What kind of technology is CRISPR?
In today’s booming research on epigenetics, stem cells, and cancer, it seems that everyone’s attention is focused on more “advanced” organisms. However, the discovery and application of the prokaryotic CRISPR system once again proved the presence of “l(fā)ower” organisms - who says these “l(fā)ittle things” can’t have sophisticated and complex systems? The CRISPR system not only enriches our understanding of the physiological mechanisms of bacteria and archaea, but more importantly, the modification and utilization of this system can bring about a technological revolution that sweeps the entire field of molecular biology and updates the existing operation mode. At the same time, it also opens a window for us to re-recognize the regulation network and mechanism of self and mutual between the entire microbial world from the perspective of CRISPR, the similarities and differences and connections between prokaryotic and eukaryotic cells, and even the evidence of co-evolution. No one can deny that this time, the “l(fā)ittle things” have indeed made a “big deal”.
I.Emerging from obscurity, gaining recognition - bacteria also have a “high-level” acquired immune system.
What exactly is CRISPR? - Its Chinese name is very long and awkward, and like English, I don’t know how to pronounce it, but it can be self-explanatory and understand its characteristics on the gene sequence, that is - clustered, regularly interspaced, short palindromic, repeated sequences (clustered regularly interspaced short palindromic repeats, CRISPR). Since it is a story, let’s sell the mystery to the end and tell you the meaning of these modifiers when we get there.
1.What is it?
The beginning of any great thing (forgive me for making this assertion, but seeing the praise is very abundant, it is not too much), the rise of any influential person, the start of any thrilling story, always inconspicuous and “unremarkable”, our protagonist CRISPR system is no exception. Many reviews have placed the origin of CRISPR in 1987, which was discovered from our most familiar E.coli, the most classic K-12 strain, but with the mentality of digging up the ancestral grave and finding out this article 26 years ago to verify it, it seems that only this point is related to our CRISPR, which is this palindromic sequence.
2.Making a name
Making a name for themselves, earning respect - bacteria also have a “high-level” acquired immune system What exactly is CRISPR? - Its Chinese name is very long and awkward, and like English, I don’t know how to pronounce it, but it can be self-explanatory and understand its characteristics on the gene sequence, that is - clustered, regularly interspaced, short palindromic, repeated sequences (clustered regularly interspaced short palindromic repeats, CRISPR). Since it is a story, let’s keep the mystery until the end and tell you the meaning of these modifiers when we get there. What is it? The origin of any great thing (forgive me for making this assertion, but seeing the praise is very abundant, it is not too much), the rise of any influential person, the start of any thrilling story, always inconspicuous and “unremarkable”, our protagonist CRISPR system is no exception. Many reviews have placed the origin of CRISPR in 1987, which was discovered from our most familiar E.coli, the most classic K-12 strain, but with the mentality of digging up the ancestral grave and finding out this article 26 years ago to verify it, it seems that only this point is related to our CRISPR, which is this palindromic sequence.
3.What do they do?
This discovery was purely accidental, and did not attract much attention from the discoverers themselves, let alone have a name for this characteristic sequence, until 2002, when it was found by computer operation that many prokaryotes (true bacteria and archaea) have similar non-repetitive sequences separated by 21-37bp palindromic repeats, and then it officially had this self-explanatory name CRISPR, clustered, regularly interspaced, short palindromic, repeated sequences (clustered regularly interspaced short palindromic repeats, CRISPR). And in addition to such characteristic sequences, there are also some CRISPR-associated genes nearby. These are the series of Cas proteins that we later said play a role in cutting and integrating exogenous fragments. So we can basically get this beautiful schematic diagram and know the three most important components in the CRISPR system - spacer (white box), palindromic repeats (double triangle), cas and other genes.
As with most scientific discoveries, the discovery of the CRISPR system went through a process of figuring out: what is it? - what is it called? - what does it do? Now we already know what it looks like and what its features are, but for a long time there were different opinions about what this strange sequence does. The function obviously has something to do with the sequence specificity of the spacer, so is it chromosomal rearrangement? modulation of expression of neighboring genes,target for DNA binding proteins,？replicon partitioning, or DNA repair? They all make sense but they are just guesses, until 2005, when data analysis from three independent groups showed that these spacers are derived from exogenous DNA, viruses or plasmids.?3?This made people wonder if this would be involved in defending against foreign DNA? In 2007, an experiment with Streptococcus thermophilus confirmed this guess, and after continuous improvement, we have this gorgeous schematic diagram below, a beautiful immune process that can only be described as elegant. A beautiful acquired immune system - who says this is the specialty of “higher” organisms? How to explain this beautiful system, I think it is easy to understand by referring to the antigen-antibody immunity we have learned. Here I use a popular analogy (as shown in Figure 3) to briefly explain it. Exogenous DNA is the bad guy, and one of the most core steps to capture the bad guy’s characteristics is - relying on the Cas complex to make the fragment characteristics proto-spacer into spacer, integrated into the CRISPR sequence, forming memory. So in the next two immune responses, spacer can quickly bind to proto-spacer. Complete this precise strike process of the target. Kill the invader. I guess you have also noticed a special “hat” PAM (also mentioned below) which appears here as a mechanism to prevent self-immunity. It is not hard to imagine that it also restricts the choice of proto-spacer at the same time.
