Tag Archives: Cas9

New tool shows early promise to help reduce the spread of antimicrobial resistance

A new tool which could help reduce the spread of antimicrobial resistance is showing early promise, through exploiting a bacterial immune system as a gene editing tool.

Antimicrobial resistance is a major global threat, with nearly five million deaths annually resulting from antibiotics failing to treat infection, according to the World Health Organisation.

Bacteria often develop resistance when resistant genes are transported between hosts. One way that this occurs is via plasmids – circular strands of DNA, which can spread easily between bacteria, and swiftly replicate. This can occur in our bodies, and in environmental settings, such as waterways.

The Exeter team harnessed the CRISPR-Cas gene editing system, which can target specific sequences of DNA, and cuts through them when they are encountered. The researchers engineered a plasmid which can specifically target the resistance gene for Gentamicin – a commonly used antibiotic.

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In laboratory experiments, the new research, published in Microbiology, found that the plasmid protected its host cell from developing resistance. Furthermore, researchers found that the plasmid effectively targeted antimicrobial-resistant genes in hosts to which it transferred, reversing their resistance.

Antimicrobial resistance threatens to outstrip covid in terms of the number of global deaths. We urgently need new ways to stop resistance spreading between hosts. Our technology is showing early promise to eliminate resistance in a wide range of different bacteria. Our next step is to conduct experiments in more complex microbial communities. We hope one day it could be a way to reduce the spread of antimicrobial resistance in environments such as sewage treatment plants, which we know are breeding grounds for resistance.”

David Walker-Sünderhauf, Lead Author, University of Exeter

The research is supported by GW4, the Medical Research Council, the Lister Institute, and JPI-AMR.

Journal reference:

Walker-Sünderhauf, D., et al. (2023) Removal of AMR plasmids using a mobile, broad host-range, CRISPR-Cas9 delivery tool. Microbiology. doi.org/10.1099/mic.0.001334.

Avanced genome editing technology could be used as a one-time treatment for CD3 delta SCID

A new UCLA-led study suggests that advanced genome editing technology could be used as a one-time treatment for the rare and deadly genetic disease CD3 delta severe combined immunodeficiency.

The condition, also known as CD3 delta SCID, is caused by a mutation in the CD3D gene, which prevents the production of the CD3 delta protein that is needed for the normal development of T cells from blood stem cells.

Without T cells, babies born with CD3 delta SCID are unable to fight off infections and, if untreated, often die within the first two years of life. Currently, bone marrow transplant is the only available treatment, but the procedure carries significant risks.

In a study published in Cell, the researchers showed that a new genome editing technique called base editing can correct the mutation that causes CD3 delta SCID in blood stem cells and restore their ability to produce T cells.

The potential therapy is the result of a collaboration between the laboratories of Dr. Donald Kohn and Dr. Gay Crooks, both members of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA and senior authors of the study.

Kohn’s lab has previously developed successful gene therapies for several immune system deficiencies, including other forms of SCID. He and his colleagues turned their attention to CD3 delta SCID at the request of Dr. Nicola Wright, a pediatric hematologist and immunologist at the Alberta Children’s Hospital Research Institute in Canada, who reached out in search of a better treatment option for her patients.

CD3 delta SCID is prevalent in the Mennonite community that migrates between Canada and Mexico.

Because newborns are not screened for SCID in Mexico, I often see babies who have been diagnosed late and are returning to Canada quite sick.”

Dr. Nicola Wright, pediatric hematologist and immunologist at the Alberta Children’s Hospital Research Institute

When Kohn presented Wright’s request to his lab, Grace McAuley, then a research associate who joined the lab at the end of her senior year at UCLA, stepped up with a daring idea.

“Grace proposed we try base editing, a very new technology my lab had never attempted before,” said Kohn, a distinguished professor of microbiology, immunology and molecular genetics, and of pediatrics.

Base editing is an ultraprecise form of genome editing that enables scientists to correct single-letter mutations in DNA. DNA is made up of four chemical bases that are referred to as A, T, C and G; those bases pair together to form the “rungs” in DNA’s double-helix ladder structure.

