FIX GENE

An anti-CRISPR for gene editing

PHYS.ORG

December 8, 2016

Researchers have discovered a way to program cells to inhibit CRISPR-Cas9 activity. "Anti-CRISPR" proteins had previously been isolated from viruses that infect bacteria, but now University of Toronto and University of Massachusetts Medical School scientists report three families of proteins that turn off CRISPR systems specifically used for gene editing.[1]

The work, which appears December 15 in Cell, offers a new strategy to prevent CRISPR-Cas9 technology from making unwanted changes.[1]

"Making CRISPR controllable allows you to have more layers of control on the system and to turn it on or off under certain conditions, such as where it works within a cell or at what point in time," says lead author Alan Davidson, a phage biologist and bacteriologist at the University of Toronto. "The three anti-CRISPR proteins we've isolated seem to bind to different parts of the Cas9, and there are surely more out there."[1]

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CRISPR inhibitors are a natural byproduct of the evolutionary arms race between viruses and bacteria.[1]

Bacteria use CRISPR-Cas complexes to target and cut up genetic material from invading viruses.[1]

In response, viruses have developed proteins that, upon infection, can quickly bind to a host bacterium's CRISPR-Cas systems, thus nullifying their effects.[1]







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Anti-CRISPR proteins are attractive experimentally because they offer one solution for preventing potential off-target effects.[1]

Research in mice has shown that such mistakes may be rare when using CRISPR-Cas9 technology, but even the occasional error could be a serious problem when being used therapeutically in humans.[1]

"CRISPR-Cas9 in ancillary cells, tissues, or organs is at best useless and at worst a safety risk," says co-author and collaborator Erik J. Sontheimer, a professor in the RNA Therapeutics Institute at the University of Massachusetts Medical School.[1]

"But if you could build an off-switch that keeps Cas9 inactive everywhere except the intended target tissue, then the tissue specificity will be improved."[1]

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"Knowing we have a safety valve will allow people to develop many more uses for CRISPR," says co-author Karen Maxwell, an assistant professor in biochemistry who is also at the University of Toronto. "Things that may have been too risky previously might be possible now."[1]





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While the work will be of great interest to those studying gene editing and gene drives, Davidson's team is also curious to follow up on the biology of how bacterial CRISPRs and viral anti-CRISPRs interact.[1]

"We didn't set out to find anti-CRISPRs, we were just trying to understand how phages incorporate themselves into bacterial genomes and stumbled onto something that I think will be important for biotechnology," Davidson says.[1]

"We were being observant and following a path that we didn't know where it could lead, and it's just been a very fun and exciting story." [1]

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Overcoming Hurdles

 In CRISPR Gene Editing

To Improve Treatment

PHYS.ORG

February 7, 2017

More and more scientists are using the powerful new gene-editing tool known as CRISPR/Cas9, a technology isolated from bacteria, that holds promise for new treatment of such genetic diseases as cystic fibrosis, muscular dystrophy and hemophilia.[2]

But to work well, the new gene-clipping tool must be delivered safely across the cell membrane and into its nucleus, a difficult process that can trigger the cell's defenses and "trap" CRISPR/Cas9, greatly reducing its treatment potential.[2]

Now, researchers in nanochemistry expert Vincent Rotello's laboratory at the University of Massachusetts Amherst have designed a delivery system using nanoparticles to assist CRISPR/Cas9 across the membrane and into the cell nucleus while avoiding entrapment by cellular machinery. Details appear in a recent issue of the journal ACS Nano.[2] br />

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The lab's experiment leader, Rubul Mout, says, "CRISPR has two components: a scissor-like protein called Cas9, and an RNA molecule called sgRNA that guides Cas9 to its target gene. Once the Cas9-sgRNA pair gets to the destination gene in the nucleus, it can interrogate its genetic mistakes and correct them with the help of the host cell's repair machinery."[2]

He points out that since CRISPR's potential was first discovered in 2012, gene editing or genome engineering has quickly become an intense research topic in biology and medicine. [2]







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The goal is to treat otherwise incurable genetic diseases by manipulating diseased genes.[2]

"However, to achieve this, biotech and pharmaceutical companies are constantly searching for more efficient CRISPR delivery methods," he adds.[2]

The new delivery method Rotello, Mout and colleagues designed involves engineering the Cas9 protein, named Cas9En, and carrier nanoparticles. Rotello says, "By finely tuning the interactions between engineered Cas9En protein and nanoparticles, we were able to construct these delivery vectors. [2]

The vectors carrying the Cas9 protein and sgRNA come into contact with the cell membrane, fuse, and release the Cas9:sgRNA directly into the cell cytoplasm."[2]

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"Cas9 protein also has a nuclear guiding sequence that ushers the complex into the destination nucleus. The key is to tweak the Cas9 protein," he adds.[2]

"We have delivered this Cas9 protein and sgRNA pair into the cell nucleus without getting it trapped on its way. We have watched the delivery process live in real time using sophisticated microscopy."[2]

Mout and colleagues say they can now deliver the Cas9 protein and sgRNA pair into about 90 percent of cells grown in a culture dish with an editing efficiency of about 30 percent. [2]

"Ninety percent cytosolic/nuclear delivery is a huge improvement compared to others methods," Mout points out.[2]

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The researchers believe that the Cas9En may also serve as a platform for delivery of a variety of other materials such as polymers, lipid nanoparticles or self-assembling peptides. Rotello says, "Now that we have achieved efficient gene editing in cultured cells, we are aimi[2]

ng to edit genes in pre-clinical animal models. We are also interested in gene editing for adoptive therapies, where a diseased cell is isolated from a patient, corrected by CRISPR in the lab, and delivered back to the patient."[2]





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Apart from gene editing, the new delivery method may have other uses. For example, another important issue in biology and medicine is tracking DNA and RNA inside cells. Recently, CRISPR has been used to aid in this research.[2]

Moumita Ray, another researcher in the Rotello lab, says, "Our method allows the precise monitoring of Cas9 protein movement inside a cell, opening new opportunities in genomic research." [2]

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[1]
An anti-CRISPR for gene editing

[2]
Overcoming hurdles in CRISPR gene editing to improve treatment

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