The latest prime editing tools address delivery and security issues

Research image of genetically modified mouse liver cells versus controls.

Safe strategy: Mice injected with a dual virus master editor show no detectable changes in liver histology (right column) compared to control mice (left column).

New CRISPR-based prime editing tools provide delivery and security gains and can even flag their own potential errors, according to a number of recent studies.

As such, they could be harnessed to modify autism-related mutations and alleviate some of the condition’s traits, says Yang Yang, associate professor of medicinal chemistry and molecular pharmacology at Purdue University in West Lafayette, Indiana. Autism often results from multiple mutations, but recent large-scale human genetic studies have identified an increasing number of autism cases caused by monogenic mutations in a single gene.

Prime editing, developed in 2019, offers advantages over conventional CRISPR systems. For example, instead of making edits by cutting two strands of DNA at the same time and using a cell’s error-prone repair machinery to repair these breaks, master editing depends on a modified reverse transcriptase enzyme that creates critically repaired single-strand breaks. more reliable, one at a time.

But despite the advantage, core DNA lengths of about 6,300 DNA letters were too large for delivery via adeno-associated viruses (AAVs), limiting their usefulness.

To solve this problem, method scientists led by David Liu of Harvard University devised a way to split the instrument and package it into two AAVs. Once inside a cell, the halves merge again.

The team used the double virus strategy in mice to install mutations in star-shaped astrocytes that previous work suggests may protect against Alzheimer’s disease and in liver cells to lower cholesterol levels and defend against coronary heart disease. Viruses have shown no signs of being toxic or delivering unwanted changes, according to a study published May 4 in natural biotechnology.

The tactic modified up to 42 percent of the cells in the animal’s cerebral cortex, 46 percent of the cells in the liver and 11 percent of the cells in the heart, surpassing previous split prime editors. It’s also the first major editing tool used in postnatal brain and heart tissue, the team notes.

gBrain editing in the brain has proven difficult largely because of its low efficiency, says Jerzy Szablowski, an assistant professor of bioengineering at Rice University in Houston, who was not involved in the study.

But this new strategy is efficient enough in the brain to lead to clinically meaningful results, says Yang, who was not involved in the work. I predict that this inspiring paper will open up a wide range of studies using the first editor to correct for disease-causing mutations in preclinical models, including those found in children with autism. Future research should move testing of this approach to nonhuman primates, she adds.

For research applications, mice designed to encode and express a master editor in each cell appear to offer another solution to the delivery problem. In this setup, the core editor remains silent until activated by an enzyme called CRE recombinase, delivered into a lipid nanoparticle via injection.

Before CRISPR and other gene-editing tools, developing a line of lab mice to investigate a single mutation could take months or years, says Tyler Jacks, David H. Koch Professor of Biology at the Massachusetts Institute of Technology , who works with Liu and pioneered this approach. With this new model, we can make virtually any mutation of interest in any gene or regulatory sequence of interest in any cell of interest. I like to say now that we are only limited by our creativity.

Jack’s lab focuses on cancer, but the editor-in-chief mouse we describe can be used to introduce mutations into genes relevant to any process, including genes involved in autism, he notes.

In the experiments, he and his team modeled lung and pancreatic cancer by injecting CRE-recombinase into selected tissues along with different guide RNAs to mutate K-RAS or p53 linked to about 30 and 60 percent of all cancers, respectively. human tumors. They detailed their findings online May 11 in natural biotechnology.

A the main concern for any gene editor is unwanted edits, particularly in the case of a gene therapy that seeks to edit large numbers of cells.

Yet another new master editing method called PE-tags makes it easier to spot off-target effects in the genome by adding a tag wherever it makes a change. Its creators led by an independent team of scientists from the University of Massachusetts Chan Medical School and their colleagues have shown that they can identify the location of a tag not only in DNA extracted from cells, but also in laboratory-grown human embryonic kidney cells and in the livers of live mice.

While prediction of off-target sites can be achieved through computational approaches, prediction always needs careful testing, says Yang, who was not part of this study. The interesting part of this article is the new strategy to be identified experimentally [a] potential off-target site in the genome.

According to the team, the first editing systems designed as therapies for Tay-Sachs disease, cystic fibrosis and Rett syndrome had 43, 20 and 24 potential off-target sites, respectively. But they could lower that number by shortening the lifespan of key editors. They detailed their findings online May 8 in Methods of nature.

For any primary editing to progress to clinical translation, understanding the off-target profile would be of paramount importance in assessing the potential side effects of that particular editing in humans, Yang says. The technology developed in this paper would help facilitate this critical step of evaluating side effects.

This new strategy has been tested primarily in human embryonic kidney cells, Yang notes. Future research should examine whether prime editing has similar or different off-target profiles in mature cells, including neurons whose genetic activity may differ significantly.

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