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  • Research improves multiplex mutagenesis to increase experimental efficiency in plant genome editing

    Research improves multiplex mutagenesis to increase experimental efficiency in plant genome editing
    27th May 2024

    Credit: Unsplash/CC0 Public Domain

    CRISPR/Cas9 remains the most powerful tool to generate mutations in plant genomes. Studying the various combinations of mutations has significantly increased the scale of experimental setups, necessitating more space to grow numerous plants.

    Researchers from VIB-UGent Center for Plant Systems Biology have improved multiplex mutagenesis, which reduces the complexity and cost of large-scale genome editing projects. Their results have been published in The Plant Journal.

    CRISPR/Cas experiments are continually increasing in scale, not only in terms of the number of mutants created through precise genome editing but also in terms of the number of genes that can be mutated simultaneously. The lab of Thomas Jacobs from VIB-UGent Center for Plant Systems Biology has developed screens to systematically mutate tens, hundreds, or even thousands of genes at a time.

    The goal is to enhance the efficiency of inheritable germline mutations, and ultimately reduce the complexity and cost of large-scale genomic editing projects. To achieve this, the team focused on two key aspects of CRISPR/Cas9 vector design: the promotor to drive Cas9 expression, and the nuclear localization signals (NLS) that direct the protein to the nucleus. By genotyping thousands of Arabidopsis plants, they found that using the RPS5A promotor to express Cas9 led to the highest mutation rate, and that flanking the Cas9 protein with bipartite NLS was the most efficient configuration to create germline mutations. Combining these two elements results in the highest observed multiplex editing efficiency, with 99% of plants harboring at least one knockout mutation and over 80% with 4 to 7 mutations

    By VIB (the Flanders Institute for Biotechnology)

    Article can be accessed on: phys.org

  • Scientists leverage machine learning to decode gene regulation in the developing human brain

    Scientists leverage machine learning to decode gene regulation in the developing human brain
    27th May 2024

    Massively parallel characterization and prediction of gene regulatory activity in the developing brain. Credit: Science (2024). DOI: 10.1126/science.adh0559

    The study, from scientists at Gladstone Institutes and University of California, San Francisco (UCSF), establishes a comprehensive catalog of genetic sequences involved in brain development and opens the door to new diagnostics or treatments for neurological conditions such as schizophrenia and autism spectrum disorder. The paper, “Massively Parallel Characterization of Regulatory Elements in the Developing Human Cortex,” appears in the journal Science

    “We collected a massive amount of data from sequences in noncoding regions of DNA that were already suspected to play a big role in brain development or disease,” says Senior Investigator Katie Pollard, Ph.D., who also serves as director of the Gladstone Institute for Data Science and Biotechnology.

    “We were able to functionally test more than 100,000 of them to find out whether they affect gene activity, and then pinpoint sequence changes that could alter their activity in disease.”

    Pollard co-led the sweeping study with Nadav Ahituv, Ph.D., professor in the Department of Bioengineering and Therapeutic Sciences at UCSF and director of the UCSF Institute for Human Genetics. Much of the experimental work on brain tissue was led by Tomasz Nowakowski, Ph.D., associate professor of neurological surgery in the UCSF Department of Medicine.

    In all, the team found 164 variants associated with psychiatric disorders and 46,802 sequences with enhancer activity in developing neurons, meaning they control the function of a given gene.

    By Gladstone Institutes

    Article can be accessed on: MedicalXpress

  • Scientists Sequence Single Cells with Long-Read Technology

    Scientists Sequence Single Cells with Long-Read Technology
    19th May 2024

    By combining two innovative approaches, researchers can now sequence the full spectrum of mutational differences between individual cells’ genomes.

