The ever-evolving world of gene editing is exciting. It has only been 11 years since Professors Jennifer Doudna and Emmanuel Charpentier published their groundbreaking book. science The paper foretells a revolution in gene editing, with CRISPR-based treatments already commercially available for patients.

CRISPR technology has been featured in thousands of scientific papers. Laboratories around the world are working to optimize various components of this technology's molecular machinery, fine-tuning and enhancing it for a wide range of applications. Here we discuss examples of recent research advances that address key bottlenecks in this field.

in vivo Gene editing to increase access to CRISPR-based therapies

Existing gene therapies that use non-viral vectors to deliver therapeutics typically ex vivo edit. The patient's cells are extracted, taken to a lab for editing, and then injected into the body. Although this approach is effective, it also has drawbacks. Prolonged hospital stays can increase the cost and limit the availability of modern gene therapies.

Kasugevy is the first U.S. Food and Drug Administration (FDA)-approved treatment utilizing CRISPR-Cas9, bringing new hope to patients with sickle cell disease. However, each treatment comes at a hefty cost of approximately $2.2 million.

“With new technology, in vivo Improving the delivery and manufacturing of gene editing treatments will be key to lowering prices,” said Nobel laureate Jennifer Doudna, who co-received the 2020 Nobel Prize in Chemistry for the development of CRISPR-Cas9 gene editing. Told.

The Doudna lab at the Innovative Genomics Institute (IGI) has been exploring innovative ways to skip the step of manipulating cells outside the body.They recently shared a method nature biotechnology Harnessing the predictability of antibody-antigen interactions provides genome editing tools in a targeted manner.

Dr. Jennifer Hamilton, a CRISPR researcher in the Doudna lab, previously discovered that once the outer envelope of the HIV-1 virus is emptied and filled with Cas9, it can edit T cells. ex vivo, converting them into CAR T cells. At the time, Hamilton referred to his “foamy” envelopes as virus-like particles, or his VLPs. Since then, she has refined her VLP and now calls it the Envelope Delivery Vehicle (EDV).

What are CAR T cells?

CAR T cells are the mainstay of cancer treatment CAR-T cell therapyHere, a patient's T cells are engineered outside the body to express chimeric antigen receptors (CARs) that can bind to and attack cancer cells.

The advantage of EDV is that it can be coated with multiple antibody fragments, increasing binding capacity and specificity when targeting specific cells.of nature biotechnology This paper is a proof-of-principle study exploring their abilities. in vivo Genome engineering.

Hamilton et al. created an EDV modified with a monoclonal antibody that targets T cells for use in mice with humanized immune systems. The EDV contained a CRISPR mechanism to knock out the natural T-cell receptor and a transgene for the B-cell-targeting receptor, which is used as a surrogate for cancer cells.

“We aimed to systemically administer a single vector that would both deliver and knock out genes in specific cell types in the body,” Hamilton said. “We used this delivery strategy to create gene-edited CAR T cells. in vivoIn hopes of streamlining the complex process used to produce gene-edited CAR T cells ex vivo. ”

“More effective delivery was possible when the particles were linked together using two antibody-ligand interactions,” Hamilton continued. “After we treat mice with T-cell targeting vectors, the targeted cell types, T-cells and hepatocytes in the liver, are absent.” Liver Cell uses delivery vehicles destined for other destinations. This was good news for the team, as they are known for their.

The IGI team hopes their research will pave the way for more widely available and cost-effective CRISPR therapies. “Although this report focuses on the manipulation of human immune cells (T cells), future research will also extend to non-immune cells, with a particular focus on targeted manipulation of tissue-resident stem cells.” in vivo,” they said.

Improving CRISPR technology to study immune cell genes

At Harvard University, scientists have been exploring ways to improve the utility of CRISPR-based gene editing in studying the human immune system.

Over the past two decades, the field of immuno-oncology has blossomed. There are now several FDA-approved treatments that target the immune system to stop cancer progression. Despite these advances, much is still unknown about the gene networks that regulate the immune system. Basic research is key here, and CRISPR technology can help.

Modeling the immune system in a dish is no easy feat, so scientists are leaning toward: in vivo Research that captures what is actually happening inside living organisms. Here, CRISPR runs into a similar hurdle in its application to gene therapy. Immune cells often need to be extracted from the organism, edited, and reinserted.

“When you put it back into mice, only certain types of immune cells are efficiently incorporated. Also, the actual process of manipulating immune cells in a dish can change the biology of the immune cells, so “If immune cells are removed from the body, we may not be able to study the things we actually want to study,” said Sharp postdoctoral fellow Dr. Martin LaFleur. This was revealed by a research lab at Harvard University. Additionally, our cells contain thousands of genes. Multiplex gene editing, which targets multiple genes simultaneously, is advantageous for immunology research, but is currently difficult.

