The latest discovery by CRISPR Queen: An anti-CRISPR protein reduces off-target effects

Release date: 2017-07-14

In December last year, two groups of scientists published several important findings that blocked the CRISPR-Cas9 active protein, and on July 12, the researchers again pointed out that using one of the anti-CRISPR proteins can reduce Cas9-mediated humans. Off-target effects in cell genome editing.

The research was published in the journal Science Advances, led by Professor Jennifer Doudna of the University of California at Berkeley, and led by Jacob Corn et al.

According to Corn, "CRISPRs are part of the bacterial immune system. A basic concept for biology is that if a creature develops a weapon, it will also prepare a defense. It turns out. Phages have evolved the way to shut down the CRISPR system, which is these anti-CRISPR proteins."

At the end of last year, Corn and the University of California, San Francisco, Joseph Bondy-Demony and others discovered several anti-CRISPR proteins present in Listeria. Two protein inhibitors, AcrIIA2 and AcrIIA4, block Cas9 from Streptococcus pyogenes. Enzyme activity, Cas9 is a DNA cleavage enzyme commonly used in genome editing.

On this basis, in order to further explore the mechanism of action of AcrIIA4, the researchers used a variety of methods, such as cryo-electron microscopy and human cell culture, to find that this anti-CRISPR protein is bound by competition and is usually used to bind DNA. The Cas9 enzyme acts on the "concave pocket" domain, which blocks the interaction of the Cas9 enzyme with DNA.

This time, for the first time, how the natural protein inhibitor AcrIIA4 interacts with the Cas9 enzyme, the researchers found that AcrIIA4 binds to the enzyme in the "sag pocket", and the binding of AcrIIA4 to the enzyme prevents the Cas9 enzyme from attaching to the target DNA and performing it. Cutting. Studies of human cells have revealed that if these cells are treated with this protein inhibitor prior to the start of gene editing, the CRIPSR-Cas complex can be substantially prevented from shearing DNA at any site.

The results also showed that approximately half of the editing of the hit target of CRISPR-Cas9 (ie, the system correctly cut the gene edits in the manner desired by the scientist) occurred within 6 hours of entry into the system. Previous studies have shown that CRISPR-Cas9 may remain in the off-target area for a period of time, but they are not sheared. By exploiting this time difference, the researchers introduced a CRISPR-Cas9 complex that was shown to cleave two genes, including genes associated with sickle cell disease, in human leukemia cells, and then after 6 hours. Added AcrIIA4. The results showed that adding AcrIIA4 at the right time prevented the gene from being cut at the wrong place, while CRISPR still had time to perform gene cleavage at the correct position.

This study points to the mechanism of action of AcrIIA4, suggesting a novel approach to regulating gene editing in mammalian cells. The most important thing is to add AcrIIA4 to human leukemia cells after several hours of introduction of CRISPR-Cas9, which can reduce the off-target effect of CRISPR-Cas9 without affecting the correct cutting of CRISPR.

“Although this research work and discovery is impressive and important, it is not very unexpected, as it is very logical for the anti-RISS protein effect. This mechanism... previously in other systems In the past, DNA simulations of phage nuclease inhibitor proteins were also found in previous studies. Professor Erik Sontheimer of the University of Massachusetts said that although Professor Sonthheimer did not participate in the study, he was the co-founder of Intellia Therapeutics. (The other founder is Jennifer Doudna), which is currently developing with natural protein inhibitors (not AcrIIA4).

Alan Davidson, a molecular geneticist from the University of Toronto (not involved in the work), said that recently, Nature magazine, Molecular Cell magazine published similar results, Davidson and Sontheime and others applied for three other patents for anti-CRISPR compounds. "These have basically the same structure, the same protein, and similar conclusions. I think the only difference in this latest paper is some data on genome editing, that is, anti-CRISPR can reduce the off-target effect when editing."

This is a very competitive field. From all aspects of the paper, this field is developing very fast.

According to Corn, these anti-CRISPR proteins can be used not only for genetic research, but also as clinical tools. They not only help to reduce the off-target effect, but also become an effective way to control the target. For example, some gene therapy methods may leave active Cas9 in the patient's cells, which needs to be turned off. "Gene editing switches are very important, but the switch is turned off. It is equally important."

But there is still a need for more effects and safety studies, and scientists need to understand what these human immune responses to CRISPR-resistant proteins are because they come from bacterial viruses. “We just did a simple anti-CRISPR protein study, and no one knows if there are other tools waiting for us.”

Reference material

Disabling Cas9 by an anti-CRISPR DNA mimic

Source: Biopass

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