How about that? Isn’t it shocking enough? No exaggeration to say that this flow chart really shocked my old thinking. Don’t look down on prokaryotes! They have such a sophisticated system that is not inferior to eukaryotic acquired immunity. We have to: 1) marvel at this wonderful world - the wisdom of the Creator and the prokaryotic little things; 2) ask ourselves - we really know too little about nature and biology. Of course, CRISPR is not the only defense system of prokaryotes. A recent NAR paper [15] also summarized and compared various immune mechanisms of true bacteria and archaea horizontally. But there is no doubt that the discovery of CRISPR greatly expanded our understanding of the physiological mechanisms of prokaryotes. According to the latest official statement [31], CRISPR systems have been found in 48% of true bacteria and 95% of archaea. It can be considered ubiquitous. In 2007, a French research group developed a CRISPRFinder [12], which allows people to upload sequences to identify whether CRISPR is contained in the genome. Thus statistics on the ubiquity of CRISPR in prokaryotes are obtained.
CRISPR systems are classified into two classes and six types based on the structure and function of the Cas proteins that are involved in the immune response. Class 1 includes type I, III and IV, and class 2 includes type II, V and VI. Each type is characterized by distinct architectures of the effector modules that include unique signature proteins.
CRISPR systems also have many types, and different bacteria contain different types of CRISPR. The figure above is a summary of the mechanisms of several types from a very good review published in 2013.Here, I also want to remind you to pay special attention to type-II, because in the following story, Cas9 will play an absolute protagonist role. Familiar, are they from the same root? After looking at the flow chart above, I think everyone will feel “familiar”. Yes, after looking at the comparison chart below, you will feel more about the similarities between prokaryotic and eukaryotic mechanisms. Perhaps, our gap is not as big as we imagined.
II.Dig deeper, use it for “me”, hit wherever you poin
Cas9 and other mechanism studies and tool transformation If the story ends here, it would be interesting, but at best it would only be a recognition of nature. In fact, the real show is just beginning. As the importance of CRISPR gradually emerged, the study of its mechanism of action also deepened step by step. Various aspects and types of protein functions became clearer. For example, how to integrate spacer into the genome? In this process, the model of Csn2 function was established.
Grafting, vaccinating bacteria!?
So someone thought, since this looks like a good immune system, since the system in E. coli seems not strong enough, can it be transplanted? The answer is yes. In 2011, on the NAR journal, a French research group?4?did just such a thing - they transplanted the Type-II CRISPR system from Streptococcus thermophilus thermophilic streptococcus into E. coli using a plasmid system, and it worked! So they happily concluded that CRISPR can be used to vaccinate bacteria, we understand their enemies, we can take the initiative to defend, preemptive. At the same time, almost “incidentally”, they also found two things,
Cas9 “one pill works” - as the only one needed, enough to work cutting protein (strong enough).
Cas9 “l(fā)eft and right arms” - works depending on McrA/HNH- and RuvC/RNaseH these two motifs. Since then Cas9 has also walked into our sight from so many types of CRISPR proteins.
So the first time, they proved that CRISPR can not only immunize itself, but also heterologously express and immunize others, this is a “vaccine”, very interesting. However, from the subsequent development of the story (if I did not miss too much in the logic I sorted out), they really wasted a treasure and used it poorly. And the importance of the results of this paper was also seriously underestimated by the researchers themselves. It is not that their happy conclusion and bacterial vaccine are not important, but that they only saw through one layer, did not see a deeper layer, did not see further. Why do I say that, because although they did not see it, someone did. And the real secret of success lies in those two incidental discoveries. “One pill works”, decisively transformed - from then on the sword pointed directly! If you want to erect a milestone in the research of CRISPR system, I think?2?he is. If you want a turning point in this story, I think he?2?is too. From here on, the legend of CRISPR will begin to unfold, and the curtain of a new generation of molecular biology revolution will soon open! On August 17, 2012, a seemingly low-key Science paper, looking at those gel pictures and data is not very interesting, here I use my language to briefly summarize what they mainly did: Yes, like a running account, to here this article looks not difficult, nor any bright spots. But did you notice carefully, to here, they have step by step to clear up every step of Cas9 as a directional endonuclease mechanism, all aspects.