LaFleur is part of a recent research initiative. innate immunology And that experimental medicine journal, has uniquely adopted CRISPR technology. Instead of modifying the target's immune cells, the researchers targeted their precursors, stem cells that are produced in the bone marrow and are the basis of all immune cell types. “We removed these stem cells from the mice, used CRISPR to knock out the gene of interest, and replaced these stem cells in the mice where the natural bone marrow stem cells had been removed,” LaFleur said.

The system, called CHimeric IMmune Editing (CHIME), allows genes to be knocked out without affecting immune cell development or function. “Previous studies have used CHIME to Point 2, which shows some promise for cancer immunotherapy, one of the focuses of the Sharp Institute. “When we removed that one gene from a subset of immune cells known as CD8+ T cells, they became better cancer fighters,” he explained.

in innate immunology, researchers are using the X -Describes CHIME-based systems. “We wanted to see if we could modify his CHIME to make it more accurate and more versatile,” he says.

“We use this to knock out two genes at once in several different cell types, introduce it to specifically target a gene in a single cell type, and find that the modified cells “We used CRISPR to disrupt the gene after it was already back in the animal. We also used it to knock out two different genes at different times,” he added. “We use a variety of tactics, including packaging multiple guide RNAs together and using tricks that only disable genes under certain circumstances, such as when mice are given a drug. We were able to demonstrate that each of these strategies is feasible.”

of experimental medicine journal This paper provides the research community with a framework for screening the function of immune genes in vivo using CRISPR. “The core of our framework is adding a genetic 'barcode' to CRISPR-edited immune cells, which allows us to track how they grow and spread within an animal. “It will be,” LaFleur said.

The Sharp lab believes that this framework and CHIME will provide a versatile tool for studying immune cells in cancer and possibly other diseases such as autoimmune diseases, ultimately leading to better treatments. I'm looking forward to it.

Fast integrated site search with CRISPR-COPIES

Beyond medical uses, CRISPR technology can also be leveraged for synthetic biology applications such as chemical and biofuel production, or genetically engineered pest resistance.

At the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) in Illinois, researchers are exploring new ways to optimize the utility of CRISPR in metabolic engineering of non-model yeast. Their goal is to economically produce biofuels and bioproducts from plant biomass, but it is difficult to produce on a large scale for several reasons.

It's the decision that's difficult where Insert edits into the genome. “Finding integration sites in a genome by hand is like looking for a needle in a haystack,” said Ashutosh Boob, a doctoral student at the University of Illinois. haystack. ”

Previously, researchers identified targets by manually screening potential integration sites. Cytocompatibility and gene expression levels at selected sites are then assessed by incorporating reporter genes, a time- and resource-intensive approach.

CRISPR-COPIES – short for COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites – is a potentially useful new tool developed by Boob and his colleagues.

“This tool leverages ScaNN, a state-of-the-art model for embedding-based nearest neighbor search for fast and accurate off-target searches, to locate genome-wide intergenic sites for most bacterial and fungal genomes. can be identified within minutes,” the researchers said.

CRISPR-COPIES has applications in synthetic biology toolkit characterization, gene therapy, and metabolic engineering. Credit: Aashutosh Boob et al.

in Nucleic acid researchBoob et al. applied new tools to characterize neutral integration sites in three species. Cupriavidus necatl, budding yeast, and HEK 293T cells. Using these integration sites, they engineered cells to overproduce 5-aminolevulinic acid, a biochemical with wide-ranging applications in agriculture and food.

“Using CRISPR-COPIES, we turn a haystack into a searchable space, allowing researchers to efficiently find all the needles that meet certain criteria,” said Boob. .

“CRISPR-COPIES is a tool that can rapidly identify chromosomal integration sites suitable for genetic engineering in any organism,” said first author and CABBI Transformation Theme Leader Steven L. of Chemical and Biomolecular Engineering (ChBE). said Dr. Huimin Zhao, who is also Miller Chair. The University of Illinois stated: “This will accelerate research in metabolic engineering of non-model yeasts for cost-effective production of chemicals and biofuels.”

The CABBI team expects both academia and industry to benefit from CRISPR-COPIES.

The CRISPR toolbox continues to expand

The versatility of CRISPR gene editing technology holds promise in addressing current limitations in medicine, agriculture, and more, providing new opportunities for innovation. The notable advances described here are by no means an exhaustive list, but merely a glimpse into the dynamic and rapidly evolving landscape of the CRISPR toolbox.

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