CRISPR-Cas Technology Applications in Genetic Engineering

Exciting developments in CRISPR/Cas9 gene therapy! Researchers have developed a biomimetic mineralized CRISPR/Cas9 RNA delivery system that addresses the challenge of efficient multiplex gene editing. This all-in-one system is inspired by natural biomineralization and allows for precise control of the coencapsulation ratio between Cas9 mRNA and multiple sgRNAs. It also provides high RNA loading capacity, improved RNA storage stability, and the nanoparticles' surface can be easily functionalized for targeted delivery in vivo at nonliver sites and rapid lysosome escape. Moreover, the nanoparticle can be easily coated with hyaluronic acid for precise and efficient accumulation at the tumor site. In a proof-of-concept, the system demonstrated significant gene-editing at each target gene and promoted apoptosis of HeLa cells in a mouse model, inhibiting tumor growth without noticeable off-target effects in liver tissue. Moreover, there were no signs of toxicity to the mice based on the hematological parameters and they had normal liver and kidney function indices with low inflammation, what was attributed to N1-methylpseudouridine triphosphate substitution of uridine, which reduces the innate immune stimulatory effects of RNA. While coencapsulation of RNA species in viral vectors is difficult due to limited loading capacity, LNPs-based nonviral delivery technologies have made their way into human clinical trials. Still, their low RNA loading and nonspecific targeting to the liver limit their wider application. For these reasons, a novel and innovative strategy like this could revolutionize RNA-based CRISPR/Cas9 gene editing and has a huge potential for advancing gene therapy and cell engineering. Learn more here: https://lnkd.in/enhjhf2h #GeneTherapy #CRISPR #Cas9 #GeneEditing #BiomedicalEngineering #Nanotechnology #nanomaterials #nanoparticles #nanomedicine

Gene Editing Points Way to Cleaner Cellular Energy. Improving Mitochondrial Efficiency In today’s world, we are striving to produce energy while at the same time minimizing damage to the environment. The body is no different. This article discusses research to understand how to increase mitochondrial energy production without also increasing harmful reactive oxygen species (ROS). Mitochondria generate energy for cells through cellular respiration, but this process also produces ROS as a byproduct. High levels of ROS can damage cells and are linked to neurodegenerative diseases, heart disease, diabetes, cancer, and aging. The researchers used CRISPR gene editing to selectively turn down genes involved in mitochondrial energy production and analyzed the effects on energy and ROS levels. They found some genes mainly affected energy, while others had larger impacts on ROS. This shows it may be possible to control energy production and ROS levels independently by targeting different genes/pathways. It advances our understanding of mitochondria and cellular energy production. By using CRISPR to turn down specific genes systematically, the researchers gained new insights into the distinct roles and pathways involved in regulating energy production versus ROS generation. This sheds light on the underlying biology. It demonstrates the possibility of decoupling energy and ROS production. Many previous efforts to boost mitochondrial energy output led to increased ROS as an unavoidable side effect. This study shows you can target certain genes/pathways to enhance energy while minimizing ROS, revealing a potential solution. It identifies specific therapeutic targets. Now that genes and pathways that preferentially affect energy or ROS levels have been pinpointed, researchers can explore manipulating those targets to optimize mitochondrial function. This could lead to new treatment strategies. It has wide applications for diseases and aging. Since faulty mitochondria play a role in many age-related diseases (neurodegeneration, heart disease, diabetes, etc.), enhancing their function safely could have far-reaching benefits. This research lays the groundwork. The results provide insights into specific therapeutic targets and pathways to optimize mitochondrial function. Overall, this work advances our understanding of improving mitochondrial efficiency, which could lead to new regenerative strategies for various degenerative diseases and aging. This technology could have monumental consequences. JP https://lnkd.in/eYT2rAwv

What drives brain diseases Every risk gene may impact one or several different cell types. Comprehending how those cell types—and even individual cells—impact a gene and affect disease progression is key to understanding how to ultimately treat that disease. This is the study’s first author, co-invented the new technique, named in vivo Perturb-seq. This method leverages CRISPR-Cas9 technology and a readout, single-cell transcriptomic analysis, to measure its impact on a cell: one cell at a time. Using CRISPR-Cas9, scientists can make precise changes to the genome during brain development, and then closely study how those changes affect individual cells using single-cell transcriptomic analysis—for tens of thousands of cells in parallel. The method also enables a level of scalability that was previously impossible—the research team was able to profile more than 30,000 cells in just one experiment, 10-20 times accelerated from the traditional approaches. In a pilot study using this new technology, the team’s interest was piqued when they saw a genetic perturbation elicit different effects when perturbed in different cell types. This is important because those impacted cell types are the sites of action for particular diseases or genetic variants. “Despite their smaller population representations, some low-abundant cell types may have a stronger impact than others by the genetic perturbation, and when we systematically look at other cell types across multiple genes, we see patterns. That’s why single-cell resolution—being able to study every cell and how each one behaves—can offer us a systematic view,” the author says. #ScienceMission #sciencenewshighlights https://lnkd.in/gKgEcBPs

