Latest ‘prime-editing’ tools tackle delivery, safety issues | Spectrum

New CRISPR-based “prime editing” tools offer improvements in delivery and security, and can even flag their own potential bugs, according to a number of recent studies.

Therefore, they could be used to modify autism-associated mutations and alleviate some features of the condition, 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 a growing number of cases of autism caused by monogenic mutations in a single gene.”

Prime editing, developed in 2019, offers advantages over traditional CRISPR systems. For example, instead of making edits by cutting two strands of DNA at once and using a cell’s error-prone repair machinery to repair those breaks, primary editing relies on a modified reverse transcriptase enzyme that more reliably creates repaired single-strand breaks. one after the other.

But despite the benefit, the major editors — about 6,300 letters of DNA in length — were too large for transmission by adeno-associated viruses (AAVs), limiting their usefulness.

To solve this problem, the inventors of the method – scientists led by Harvard University’s David Liu – devised a way to split the tool and pack it into two AAVs. Once you’re in a cell, the halves come back together.

The team used the dual-virus strategy in mice to install mutations in stellate astrocytes, which previous work suggested might protect against Alzheimer’s disease, and in liver cells to lower cholesterol and protect against coronary heart disease protect heart disease. The viruses showed no signs of being toxic or causing unintended changes, according to a study published May 4 in natural biotechnology.

The tactic processed up to 42 percent of the cells in the animals’ cerebral cortex, 46 percent of the cells in the liver and 11 percent of the cells in the heart, beating previous split-prime editors. It is also the first premium editing tool used in postnatal brain and heart tissue, the team notes.

GProcessing enes in the brain has proven difficult “mainly because of their low efficiency,” says Jerzy Szablowski, an assistant professor of bioengineering at Rice University in Houston, who did not take part in the study.

But this new strategy is efficient enough in the brain to “lead to clinically significant results,” says Yang, who was also not involved in the work. “I expect this inspiring article will open a wide range of studies using the Prime Editor to correct disease-causing mutations in preclinical models, including those found in children with autism.” Future research should be tested shifting this approach to nonhuman primates, he adds.

For research applications, mice engineered to encode and express a main editor in each cell appear to offer another solution to the transmission problem. In this setup, the main editor remains silent until activated by an enzyme called CRE recombinase, which is injected into a lipid nanoparticle.

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

Jacks’ lab is focused on cancer, but “the prime editor mouse we’ve described can be used to introduce mutations into genes relevant to any process, including genes involved in autism,” he notes.

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

A A major problem with any gene editor is unwanted changes – especially in the case of gene therapy aimed at editing large numbers of cells.

Another new prime-editing method called PE-Tag makes it easier to detect off-target effects across the genome by adding a tag at each point where a change is made. Its creators – led by an independent team of scientists from the University of Massachusetts Chan Medical School and their colleagues – showed that they could determine the position of a tag not only in DNA extracted from cells, but also in laboratory-grown human embryonic kidney cells and in of DNA were able to identify livers from living mice.

“While predicting off-target locations can be achieved through computational approaches, prediction always requires careful testing,” says Yang, who did not participate in this study. “The exciting part of this work is the new strategy for experimental identification [a] potential off-target site in the genome.”

The team found that prime-editing systems developed 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 senior editors. They detailed their findings online on May 8 natural methods.

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

This new strategy was mainly tested on human embryonic kidney cells, Yang notes. Future research should examine whether prime editing exhibits similar or different off-target profiles in mature cells, including neurons, whose genetic activity can differ significantly.