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What’s the Latest in CRISPR Gene-Editing Technology?

A graphic representing the human genome.
Credit: iStock.
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Excitement is a constant in the ever-evolving world of gene editing. It’s been a mere 11 years since Professors Jennifer Doudna and Emmanuelle Charpentier published their landmark Science paper that foreshadowed a revolution in gene editing, and already there are CRISPR-based therapies on the market for patients.

CRISPR technology has featured in thousands of scientific papers. Laboratories across the world are striving to optimize various components of the technique’s molecular machinery, tweaking and enhancing it for a broad range of applications. Here, we discuss examples of recent research advancements that tackle core bottlenecks in the field.

In vivo gene editing for increased accessibility to CRISPR-based therapies

Existing gene therapies that use non-viral vectors for therapeutic delivery typically require ex vivo editing. A patient’s cells are extracted, transported to a laboratory for editing and then infused back into the body. This approach, while effective, has its drawbacks. Extended hospital stays contribute to the costly nature of modern gene therapies, which could limit their accessibility.

Casgevy, the first US Food and Drug Administration (FDA)-approved therapy that utilizes CRISPR-Cas9, offers new hope for patients with sickle cell disease. But it comes with a hefty price tag – approximately $2.2 million dollars for a one-time treatment.

"New technologies allowing in vivo delivery of gene-editing therapies and improved manufacturing will be key to driving prices down,” said Nobel Laureate Jennifer Doudna, who shared the 2020 Nobel Prize in Chemistry for her work developing CRISPR-Cas9 gene editing.

The Doudna laboratory at the Innovative Genomics Institute (IGI) has been exploring innovative ways to skip the step of engineering cells outside of the body. They recently shared a method in Nature Biotechnology that harnesses the predictability of antibody–antigen interactions to deliver genome editing tools in a targeted manner.

Dr. Jennifer Hamilton, a CRISPR researcher in Doudna’s laboratory, previously discovered that the exterior envelope of an HIV-1 virus – once emptied and filled with Cas9 – was capable of editing T cells ex vivo, converting them to CAR T cells. At that time, Hamilton referred to her “bubble” envelopes as virus-like particles, or VLPs. Since then, she has modified the VLPs to the extent that she now calls them enveloped delivery vehicles, or EDVs.

What are CAR T cells?

CAR T cells are the major players in the cancer treatment CAR T-cell therapy, where a patient’s T cells are engineered outside of the body to express a chimeric antigen receptor (CAR), which can bind to and attack cancer cells.

An advantage of EDVs is that they can be coated with several antibody fragments, which increases their binding capacity and specificity when targeting specific cells. The Nature Biotechnology paper is a proof-of-principle study exploring their capacity for in vivo genome engineering.

Hamilton and colleagues created EDVs that had been decorated with monoclonal antibodies targeting T cells for application in a mouse with a humanized immune system. The EDVs contained the CRISPR machinery to knock out the native T-cell receptor, and a transgene for a receptor that targets B cells, which were used as a proxy for cancer cells.

“We aimed to systemically administer a single vector that would do both gene delivery and gene knockout in specific cell types inside the body,” said Hamilton. “We used this delivery strategy to make gene-edited CAR T-cells in vivo, in the hopes that we’d be able to streamline the complex process used to manufacture gene-edited CAR T-cells ex vivo."

"We were able to get more effective delivery when the particles bound using two antibody ligand interactions,” Hamilton continued. “After treating mice with T-cell-targeted vectors, we observed genome engineering in our cell type of interest, T cells, and not in liver hepatocytes.” Liver cells are renowned for taking up delivery vehicles that are intended for other destinations, so this was good news for the team.

The IGI team hope their research paves the way for CRISPR therapies that are more widely available and cost-effective. “Although this report focuses on the engineering of human immune cells (T cells), future work will be extended to non-immune cells, with a particular focus on the targeted engineering of tissue-resident stem cells in vivo,” they said.

Refining CRISPR technology to study immune cell genes

Over at Harvard University, scientists have been searching for ways to improve the utility of CRISPR-based gene editing for studying the human immune system. 

In the last 20 years, the field of immuno-oncology has blossomed – there are now several FDA-approved therapies that target the immune system to combat cancer progression. Despite this progress, there is still so much that we don’t know about the genetic networks that coordinate the immune system. Basic research is important here, and CRISPR technology can offer a helping hand.

Modeling the immune system in a dish is no easy feat, so scientists lean towards in vivo studies to capture what’s actually happening within a living organism. Here, CRISPR hits a similar roadblock to its application in gene therapy: immune cells often need to be extracted from the organism, edited and then re-inserted.