CAIN - A CRISPR tool that "overrides Mendelian inheritance" to enable 99% gene transmission 🧬Darwinian natural selection directs gene inheritance by Mendel's laws, where an equal chance is typical for alleles to pass on to the next generation. 🧬Artificial selection (and sometimes natural selection) enables a "super-Mendelian inheritance" where certain genes are inherited at rates greater than the expected 50%, potentially allowing these genes to spread through populations even if they are detrimental to organisms. Could we use this mechanism to manipulate natural populations by introducing alleles that benefit us even if they harm some plant species, or to eliminate species that are not useful in our fields? 🧬Researchers now developed a plant gene drive system called "CRISPR-Assisted Inheritance utilizing NPG1 (CAIN)", which uses a toxin-antidote mechanism in the male germline to override Mendelian inheritance in plants. 🧬CAIN uses a CRISPR-Cas9 construct that disrupts the key gene responsible for pollen germination (NPG1), which is the "toxin" part of the tool . The "antidote" is a recoded, CRISPR-resistant NPG1 gene that rescues functionality, but only in pollen cells containing the gene drive. 🧬In the proof of concept study, researchers were careful to prevent unintended spread of this mechanism to natural populations. So they use Arabidopsis thaliana , a self-pollinated model plant species, and reported a remarkably high transmission rate of the gene drive—between 88% and 99%—over two generations. 🧬CAIN can thus be a paradigm-shifting tool that will reshape ecological agricultural practices and can potentially balance crop protection and environmental considerations to minimize the loss of biodiversity while optimizing productivity to enable high transmission of desired traits to boost food productivity The publication: https://lnkd.in/gtx5fgyP The article: https://lnkd.in/gd4Sd28C

Happy Monday all! Check out this exciting new CSHL preprint by Curtis et al., "Genome-wide CRISPRa screens nominate modulators of CAR T cell survival within distinct tumor cytokine milieus." Abstract: Chimeric Antigen Receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies and translating this success to other cancers remains an ongoing clinical objective. Next-generation T cell products in development aim to genetically modulate many facets of cell behavior, for which gene-nominating platforms provide a useful framework for prioritization. Among competing screening approaches, CRISPR activation (CRISPRa) technology permits gain-of-function (GoF) gene surveys at genome-wide scale, but routine implementation in primary T cells has been stymied by high cell requirements (∼107 - 108) and abbreviated activity. Here, we describe a novel cell manufacturing schema using an all-in-one transposon-based gene delivery system coupled with CAR-restricted cell expansion to generate yields (109) of primary T cells bearing CAR and CRISPRa transgenes that are well above the threshold needed for genome-scale screening. CRISPRa activity is sustained via the inclusion of divergent, duplicate Elongation Factor 1α core/human T-cell leukemia virus (EF1α-HTLV) hybrid promoters; while guide RNA representation is preserved through late lentiviral transduction, thus preventing bottlenecking and premature candidate pruning. CRISPRa-CAR T cells manufactured via this pipeline retain potent on-target gene-overexpression (>85% target+) across varied cell subsets (e.g. Tim-3+Lag3+ or serial-challenge) and timescales (>14 days). When deployed to survival-based genome-wide selection landscapes, CRISPRa-CAR pools nominate known and novel endogenous genes capable of enhancing CD8+ CAR T survival in cytokine-rich (e.g. MYC, FUT6, IRF4, GSE1) and cytokine-depleted (e.g. CSF2RB, STAT6, IRF4, GSE1) settings of tumor challenge. This system will have broad utility for therapy-enhancing gene discovery. #drugdiscovery #cancerresearch #cartcelltherapy #immunooncology #immunotherapy #crispr #scientificresearch

