Cute Dragon Lab successfully analyzed the molecular structure of “the smallest Cas9 in history”

CRISPR ushered in a new era of treatment for genetic diseases. Tools such as the popular CRISPR-Cas9 have been designed in the lab to cure many genetic diseases, but these tools are too large to deliver effectively to patients. A major work in the scientific community has focused on trying to miniaturize CRISPR tools small enough to accommodate current delivery methods, such as adenovirus-associated virus (AAV) vectors. When it comes to miniaturizing Cas9 tools, there is currently no manual boot method that has been very successful.

Cornell University’s Cute Dragon Lab solved this size problem by successfully resolving the molecular structure of the “smallest Cas9 in history” — a type based on iscB, an ancient relative of Cas9 found in transposons, which is only about one-third the size of Cas9.

The work, titled “Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9,” was published in the May 26 issue of the journal Science.

A glimpse of “a thousand years at a glance”?

In recent years, studies have shown that The Isc family of cognates is widespread in Cas9. Zhang’s lab at Harvard University has shown that these Isc families may be the ancient ancestors of the Cas9 protein, with a smaller size and wider distribution in the biological world, and even identified multiple ISCB loci in the chloroplasts of a terrestrial green algae in eukaryotes. As the most powerful gene editing tool at present, Cas9 won the Nobel Prize within a few years after discovering its function, and its function and brilliance needless to say, but from the perspective of evolutionary biology, how did such a powerful Cas9 evolve? Can we trace our ancestors back to Cas9? In other words, can we see what the ancestors of Cas9 really looked like?

“These systems inside bacteria are constantly being selected every minute – nature has basically rolled the dice billions of times and come up with these very powerful tools. Now, by capturing their ancestral faces in high resolution, we can better define and utilize them…”Professor Cute Dragon said.

From the captured high-resolution images, the HNH domain of IscB and Cas9, the Bridge-RuvC domain, maintains a high degree of homology with Cas9. And the gRNA part of IscB (the chimera of guide RNA and ωRNA) and the sgRNA of Cas9 are also highly coincident, and these high-resolution image comparisons fully confirm that IscB is the evolutionary source of Cas9. From this point of view, it took millions of years for IscB to evolve to Cas9, and now we have captured its conformation, which is called “one eye ten thousand years”, even if you shine again, you still have to look at me for thousands of years.

Millions of years of “bucket to star shift”?

Now everyone uses Cas9, such as SpyCas9, with a length of 1384 amino acids, while IscB has only 496 amino acids, and this work can be optimized to finally get a version of 450 amino acids but the activity is unchanged. This version is less than a third of Cas9’s. Therefore, a question that lies in front of everyone is how such a small ancestor was assembled, and what did it rely on to complete the similar functions of Cas9? Or which “genes” does Cas9 inherit from IscB?

Through high-resolution structural analysis, the Cute Dragon Lab found that although The protein size of IscB is small, it is fused into the guide RNA through the ωRNA system, and then the remaining ωRNA is replaced with part of the Cas9 protein to achieve a smaller size. By replacing the protein components in the larger Cas9 with omegaRNA, the IscB protein remains the center of the core chemical (DNA cleavage) reaction. Although IscB has a longer RNA backbone (omegaRNA is 100 bases longer than Cas9’s sgRNA), structural comparison found that IscB used this extra 100 nt RNA to replace Cas9’s function of nearly 1,000 amino acids, which is amazing that “at the beginning of life, I had already figured out how to perform life activities more energy-efficiently.”

Of course, the evolution of how is the omegaRNA of IscB was later replaced by Cas9 into a protein is still unknown, and we can only say that the change of macromolecules has been completed through millions of years of “bucket to star shift”. IscB or Cas9, as the immune guardians of different stages of life’s evolution, have not been vigorous, and have been satisfied. No matter how you fight and move the stars, or fight to the point of running forward… If one day, you don’t remember anymore, I don’t remember anymore, but someone will definitely find each other for us… In fact, life is the same.