    Credits: The scientist

    Traditional sequencing is often likened to making a smoothie: researchers blend a bunch of cells, obtain an average sequence, and draw conclusions on the ingredients that comprise the slush. More recently, scientists have gained the ability to perform single-cell sequencing, which can reveal rare variations between cells and the evolution of cell lineages. But current methods require reading the genome in short sections and therefore often fail to capture complex repetitive regions, which scientists are increasingly linking to health and disease. Long-read technologies could overcome this pitfall; however, their methods require much more DNA than can be extracted from a single cell. Single-cell, long-read sequencing has remained frustratingly out of reach.

    That is, until now. By combining “two very innovative approaches” a cutting-edge DNA amplification technique with the latest advances in DNA sequencing a team of scientists have applied long-read technology to single cells, says Alexander Hoischen, a researcher of genomic technologies at Radboud University Medical Center in The Netherlands who was not involved in the research. “This was unthinkable just two or three years ago,” says Hoischen.

    The feat may allow for a more detailed look at mutations underlying all sorts of diseases, experts tell The Scientist.

    Over the past decade, reads the product of DNA sequencing have been getting longer. Long-read sequencing has allowed scientists to sequence troublesome “dark regions” of the genome that are inaccessible to short-read technologies, either due to an abundance of guanines and cytosines, or duplicated regions not easily mapped to a chromosome.

    However, long-read sequencing requires a ton of DNA. Several micrograms of genetic material are needed, but “a single cell contains just six picograms,” says Joanna Hård, a computational biologist at ETH Zurich in Switzerland. “So substantial amplification is required before you can sequence it” using long-read methods, she says.

    And that’s where things get tricky, says Hård, as the primary methods used to amplify DNA are prone to “amplification bias”: the tendency for certain sequences to be ramped up at the expense of others. Now, Hård and colleagues have obtained long reads from individual cells using an improved DNA amplification method. Though not yet peer-reviewed, the results were reported in a preprint uploaded to bioRxiv on January 23. To minimize amplification bias, the team used a technique called droplet-based multiple displacement amplification. It works by trapping DNA fragments in droplets that contain a limited supply of reagents, preventing over-amplification of certain regions. “There is a more even amplification, so you get better representation of the genome,” Adam Ameur, a bioinformatician at Uppsala University in Sweden, tells The Scientist.

     

    By Holly Barker, PhD

    Article can be accessed on: The Scientist

  • A New Delivery System Offers Hope for Cystic Fibrosis

    A New Delivery System Offers Hope for Cystic Fibrosis
    19th May 2024

    Credits: The scientist

    CRISPR-carrying lipid nanoparticles enabled researchers to correct a rare nonsense mutation in the lungs of a cystic fibrosis mouse model.

    From its first description in 1935 until now, clinical outcomes for patients with cystic fibrosis have undergone a dramatic transformation. While the rare genetic disease was once an early death sentence that often prevented patients from reaching adulthood, advancements in cystic fibrosis therapeutics have greatly extended the lifespans of those suffering from the disease over the years. However, these therapies are not effective in all patients.

    In a study published in Nature Communications, scientists reported a novel strategy to deliver the CRISPR-Cas9 gene-editing system into the lungs of a cystic fibrosis mouse model and correct the underlying mutations. Once developed and tested, this approach could allow clinicians to treat every patient with cystic fibrosis, including those who were previously untreatable. Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel on the cell surface that helps control the concentration of salt and water within bodily secretions. Impaired or absent CFTR activity leads to the characteristic thick and sticky mucus associated with the disease and consequently, an increase in the frequency of respiratory infections. Clinicians have developed small molecule drugs, such as Trikafta, that effectively treat 90 percent of patients.

    “The problem is that these drugs are only for symptom management,” said Yehui Sun, a graduate student in Daniel Siegwart’s laboratory at the University of Texas Southwestern Medical Center and author of this study. “[These drugs cannot] cure the root of the disease because it is a genetic disease.” The patient’s cells must produce the CFTR protein for the existing therapies to work, which leaves patients with nonsense mutations without options.

