Discovered a new self-destructing CRISPR system

CRISPR is the acronym –proposed by the Spaniard Francis Mojica– for what in Spanish are ‘clustered and regularly spaced short palindromic repeats’, in fact bacterial immune systems. Over the past two decades, it has captured the imagination of scientists and laypeople alike for its gene-editing potential, winning Nobel laureates Jennifer Doudna and Emmanuel Charpentier.

Now, researchers from the United States and Germany describe in two Nature articles the structure and function of a newly discovered CRISPR immune system that, unlike other better known ones that turn off foreign genes to protect bacterial cells, these ‘turn off’ the who are infected to prevent them from spreading the infection.

“With this new system, known as Cas12a2, we are looking at structure and function unlike anything seen so far in CRISPR systems,” says Ryan Jackson, professor at Utah State University (USU) in the US. UU and co-author of both papers.

CRISPR-Cas12a2 ‘turns off’ cells that are infected to prevent them from transmitting the infection

Also participating in the study are other scientists from USU, the University of Texas (UT) at Austin, Germany’s Helmholtz Institute for Research on RNA-Based Infections and the American biotechnology company Benson Hill, which first identified the amino acid sequence of this romance. bacterial immune system.

In some ways, it’s similar to the better-known CRISPR-Cas9, which binds to target DNA and cuts it – like molecular scissors – effectively disabling a specific gene or sequence. However, CRISPR-Cas12a2 binds to a different target and that binding has a very different effect.

Incredibly, Cas12a2 nucleases bend the normally straight piece of double-stranded DNA by 90 degrees, a jaw-dropping structural change.

Ryan Jackson (USA)

“Cas12a2 combines with a guide RNA to form a ribonucleoprotein complex”, explains Jackson to SINC. “Then – he continues – this guide RNA directs the complex to the target RNA [vírico] with base pairing. When the complex is bound, the protein component (Cas12a2) undergoes a major conformational change, which opens up a protein surface (active site of the enzyme) that binds, folds, and cleaves DNA. It cleaves it again, causing the cells with the target RNA to go dormant or die.”

DNA folding without ceremony

“Incredibly, Cas12a2 nucleases bend the normally straight piece of double-stranded DNA by 90 degrees to force the ‘backbone’ of the helix towards the active enzymatic site, where it is cut,” he notes, “an amazing structural change to see , a phenomenon that leaves scientific colleagues with their mouths open”.

“Cas12a2 grabs both ends of the DNA double helix and bends it very tightly,” adds another of the authors, Jack Bravo of UT Austin, “so the helix opens up in the middle and that allows this site to actively destroy DNA fragments. DNA that becomes monostranded. This is what sets Cas12a2 apart from all other DNA targeting systems.”

These conformational changes expose the RuvC active site (light green), relieving self-inhibition and allowing duplex capture. 3/ pic.twitter.com/mLGMtxxMxU

—Jack Bravo (@jpkbravo) June 14, 2022

Using cryo-electron microscopy (cryo-EM), the authors demonstrated this unique behavior of CRISPR-Cas12a2, which triggers the degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA. It is a natural defense strategy for bacteria and archaea to limit the spread of viruses and other pathogens.

Cas12a2 nuclease is capable of digesting double-stranded and single-stranded DNA, as well as single-stranded RNA.

Differences with Cas9

To compare Cas9, “it uses a guide RNA to bind to double-stranded DNA, but Cas12a2 uses it to bind to single-stranded RNA,” says Jackson, “and once Cas9 binds to DNA, it does a single break at a very specific site, while binding of complementary RNA turns Cas12a2 into an active nuclease that makes many cuts in both DNA and RNA. It’s a nonspecific activity no matter what the base sequence of these nucleic acids is.”

“Furthermore – he adds –, in nature, Cas9 is usually programmed to go to the DNA of the virus and cut it. When successful, it allows the cell to survive. Cas12a2 is also programmed to attack viruses, but when it binds to the viral RNA, it begins to cut all the cell’s DNA, which “turns it off” instead of protecting it. Cell closure protects the bacterial colony from viral spread.

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Therapeutic and diagnostic applications

“If Cas12a2 could be harnessed to identify, attack and destroy cells at the genetic level, the potential therapeutic applications would be significant”, says the researcher. “We are just scratching the surface, but we believe that Cas12a2 could lead to additional and improved CRISPR technologies that would greatly benefit society.”

So far, these studies have involved bacterial cultures and purified proteins in biochemical experiments, but the team is currently working on trying to reuse Cas12a2 in other types of cells, including human ones. “We’ve already repurposed Cas12a2 to be able to quickly detect the RNA, which can be used in disease diagnosis,” says Jackson.

These studies were with bacterial cultures, but the team is trying to reuse Cas12a2 in other types of cells, including humans.

According to the authors, thanks to its ability to target so many types of genetic material, this discovery has potential for the development of new inexpensive and highly sensitive diagnostic tests for a wide range of viral infectious diseases such as covid-19, influenza, Ebola and Zika.

“If a new virus appeared tomorrow, all you had to do was figure out its genome and change the guide RNA in a test to have a test against it,” says co-author David Taylor of UT Austin.

Such a diagnosis would require a separate investigation and would likely involve taking saliva or a nasal swab from a patient to mix with the team’s modified Cas12a2 protein, a fragment of guide RNA that would act as a photo to identify a particular virus, and a fluorescent sample. . probe designed to light up when its single-stranded DNA is cut. In the future it may be a reality.

Sacrifice the individual for the good of the colony

Researcher Lluís Montoliu, from the National Center for Biotechnology at CSIC, is an expert in CRISPR, co-author of works such as the one published this week on the ‘resuscitation’ of ancestors using this gene editing tool. It evaluates advances in Cas12a2 as follows:

“There are many types of CRISPR-Cas systems, both in bacteria and in archaea, both prokaryotes. More than 80% of archaea are believed to have CRISPR systems and approximately 50% of bacteria. The Cas12a2 system is special in that it is a nuclease capable of digesting both double-stranded and single-stranded DNA as well as single-stranded RNA after being activated by the small RNA guide molecule.

Furthermore, these digestions, these cuts are made in a non-specific way, without attending to predetermined sequences (as Cas9 does). The reason why it behaves this way was discovered by these researchers when they discovered that it is a system that aborts any possible infection by disconnecting the infected cell, before the viruses can grow and multiply inside it.

In other words, when detecting the entry of the complementary virus in the RNA guide, instead of simply cutting the genome of the invading virus (as Cas9 does), Cas12a2 activates a general and non-specific cutting capacity for any DNA molecule and RNA of the cell, including its own genome and its own RNA, which leads to death, molecular suicide of this bacterium, which prevents the spread of infection through its inactivation.

An ingenious solution adopted by these prokaryotes in which the individual never counts, but the colony. In which it is perfectly possible to sacrifice some individuals of the colony to preserve the others. The multiple solutions that bacteria have explored to deal with the viruses that stalk them never cease to amaze us. And CRISPR systems, because of their versatility, are perfect for developing these unique and effective strategies.

References:

Jack PK Bravo and others:”RNA targeting releases indiscriminate nuclease activity of CRISPR-Cas12a2“. Oleg Dmytrenko and others: “Cas12a2 causes abortive infection through RNA-triggered dsDNA destruction“. Nature, 2023

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