By the way, there is another overlooked (at least in this paper did not make a big move) surprise - two “l(fā)eft and right arms” domains each cut a chain, HNH domain cuts complementary DNA strand, RuvC-like domain cuts noncomplementary DNA strand. Then they said, they cleared up all aspects, but is this just a mechanism study? Wrong! They have a very clear purpose - sword pointed directly! Can be artificially customized specific endonuclease! For my use, hit wherever you point. They then used these findings to design and do an in vitro cutting of the GFP gene experiment, and sure enough succeeded. And they are all cut according to the expected design of the cut, is this not “want to cut where to cut where, mom no longer have to worry about my topic”?. At the same time, it is inferred that due to the restrictive PAM sequence NGG is very common, basically the use of restrictions is very small - expected to be able to achieve see people kill people, see ghosts kill ghosts! And two must-have RNAs can already “double swords together”, which can make the construction of RNA more convenient and more stable. A new “killer” is about to emerge.
III “Continental” missiles, kill all living beings?
Gene editing, synthetic biology tools in a variety of eukaryotic and prokaryotic cells The prelude just now probably only excited the people who are interested in this field (and many people also started to enter the field from paying attention to this paper, starting to grab the beach), but what really made CRISPR system enter everyone’s vision, issued a voice that can not be ignored, triggered a shock is the next two papers. Although we tell this story from the beginning you feel as if there is this paper logically, but at first glance, the results of the two papers are really tempting, too beautiful. Indeed, in just five months, they took a big step forward, an important big step. Although it is two papers, the meaning is one, they are very interesting, back to back, printed in the same period of Science, one before and one after. The first paper?is Cong Le’s senior’s paper, and the second paper is also not small, it is George M. Church’s group’s work. (It should be said that you can see that the second paper was almost “forced” out by the first paper, there is a kind of rushing to publish without well-organized feeling, but actually did a lot of things, specifically later. There is also a tragedy of the same third paper , came late, had to nature sub-journal.) The breakthrough of these two papers is - in human cells, successfully achieved Cas9-mediated genome directional editing. (Note that it is editing, not simply cutting. Well, Genome Editing, doesn’t it sound high-end and classy.) “Continental” missiles, deep into the enemy mirror, precise guidance, successfully completed.
This is a very vivid metaphor, used to describe how the CRISPR/Cas9 system enters the cell nucleus and edits the target gene. Cas9 protein can be seen as the warhead of the missile, it can recognize and cut the DNA double strand. sgRNA can be seen as the guidance system of the missile, it can guide Cas9 to the correct position by complementary pairing with the target DNA. In order to make this missile system enter the cell nucleus, it also needs to add a nuclear signal (NLS) to Cas9 protein, just like adding a rocket booster to the missile. With these components, CRISPR/Cas9 system can achieve efficient, precise and flexible gene editing in cells.
An article in April 2013 showed that the infectivity of the bacterium Francisella novicida depends on its own CRISPR system. When growing in mammalian cells, the bacterium shuts down its own lipoprotein synthesis gene through the CRISPR system to avoid being detected and destroyed by the host immune system. “If the host’s immune cells are likened to sharks in the sea, then for them, bacterial lipoproteins are like blood in the sea, full of attraction. Therefore, in order not to be detected by the immune system, bacteria must shut down lipoprotein production.” Amazing! “In addition to cutting phage genes, Cas9 can also regulate bacterial genes, which is a new discovery,” It seems that CRISPR system is a center of a comprehensive regulatory network between prokaryotes and between prokaryotes and eukaryotes, a means and method for prokaryotes to resist adverse external environments, not as simple as cutting exogenous genes. It also gives us an opportunity to re-understand the wonderful “microbial world” from the perspective of CRISPR. Of course, there are more reports on CRISPR?to guide us to continue to pay attention.
CRISPR is a remarkable discovery that has revolutionized the field of gene editing and opened up new possibilities for biotechnology and medicine. By harnessing the natural defense mechanism of bacteria and archaea, scientists have developed a powerful tool that can precisely and efficiently modify the genomes of various organisms, from bacteria to plants and animals, including humans. CRISPR has many potential applications, such as creating disease-resistant crops, developing new therapies for genetic diseases, and even altering human traits. However, CRISPR also poses ethical and social challenges, such as the risk of unintended consequences, the possibility of misuse or abuse, and the impact on human dignity and identity. Therefore, CRISPR requires careful regulation and oversight, as well as public engagement and education, to ensure its responsible and beneficial use for humanity and the environment.
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