What’s the use of discovering the most mini Cas9?

“There is a lot of interest in miniaturizing Cas9 to expand its use. For example, a Cas9-based genome editor needs to be packaged into a mature delivery tool, such as an adenovirus-associated virus (AAV) vector. Neither structure-guided nor directed evolution has been particularly successful in miniaturizing RNA-guided nucleases. Professor David R. Liu of Harvard University said.

Gene editors need to be small so that they can put them into viral vectors and then further deliver them into the cell. Professor Cute Dragon said: “There are a lot of super eye-catching applications that further require editors to fuse with other enzymes and functions, and they are on a much larger scale. “If we can put them into viral vectors, then we can achieve a better way of delivering them to patients.”

The miniaturization of gene editing has always been a tireless task for scientists. The capacity of the popular AAV delivery tool is only 4.7 kb. And Cas9 itself occupies close to 4kb, coupled with the sgRNA expression region, and now open up more and more complex synergies (such as cytosine deaminase), it is easy to exceed the upper limit of AAV, and “inch-by-step”. If a super-small replacement with the same editing capabilities can be found, it could spark a future gene-editing revolution.

The IscB is only 1.3 kb, and the guide-ωRNA totals only about 0.2 kb. So there’s still a huge amount of room for scientists to fill it. As long as you use your imagination, this space will always have your share.

What is valuable is that Zhang Feng’s team has confirmed that LSCB can achieve the maximum insertion/deletion rate with 16 nt guides, coupled with its ultra-small size, so it is indeed expected to become a candidate for the next generation of genome editing tools.

And these jobs at cute dragon labs undoubtedly cast a vote for the candidate. “It’s about understanding the structure of molecules and how they react chemically,” said co-first author Gabriel Schuler, a phD student in the field of microbiology. “Studying these ancient Cas9 structures gives us a new starting point to generate more powerful and easy-to-use gene-editing tools.”

How does the most mini Cas9 hedge (off target)?

In addition, as a basic trait of gene editing tools, it is low off-target and high efficiency. So in what way to avoid off-target and prevent self-harm? Through the comparison of multiple states, the researchers found that when the guide sequence of 16 bases and target DNA were not fully complementary, the Non-target strand would hinder the HNH nuclease from binding to the target strand in the form of steric hindrance, and the result was that neither strand would be cut, thus ensuring safety. The guide sequence and target DNA are fully complementary paired, and after determining that it is the “right person”, the state’s larger R-loop space will allow the HNH domain to bind to the target strand, which will trigger the cleavage, and the movement of the HNH will push the Non-target strand to the RuvC nuclease, triggering the second step of cleavage. Step by step, not impatient, everything is agreed upon, can not be changed.

IscB functional mechanism diagram: in the absence of a determined target DNA substrate (left), non-target strand DNA (NTS) hinders HNH nucleases from approaching the target strand (TS) through steric hindrance, thereby preventing off-target miscision; when the correct substrate is determined (in the middle), a larger R-loop allows HNH nucleases to bind to TS and cleave; HNH will further push NTS to RuvC nuclease. A second step of cutting is performed, culminating in a double-strand break.


Co-first author Chunyi Hu, a postdoctoral researcher in the Department of Molecular Biology and Genetics, said a better understanding of the function of RNA was a motivation behind the study. “There are also many mysteries — such as why do transposons use RNA to guide the system? What role does RNA play? Can RNA itself evolve to a smaller size? There’s a lot more work to be done in the future…”

One challenge researchers still face is that while IscB-ωRNA is so active in test tubes that it can complete the cutting of double-stranded DNA in minutes, it is not as effective at altering chromosomal DNA in human cells. The next step in their research will be to use molecular structures to explore and optimize what they have identified as causes of low cell activity in humans. (Source: Science Network)

Related paper information:DOI: 10.1126/science.abq7220

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