While other gene editing platforms, like CRISPR-Cas9, cut both strands of the chromosome to make changes to DNA, base editing chemically changes one DNA base letter into another -; an A to a G, for example -; leaving the chromosome intact.

“I had a very steep learning curve in the beginning, when base editing just wasn’t working,” said McAuley, who is now pursuing an M.D.-Ph.D. at UC San Diego and is the study’s co-first author. “But I kept pushing forward. My goal was help get this therapy to the clinic as fast as was safely possible.”

McAuley reached out to the Broad Institute’s David Liu, the inventor of base editing, for advice on how to evaluate the technique’s safety for this particular use. Eventually, McAuley identified a base editor that was highly efficient at correcting the disease-causing genetic mutation.

Because the disease is extremely rare, obtaining patient stem cells for the UCLA study was a significant challenge. The project got a boost when Wright provided the researchers with blood stem cells donated by a CD3 delta SCID patient who was undergoing a bone marrow transplant.

The base editor corrected an average of almost 71% of the patient’s stem cells across three laboratory experiments.

Next, McAuley worked with Dr. Gloria Yiu, a UCLA clinical instructor in rheumatology, to test whether the corrected cells could give rise to T cells. Yiu used artificial thymic organoids, which are stem cell-derived tissue models developed by Crooks’ lab that mimic the environment of the human thymus -; the organ where blood stem cells become T cells.

When the corrected blood stem cells were introduced into the artificial thymic organoids, they produced fully functional and mature T cells.

“Because the artificial thymic organoid supports the development of mature T cells so efficiently, it was the ideal system to show that base editing of patients’ stem cells could fix the defect seen in this disease,” said Yiu, who is also a co-first author of the study.

As a final step, McAuley studied the longevity of the corrected stem cells by transplanting them into a mouse. The corrected cells remained four months after transplant, indicating that base editing had corrected the mutation in true, self-renewing blood stem cells. The findings suggest that corrected blood stem cells could persist long-term and produce the T cells patients would need to live healthy lives.

“This project was a beautiful picture of team science, with clinical need and scientific expertise aligned,” said Crooks, a professor of pathology and laboratory medicine. “Every team member played a vital role in making this work successful.”

The research team is now working with Wright on how to bring the new approach to a clinical trial for infants with CD3 delta SCID from Canada, Mexico and the U.S.

This research was funded by the Jeffrey Modell Foundation, the National Institutes of Health, the Bill and Melinda Gates Foundation, the Howard Hughes Medical Institute, the V Foundation and the A.P. Giannini Foundation.

The therapeutic approach described in this article has been used in preclinical tests only and has not been tested in humans or approved by the Food and Drug Administration as safe and effective for use in humans. The technique is covered by a patent application filed by the UCLA Technology Development Group on behalf of the Regents of the University of California, with Kohn and McAuley listed as co-inventors.

Journal reference:

McAuley, G.E., et al. (2023) Human T cell generation is restored in CD3δ severe combined immunodeficiency through adenine base editing. Cell. doi.org/10.1016/j.cell.2023.02.027.

Newly discovered CRISPR immune system shuts down infected cells to thwart infection

Thought LeadersRyan JacksonAssistant Professor Utah State University 

In this interview, News Medical speaks to Assistant Professor Ryan Jackson about his latest work, published in tandem Nature papers, detailing the discovery of a new CRISPR immune system. 

Please can you introduce yourself and tell us about your professional background?

I am an Assistant Professor at Utah State University (USU). I use biochemical and structural techniques to understand how the molecules that perform the reactions of life function. I’ve been working in the CRISPR field since 2011. I started as a postdoc in Blake Wiedenheft’s lab at Montana State University, and in 2016 I started my own research lab at USU. I earned both of my degrees (a B.S. in Biology and a Ph.D. in Biochemistry) from USU, so joining the faculty was like coming home. My research lab specializes in determining the structure and function of newly discovered and obscure CRISPR systems.

Due to its gene editing potential, CRISPR has caught the imagination of the general public and the scientific community. Could you briefly tell us what CRISPR is and its potential medical significance?

