recombination/repair intermediates and has not yet forgotten how to do it. Therefore, the expression of cas genes increases the resistance of some bacteria to radiation, and disabling the CRISPR system leads to an increase in their sensitivity to DNA-damaging factors and disruption of chromosome divergence.;
• in the remodeling (reorganization) of the genome. Cas proteins sometimes make mistakes and instead of enemy DNA, they make fragments of their genome as spacers. If the "autoimmune" reactions that followed do not lead to cell death, then most often major rearrangements occur aimed at partially or completely getting rid of the "failed" CRISPR system. Sometimes rearrangements can increase the fitness of the host to a niche – for example, by duplicating useful genes;
• in the competition of mobile genetic elements with each other, if they carry these very CRISPR-Cas systems;
• in the introduction of bacteria into a "dormant", inactive state. This function is essentially also immune, but extreme. It is assumed that in selected CRISPR systems, one of the Cas proteins can serve as a "toxin" and its partner as an "antitoxin", and when a phage enters the cell, the "toxin" is released and begins to destroy any RNA. But the cell that "freezes" at the same time has time to frantically collect spacers. If this does not work, then the raging Cas-toxin leads the cell to suicide. It has not been possible to prove this principle yet [7]. But we have already seen such a dramatic finale somewhere: this is exactly what the type VI system does. This outcome of phage invasion is called abortive infection. It's bad for the cell, good for the population...

The infographic was made jointly with Pavel Chirkov, Master of the Faculty of Political Science of St. Petersburg State University. You can download it in one file here

Literature

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Portal "Eternal youth" http://vechnayamolodost.ru  28.11.2016