    Since its initial discovery, researchers believed that they could use CRISPR-Cas9 to help cure genetic diseases. However, the lack of effective delivery carriers that could target specific organs held back this approach. To solve this problem, Siegwart and his team previously developed an advanced delivery strategy called selective organ targeting lipid nanoparticles (SORT LNP). By modifying the composition and biophysical properties of these nanoparticles, the researchers selectively targeted cells in the lungs, livers, or brains of mice after intravenous administration. Building on this work, Sun, Siegwart, and their team further optimized the formulation of their lung SORT LNP and improved its delivery, efficacy, and lung-targeting specificity, while producing minimum toxicity. After encapsulating the CRISPR system components including Cas9 mRNA, mutation-specific single guide RNA, and donor single stranded DNA template, they injected the lipid nanoparticles intravenously into a cystic fibrosis mouse model harboring the nonsense mutation, G542X. Through next-generation sequencing (NGS) of DNA extracted from its lung tissue, the researchers determined that their gene editing strategy successfully corrected the G542X mutation in murine lungs.

    By Charlene Lancaster, PhD

    Article can be accessed on: The Scientist

     

  • Study shows how depletion of mitochondria in axons can directly lead to protein accumulation

    Study shows how depletion of mitochondria in axons can directly lead to protein accumulation
    6th May 2024

    Credit: Tokyo Metropolitan University

    Researchers from Tokyo Metropolitan University have identified how proteins collect abnormally in neurons, a feature of neurodegenerative diseases like Alzheimer’s. The research is published in the journal eLife.

    The researchers used fruit flies to show that depletion of mitochondria in axons can directly lead to protein accumulation. At the same time, significantly high amounts of a protein called eIF2β were found. Restoring the levels to normal led to a recovery in protein recycling. The findings promise new treatments for neurodegenerative diseases. Every cell in our bodies is a busy factory, where proteins are constantly being produced and disassembled. Any changes or lapses in either the production or recycling phases can lead to serious illnesses. Neurodegenerative diseases such as Alzheimer’s and amyotrophic lateral sclerosis (ALS), for example, are known to be accompanied by an abnormal build-up of proteins in neurons. However, the trigger behind this accumulation remains unknown.

    A team led by Associate Professor Kanae Ando of Tokyo Metropolitan University has been trying to determine the causes of abnormal protein build-up by studying Drosophila fruit flies, a commonly studied model organism that has many key similarities with human physiology. They focused on the presence of mitochondria in axons, the long tendril-like appendages that stretch out of neurons and form the necessary connections that allow signals to be transmitted inside our brains. It is known that the levels of mitochondria in axons can drop with age, and during the progress of neurodegenerative diseases.

    By Tokyo Metropolitan University

    Article can be accessed on: MedicalXpress

  • Novel triple drug combination effective against antibiotic-resistant bacteria

    Novel triple drug combination effective against antibiotic-resistant bacteria
    6th May 2024

    Graphical abstract. Credit: Engineering (2024). DOI: 10.1016/j.eng.2024.02.010

    Scientists at the Ineos Oxford Institute (IOI) have found a new potential combination therapy to combat antimicrobial resistance (AMR) by targeting two key bacterial enzymes involved in resistance. The study, “The Triple Combination of Meropenem, Avibactam, and a Metallo-β-Lactamase Inhibitor Optimizes Antibacterial Coverage Against Different β-Lactamase Producers,” has been published in Engineering.

    This new study looked at a combination of three drugs: the β-lactam antibiotic meropenem, a newly developed MBL inhibitor called indole-2-carboxylate 58 (InC58), and an SBL inhibitor called avibactam (AVI).

    “This study builds on our previous work to develop broad spectrum metallo β-lactamase inhibitors. Here we combatted multiple resistance mechanisms simultaneously to great effect, and this is a great example of how chemistry and microbiology teams can collaborate to develop new potential therapies. This combination therapy works very well in the lab and the next challenge will be to show that this works in infection models and ultimately in a hospital setting,” said Dr Alistair Farley, IOI Scientific Lead and a co-author for the study.