“Only certain immune cell types can be incorporated efficiently when transferred back into a mouse. Also, the actual process of manipulating immune cells in a dish can change their biology, so you may not be studying what you actually want to study once they’re removed from the body,” Dr. Martin LaFleur, a postdoctoral fellow in the Sharpe Laboratory at Harvard University, said. What’s more, our cells contain thousands of genes; multiplexed gene editing, where several genes are simultaneously targeted, would be advantageous for immune studies, but is currently difficult.

LaFleur was part of a recent research initiative, presented in Nature Immunology and the Journal of Experimental Medicine, that adopted CRISPR technology in a unique way. Instead of modifying the immune cells of interest, the researchers targeted their precursors – stem cells that are produced in bone marrow and are the foundations for all immune cell types. “We removed those from mice and used CRISPR to knock out the genes we were interested in, and then replaced these stem cells in mice whose native bone marrow stem cells had been removed,” said LaFleur.

The system is called CHimeric IMmune Editing, or CHIME, and it enables the knockout of genes without impacting the development or function of immune cells. “In an earlier study, we used CHIME to knock out a gene called Ptpn2, which has shown some promise for cancer immunotherapy, one of the focuses of the Sharpe Lab. When we deleted that one gene in a subset of immune cells known as CD8+ T cells, they became better cancer fighters,” LaFleur explained.

In Nature Immunology, the researchers describe X-CHIME-based systems: for interrogation of gene function, either combinatorially (C-CHIME), inducible (I-CHIME), lineage-specifically (L-CHIME) or sequentially (S-CHIME). “We wanted to see if we could modify CHIME and make it both more precise and more versatile,” said LaFleur.

“We used it to knock out two genes at once in several different cell types, we deployed it to target genes specifically in a single cell type, we used CRISPR to disrupt genes in modified cells once they were already back inside the animal, and we also used it to knock out two different genes at different points in time,” he added. “We used different tactics, such as packaging multiple guide RNAs together and using a trick that disables genes only under certain circumstances, such as when mice receive a drug. We were able to demonstrate that each of these strategies is feasible.”

The Journal of Experimental Medicine paper provides the research community with a framework for using CRISPR to screen the function of immune genes in living organisms. “Central to our framework is adding a genetic ‘barcode’ to CRISPR-edited immune cells so we can track them as they multiply and spread within animals,” LaFleur said.

The Sharpe lab hopes that the framework – and CHIME – can offer versatile tools for studying immune cells in cancer, and perhaps other conditions such as autoimmune diseases, ultimately leading to better therapies.

Fast-tracking integration site searching with CRISPR-COPIES

Beyond its use in medicine, CRISPR technology can be harnessed for synthetic biology applications, such as the production of chemicals and biofuels, or genetically engineering pest-resistance.

At the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) in Illinois, researchers are seeking new ways to optimize CRISPR’s utility for metabolic engineering of non-model yeasts. Their goal is to economically produce biofuels and bioproducts from plant biomass, which is challenging to do at a large scale for several reasons.

One difficulty is deciding where in the genome to insert edits. “Finding the integration site in the genome manually is like searching for a needle in a haystack,” said Aashutosh Boob, a PhD student at the University of Illinois: “Finding the integration site in the genome manually is like searching for a needle in a haystack.”

In the past, researchers would identify targets by manually screening for potential integration sites. They would subsequently assess the chosen site's cellular fitness and gene expression levels by integrating a reporter gene, an approach that is time-consuming and resource heavy.

CRISPR-COPIES – which stands for COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites – is a new tool developed by Boob and colleagues that could help.

“This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search, and can identify genome-wide intergenic sites for most bacterial and fungal genomes 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 Acids Research, Boob and colleagues applied their new tool to characterize neutral integration sites in three species: Cupriavidus necator, Saccharomyces cerevisiae, and HEK 293T cells. Using these integration sites, they engineered cells that overproduced 5-aminolevulinic acid, a biochemical that has wide-reaching applications in agriculture and food.

“With CRISPR-COPIES, we transform the haystack into a searchable space, empowering researchers to efficiently locate all the needles that align with their specific criteria,” Boob said.

“CRISPR-COPIES is a tool that can quickly identify appropriate chromosomal integration sites for genetic engineering in any organism,” Dr. Huimin Zhao, lead author, CABBI conversion theme leader and Steven L. Miller Chair of Chemical and Biomolecular Engineering (ChBE) at the University of Illinois, said. “It will accelerate our work in the metabolic engineering of non-model yeasts for cost-effective production of chemicals and biofuels.”

The CABBI team anticipate that both academia and industry will benefit from CRISPR-COPIES.

The CRISPR toolbox continues to expand

CRISPR gene-editing technology’s versatility holds promise for addressing current limitations in medicine, agriculture and beyond, and provides new opportunities for innovation. The notable advancements discussed here are by no means an exhaustive list, they merely offer us a glimpse into the dynamic and swiftly evolving landscape of the CRISPR toolbox.