𝗥𝗲𝗺𝗲𝗺𝗯𝗲𝗿 𝘁𝗵𝗲 𝗔𝗟𝗦 𝗜𝗰𝗲 𝗕𝘂𝗰𝗸𝗲𝘁 𝗖𝗵𝗮𝗹𝗹𝗲𝗻𝗴𝗲? A decade ago, millions of people dumped freezing water on themselves to raise awareness for ALS, a brutal neurodegenerative disease that robs people of muscle control and takes lives far too early. Well… science just landed a major blow in the fight against ALS. Researchers have used CRISPR gene editing to target and shut down ALS-related genes in mice. How? They packaged the CRISPR system into a neutralized virus that can cross the blood-brain barrier, a notoriously hard target, and deliver precise DNA edits directly to neurons. The result? >Dramatic reductions in muscle degeneration >Significant extension of the mouse’s lifespan This is a massive leap forward in neurogenetics and therapeutic gene editing. Human trials are still a ways off. But this kind of breakthrough shows what’s possible when cutting-edge neuroscience meets smart delivery systems. The Ice Bucket Challenge brought the world’s attention to ALS. CRISPR might just bring us the cure.

The CRISPR Journal published today our report describing “Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas.”  https://lnkd.in/eKRy7q8u It truly takes a village to bring resistance to the PRRS virus into the commercial population of pigs. It started with brilliant research by scientists at the University of Missouri-Columbia and Roslin Institute, who identified the CD163 protein as the key entry point for the virus and specific edits needed for resistance to the virus. This was followed by many years of dedicated work by scientists at Genus PLC and its pig division, Pig Improvement Company (PIC) - North America, to incorporate the precise edit into the commercial population of pigs without any off-target effects. Rodolphe Barrangou, professor of food, bioprocessing, and nutrition sciences at North Carolina State University and editor-in-chief of The CRISPR Journal, said it well: “This is a milestone study illustrating the potential of CRISPR-based technologies for commercial livestock breeding.”

Some hereditary genetic defects cause an exaggerated immune response that can be fatal. Using the CRISPR-Cas9 gene-editing tool, such defects can be corrected, thus normalizing the immune response, as researchers led by Klaus Rajewsky from the Max Delbrück Center now report in Science Immunology. Familial hemophagocytic lymphohistiocytosis (FHL) is a rare disease of the immune system that usually occurs in infants and young children under the age of 18 months. The condition is severe and has a high mortality rate. It is caused by various gene mutations that prevent cytotoxic T cells from functioning normally. These are a group of immune cells that kill virus-infected cells or otherwise altered cells. If a child with FHL contracts a virus—such as the Epstein-Barr virus (EBV), but also other viruses—the cytotoxic T cells cannot eliminate the infected cells. Instead, the immune response gets out of control. This leads to a cytokine storm and an excessive inflammatory reaction that affects the entire organism. “Doctors treat FHL with a combination of chemotherapy, immunosuppression and bone marrow transplantation, but many children still die of the disease," says Professor Klaus Rajewsky, who heads the Immune Regulation and Cancer Lab at the Max Delbrück Center. He and his team have therefore developed a new therapeutic strategy. Using the CRISPR-Cas9 gene-editing tool, the researchers succeeded in repairing defective T cells from mice and from two critically ill infants. The repaired cytotoxic T cells then functioned normally, with the mice recovering from hemophagocytic lymphohistiocytosis. https://lnkd.in/gTeg9Qdf

Researchers at North Carolina State University present a multiplex CRISPR genome editing strategy to modify lignin biosynthesis genes and reduce the lignin content of Populus trichocarpa, a species of poplar. CRISPR editing increased the wood carbohydrate-to-lignin ratio up to 228% that of wild type, leading to more-efficient fiber pulping. 

FDA approves two gene-modified autologous cell therapies for sickle cell disease, including first that uses CRISPR – paving the way for expanded use of personalized gene-engineered cell therapies. Bespoke autologous cell therapies have a niche as living therapeutics in clinical use cases where immune function and compatibility are paramount for potency. Enter the FDA approval of bluebird bio’ Lyfgenia as well as Vertex Pharmaceuticals’ Casgevy, both for SCD. Although the FDA approval of a gene engineered autologous cell pharmaceutical was first achieved with Novartis’ Kymirah in 2017, the FDA’ approval of Vertex’ Casgevy speaks to its assent of CRISPR-mediated gene editing as meeting safety equipoise. This also paves the way for FDA-sanctioned commercial manufacturing schemes for use of CRISPR gene-engineering technology – in addition to lentiviral transduction – as commercially viable means of manufacturing bespoke personalized gene-engineered cell therapies for use in other catastrophic illnesses with unmet medical needs. Disruptive gene-engineering technologies coupled with open market competition will be drivers of sustainable and accessible living therapeutics treatments and cures now and in future. Congratulations to the legion early innovative scientific discoverers and the risk-taking moxxi of CGT commercial developers to bring these living  pharmaceutical platforms over the marketing approval goal line.