    The team tested the effectiveness of the combination of all three compounds, compared to a combination of meropenem with either InC58 or AVI alone, on 51 strains of meropenem-resistant bacteria. Researchers compared the minimum inhibitory concentration (MIC) of the different drug combinations. The study found that the triple-drug combination was more effective at stopping growth of bacteria in the lab than either of the dual-drug combinations.

    By  University of Oxford

    Article can be accessed on: phys.org

  • Chromatin accessibility: A new avenue for gene editing

    Chromatin accessibility: A new avenue for gene editing
    23rd April 2024

    TFDP1, a modulator of genome accessibility. Credit: Kanazawa University

    In a study recently published in Nature Genetics, researchers from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University explore chromatin accessibility, i.e., endogenous access pathways to the genomic DNA, and its use as a tool for gene editing. Researchers from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, led by Yusuke Miyanari, have used advanced genetic screening methods to unravel chromatin accessibility and its pathways.

    For the investigation, the team used a combination of two technologies CRISPR screening and ATAC-see. While the former is a method to suppress the function of a desired set of genes, the latter is a means to identify which ones are essential for chromatin accessibility. Thus, using this method all genes playing a crucial role in chromatin accessibility could be pinned down.

    With the help of these assays, novel pathways and individual players involved in chromatin accessibility were uncovered some playing a positive role and some negative. Of these, one particular protein, TFDP1, showed a negative effect on chromatin accessibility. When it was suppressed, a significant increase in chromatin accessibility was observed, accompanied by nucleosome reduction. A deeper dive into the mechanism of TFDP1 revealed that it functions by regulating the genes responsible for production of certain histone proteins. The team then focused their study toward exploring biotechnological applications of their findings. After suppressing TFDP1, two different approaches were tried. The first approach involved gene editing using the CRISPR/Cas9 tool. This revealed that deletion of TFDP1 made the gene editing process easier.

    By Nano Life Science Institute (NanoLSI)

    Article can be accessed on: phys.org

  • New stem cell model can help personalize stem cell treatment for immunodeficiency patients

    New stem cell model can help personalize stem cell treatment for immunodeficiency patients
    22nd April 2024

    Graphical abstract. Credit: Journal of Allergy and Clinical Immunology (2023). DOI: 10.1016/j.jaci.2023.11.914

    A collaborative research team has pioneered a new stem cell model to help personalize treatment for patients suffering from rare forms of immunodeficiency. The research findings were published in the Journal of Allergy and Clinical Immunology.

    Primary immunodeficiencies, also known as “inborn errors of immunity,” are debilitating diseases that compromise the immune system, leaving patients highly vulnerable to infections, autoimmunity, and even cancer. To date, about 500 primary immunodeficiencies are known, but the list is growing yearly as new diseases emerge. One example is a rare disorder called STAT1-Gain-of-Function (STAT1-GoF) disease. Patients with STAT1-GoF are born with an inheritable defect in their immune system, making them susceptible to life-threatening infections, autoimmune disorders, aneurysms, and cancers.

    Collaborating with partners at the Centre for Translational Stem Cell Biology (CTSCB) and the University of Cambridge, HKUMed has pioneered a new stem cell platform to help patients with primary immunodeficiencies. The research team led by Dr. Philip Li Hei, Professors Liu Pengtao, and Chak-sing Lau from the LKS Faculty of Medicine of the University of Hong Kong (HKUMed) took blood samples from patients and re-engineered the patients’ cells into Expanded Potential Stem Cells (EPSCs), which can be used as personalized disease models, enabling various therapies to be tested to identify the most effective and safest treatment options without causing unnecessary risk to the patients.