CRISPR was discovered by scientists studying bacterial immune systems. In nature, CRISPR systems protect bacteria from viruses. But once scientists discovered that the system could be reprogrammed to cut and edit DNA sequences in a variety of cells, including plants and humans, it has been primarily understood to be a tool for gene editing.

Image Credit: ART-ur/Shutterstock.com

Image Credit: ART-ur/Shutterstock.com

The potential medical significance of CRISPR-based tools is vast, including tools for research to identify the roles of genes in disease and tools for the fast diagnosis of disease. Probably the most amazing potential is the ability to edit the DNA of cells that make humans sick – potentially curing genetic diseases. A recent example of this is Victoria Gray and the use of CRISPR to cure her sickle cell anemia.

You have recently published two papers in the journal Nature. Please could you describe how you conducted each study and the main findings from each paper? How does this new system, Cas12a2, differ from better-known CRISPR systems, such as CRISPR-Cas9?

We used microbiology, biochemistry, and structural methods to determine how a distinct CRISPR system functions. We discovered that, unlike the better-known systems like Cas9, which protect cells from viral-induced death, activation of our system shuts down cells that are infected. This “abortive” mechanism kills or slows down the growth of the cell before the virus can replicate and infect other bacteria.

We also determined that, unlike CRISPR systems that target DNA (e.g., Cas9), our system (Cas12a2) targets RNA. Instead of making a single specific cut in the targeted DNA, like Cas9, when Cas12a2 binds RNA, it drastically changes its shape in a way that allows it to bind and cut any sequence of DNA or RNA. This cutting activity destroys the virus genome but also degrades the genome of the host cell, shutting down the cell before the virus can replicate.

How is Cas12a2 able to thwart infection, and what are the potential applications of such an ability?

Cas12a2 recognizes virus RNA and then kills or shuts down the infected cell before more viruses can be made. Although much more needs to be done, we can envision repurposing Cas12a2 to recognize infected human cells and then causing them to die before they can make new viruses. Effectively stopping an infection in its tracks. We envision that such therapies could extend to any cells with a specific genetic marker that could be recognized by Cas12a2, for example, cancer.

USU Biochemists Describe Structure and Function of Newly Discovered CRISPR Immune System

What are the potential therapeutic and diagnostic applications of Cas12a2?

The therapeutic applications would be any application where the death of a cell with a specific RNA signature would treat a disease state. Such applications could treat communicable diseases as well as more complicated diseases such as cancer.

The diagnostics applications are more obvious and are more likely to be realized in the near future. Cas12a2 can be programmed to identify RNA from any disease. The RNA-induced activation of DNA cutting can be leveraged to amplify RNA detection. Thus, this tool could be used to diagnose any disease quickly.

How significant are your findings to the current understanding of CRISPR systems and their potential applications?

Our results are significant because no other CRISPR-associated (Cas) protein binds RNA and then becomes a single- and double-stranded DNA and RNA cutter that cuts any sequence.

Image Credit: Panuwach/Shutterstock.com

Image Credit: Panuwach/Shutterstock.com

Other proteins have parts of this activity but not both. For example, Cas13 enzymes bind RNA and then become RNA cutters, and Cas12a enzymes bind double-stranded DNA and become single-stranded DNA cutters. Also, some CRISPR systems link RNA-binding to DNA degradation (e.g., type III CRISPR systems activating NucC DNAses), but this activity relies on multiple proteins and second messenger intermediates.   

As far as we know, Cas12a2 is the only single-protein Cas enzyme that binds RNA and then becomes a single-stranded DNA, double-stranded DNA, and single-stranded RNA cutter.

What is next for you and your research?

We are working to see how Cas12a2 can be repurposed, and we continue to research the structure and function of other newly discovered and obscure CRISPR systems. We anticipate that our work will lead to many more interesting discoveries with potential applications in science and medicine.

Where can readers find more information?

About Assistant Professor Ryan Jackson

I am an Assistant Professor of Biochemistry at Utah State University. I have been using structural biology and biochemistry to determine the function of the molecules of life since 2007. I am most interested in understanding how protein and RNA molecules interact in life science reactions.

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