BreakTag opens the way for precise, predictable & personalised genome editing
In a recent study published in Nature Biotechnology, the team of Vassilis Roukos at the Institute of Molecular Biology (IMB) in Mainz, Germany has developed a new method that simultaneously assesses the location and structure of DNA double-strand breaks made by the genome-editing tool CRISPR/Cas9. This methodology, which they named BreakTag, allows scientists to design and test better CRISPR tools that can be used in personalised genome editing to treat currently incurable genetic diseases.
Like a pair of molecular scissors, CRISPR/Cas9 is a powerful tool for editing genomes, which scientists use to cut DNA at specific locations. This revolutionary technology is already successfully being used to edit the DNA of patients to correct mutations that cause debilitating diseases such as sickle cell disease and β-thalassemia. Importantly, it holds the promise of curing many more devastating genetic diseases that currently cannot be treated effectively. These include cystic fibrosis, muscular dystrophy, several blood disorders, immunological and neurodegenerative diseases, and some cancers.
However, the CRISPR/Cas9 technology also has significant risks: the Cas9 enzyme sometimes cuts at unintended, off-target sites. Moreover, the changes produced in the DNA when it cuts are not consistent, ranging from insertions to deletions of various sizes. This unpredictability can mean that the mutation causing the disease is not corrected. Even worse, it can cause more mutations at unintended locations. There is a possibility that latter can, in turn, result in adverse side effects, including secondary diseases such as cancer. Such risks must of course be minimised as much as possible when treating human patients.
To improve the safety of genome editing technology, we need to understand how to make CRISPR/Cas9 tools that cut in a more precise and predictable fashion. Scientists thought that some of Cas9’s unpredictability is because the enzyme sometimes makes DNA cuts with blunt ends (i.e. no overhanging DNA nucleotides) while other times the cuts have staggered ends (i.e. an overhang of 1-3 single-stranded nucleotides), and these might be repaired differently by the cell. However, no one was sure what caused this or whether making Cas9 enzymes that predominantly cut in one way would improve the predictability of genome editing, because there was no way to measure the location and structure of Cas9 cuts on a large scale.
This is where Vassilis Roukos comes in. His team at IMB and the Medical School of the University of Patras (Greece) have developed a fast and extremely sensitive high-throughput technology called BreakTag, which—for the first time—can do two things simultaneously: first, it can detect the location of CRISPR/Cas9 cuts (i.e. double-strand DNA breaks) throughout the genome, and second, it can determine whether they are blunt or staggered.
Using BreakTag, the researchers analysed the structure of CRISPR/Cas9 cut sites at more than 150,000 locations in the human genome. By comparing the BreakTag data of sites that were cut in a blunt versus staggered fashion with known CRISPR/Cas9 repair outcomes, the researchers noticed that staggered cuts often produce single-nucleotide insertions in the DNA, whereas blunt cuts produce more variable changes. Indeed, over 90% of sites with a single-nucleotide insertion as the most common repair outcome originated from a staggered cut. This meant that, if there was a way to make Cas9 preferentially make staggered cuts, scientists would be able to more reliably edit single-nucleotide insertions into genomic DNA. Importantly, single-nucleotide insertions are the exact type of genome editing needed to correct single nucleotide deletion mutations that are the underlying cause of diseases such as cystic fibrosis and muscular dystrophy, which affect hundreds of thousands of people worldwide.
Analysing their BreakTag data with artificial intelligence (AI) tools, Vassilis and his team discovered that the sequence of the targeted cut site is a major factor that determines whether Cas9 makes a blunt or staggered cut. Specifically, a guanine in position 17 of the target sequence (also called the protospacer sequence) means Cas9 is more likely to cut with a blunt end, while a guanine at position 18 is more likely to induce a staggered cut. Vassilis says, “This discovery is a significant advance towards making genome editing safer and more reliable. By specifically selecting Cas9 target sites with a guanine at position 18 and no off-targets, we can now more precisely correct frameshift mutations with a greatly reduced chance of adverse side effects. This takes us one step closer to bringing CRISPR genome editing into the clinic more broadly and using it to cure many more patients.”
The researchers also found that small genetic differences between individuals (i.e. different patients) could influence the way Cas9 cuts DNA and the genome editing outcome, especially if such differences affected whether a guanine is present at position 17 or 18. “Our research shows that researchers or doctors could also enhance the precision and predictability of CRISPR/Cas9 gene editing by looking at the exact sequence of the target site in the patient being treated and choosing a target site accordingly”, explains Gabriel Longo, co-first author of the study.
On a wider level, the new BreakTag technology is extremely useful because it opens the way for researchers to design even better genome editing nucleases and experimentally test how precise and predictable they are. For example, Vassilis and his team used BreakTag to compare the activity, specificity and ratio of blunt/staggered cuts for different Cas9 variants and found that some had a higher frequency of staggered cuts and single-nucleotide insertions. Doctors treating a patient with a frameshift mutation could therefore choose to use one of these Cas9 variants to maximise the chances of correcting the mutation.
“Researchers and companies could use AI tools to develop new Cas9 variants designed to make specific types of DNA cuts and then experimentally assess how accurate and predictable they are with BreakTag to quickly determine which ones are the best for correcting a specific mutation. This would allow doctors to use CRISPR/Cas9 to treat even more diseases effectively with fewer adverse side effects”, says Sergi Sayols, who is also a co-first author of the study.
Further details
Further information can be found here: https://www.nature.com/articles/s41587-024-02238-8
A patent application has been filed for the BreakTag technology (WO 2024/033378 A1), https://patents.google.com/patent/WO2024033378A1/en
Vassilis Roukos is an Affiliated Group Leader at the Institute of Molecular Biology. Further information about research in the Roukos lab can be found at www.imb.de/roukos.
About the Institute of Molecular Biology gGmbH
The Institute of Molecular Biology gGmbH (IMB) is a centre of excellence in the life sciences that was established in 2011 on the campus of Johannes Gutenberg University Mainz (JGU). Research at IMB focuses on the cutting-edge fields of epigenetics, genome stability, ageing and RNA biology. The institute is a prime example of successful collaboration between a private foundation and government: The Boehringer Ingelheim Foundation has committed 154 million euros to be disbursed from 2009 until 2027 to cover the operating costs of research at IMB. The State of Rhineland-Palatinate has provided approximately 50 million euros for the construction of a state-of-the-art building and is giving a further 52 million in core funding from 2020 until 2027. For more information about IMB, please visit: www.imb.de.
Boehringer Ingelheim Foundation
The Boehringer Ingelheim Foundation is an independent, non-profit organization that is committed to the promotion of the medical, biological, chemical, and pharmaceutical sciences. It was established in 1977 by Hubertus Liebrecht (1931–1991), a member of the shareholder family of the Boehringer Ingelheim company. Through its funding programmes Plus 3, Exploration Grants and Rise up!, the Foundation supports excellent scientists during critical stages of their careers. It also endows the international Heinrich Wieland Prize, as well as awards for up-and-coming scientists in Germany. In addition, the Foundation funds institutional projects in Germany, such as the Institute of Molecular Biology (IMB) and the European Molecular Biology Laboratory (EMBL) in Heidelberg. www.boehringer-ingelheim-stiftung.de/en
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