    By The University of Hong Kong

    Article can be accessed on: MedicalXpress

  • Study uses metabolomics to identify novel diagnostic markers for chronic obstructive pulmonary disease

    Study uses metabolomics to identify novel diagnostic markers for chronic obstructive pulmonary disease
    18th April 2024

     Credit: Dr. Dr. Tiantian Zhang and Dr. Hongmei Zhao from Peking Union Medical College (ars.els-cdn.com/content/image/1-s2.0-S2772558823000579-gr2.jpg)

    Chronic obstructive pulmonary disease (COPD) is a chronic lung disease with irreversible airflow limitation and a leading cause of death worldwide. COPD is characterized by chronic bronchitis and emphysema and is associated with malnutrition, muscle weakness, and an increased risk of infection. Although pulmonary tests are considered as the gold standard for COPD diagnosis, they cannot detect early stages of COPD, leading to underdiagnosis. This emphasizes the need for specific biomarkers for early diagnosis, classification, and clinical interventions.

    Recent studies suggested that changes in lipids, amino acids, glucose, nucleotides, and microbial metabolites in lungs and intestine can effectively diagnose early COPD. Metabolomics, a discipline that analyzes different metabolites from body fluids, has emerged as a prominent technique for COPD assessment. However, there are no studies that identify and summarize the metabolites that significantly change during COPD.

    A recent review by Dr. Wenqian Wu, Dr. Zhiwei Li, Dr. Tiantian Zhang, and Dr. Hongmei Zhao from the Peking Union Medical College, along with Dr. Yongqiang Wang from 302 Hospital of China Guizhou Aviation Industry Group, and Dr. Chuan Huang at the Chinese Academy of Medical Sciences, provided an in-depth account of the advances in metabolomics of COPD over the last five years, highlighting some potential diagnostic markers and therapeutic targets.

    By Cactus Communications

    Article can be accessed on: MedicalXpress

  • MEGA CRISPR: Engineering Better Immunotherapies with RNA Editing

    MEGA CRISPR: Engineering Better Immunotherapies with RNA Editing
    12th April 2024

    Credits: The scientist

    Many foundational research technologies have transformed cellular therapies, moving treatments from concept to clinic. In the past decade, chimeric antigen receptors (CAR) and genome editing are two standouts that led to breakthrough CAR T cell therapies for leukemia and lymphoma. Scientists engineer these treatments with virus-mediated gene insertion ex vivo, which instructs T cells to express synthetic receptors that detect tumor-specific antigens and guide cancer cell eradication after transplantation. Researchers investigate clustered regularly interspaced short palindromic repeats (CRISPR) editing to improve CAR T cell therapies and expand their applicability to more cancer types. However, CRISPR-associated nuclease 9 (Cas9)-based genome cutting tools face unique safety and efficacy limitations due to the permanent nature of DNA editing. To circumvent these limitations, bioengineer Lei (Stanley) Qi and physician immunologist Crystal Mackall at Stanford University developed an RNA editing tool called multiplexed effector guide arrays (MEGA). In a study published in Cell, the team used Cas9’s cousin, Cas13, and a pooled array of guide RNAs to simultaneously edit multiple gene transcripts in primary human T cells without targeting or cutting genomic DNA. This multi-targeting method addresses an unmet need in cell therapy optimization by allowing the researchers to dynamically regulate several pathways per T cell, rather than add or ablate individual genes completely, one at a time. The researchers screened for genes that synergistically affect T cell function and knocked down redundant transcripts that drive T cell exhaustion in culture and in mice.

    “Our years of experience on gene editing at the DNA level makes us realize that this technology, while very powerful, still has some intrinsic challenges, which possibly could only be addressed if we find methods to engineer RNA,” said Qi. Among these difficulties are off-target cuts and accumulation of genomic instability through multiple DNA edits. “RNA is completely different. If we target RNA, we do not touch the DNA all, and RNA editing is reversible,” Qi explained. Another advantage is Cas13’s ability to process several unique guide RNAs from a single array, which allowed the researchers to target multiple RNA transcripts at once in the same cell.

     

    By Deanna MacNeil, PhD

    Article can be accessed on: The Scientist