Scientists from the Medical Research Council have found that the distortion of DNA form may lead to changes in the target group when editing a gene
DNA distortions that occur routinely during gene expression and other cellular processes could lead to changes in the genome using CRISPR-Cas9, according to a new study. Scientists from medical research have argued that their findings can help pave the way for improving the accuracy of editing genes for clinical applications.
During gene expression, DNA is stretched and distorted from its usual form. While this is necessary for the proper functioning of the cell's own machine, it can pose a challenge for editing the CRISPR-Cas9 gene by increasing the risk of non-target edits, potentially introducing harmful changes. The results of scientists from the London Institute of Medical Sciences and AstraZeneca have been published today in the journal Nature Structural and Molecular Biology.
CRISPR-Cas9, a tool for editing genes that allows researchers to detect and edit DNA strands, has gained worldwide recognition for its many purposes as scientists use technology in a number of sectors, including medicine, drug discovery and agriculture.
In the study, the accuracy and precision of CRISPR-Cas9 was investigated using a new approach: scientists used optical tweezers – a tool that uses laser beams to manipulate DNA – to mimic distortions that DNA naturally passes while being read by the machine The cell then uses CRISPR-Cas9 to edit the gene and track its accuracy using fluorescence microscopy.
The results show that CRISPR is accurate when the DNA is free and relaxed. However, when distortions – in this case from severely stretched – the accuracy decreases and changes outside the target group are observed. Understanding this effect will help design CRISPR systems with increased precision, along with methods to assess this risk.
Principal investigator Professor David Rueda of LMS at MRC and Imperial College London said: "CRISPR-Cas9 has gained popularity as a potential tool to prevent or combat diseases caused by DNA mutations, and we wanted to investigate how accurate it is. When we manipulated DNA structures, mimicking the natural distortions that DNA undergoes, we found that edits of unwanted objects were made. In this scenario, CRISPR-Cas9 may show up to 50% targeting discrepancy, which would be a concern about using this tool to edit genes in clinical conditions.
"On the other hand, what is very positive is that we have developed a system that works to present CRISPR-Cas9 clearly so we can study why mistakes happen and adapt the technology to improve accuracy and specificity. "
Dr. Emanuela Quoomo, Assistant Director of Discovery Sciences at AstraZeneca, said: "At AstraZeneca we are working to understand the CRISPR mechanism of action so we can design improved enzymes for research and potential therapeutic purposes in the future. This study adds to our study of CRISPR-Cas9 and highlights future areas of research to better understand the relationship between DNA structure and off-site effects. "
Dr. Lindsay Wilson, Program Manager for Genetics, Epigenetics and Genomics at the MRC, said: "This study is critical because it helps us understand the risks of using CRISPR, for example, for human cell editing. Ultimately, this can lead to risk reduction strategies. MRC supports the use of scientifically and ethically stringent laboratory studies using genome editing, including research into understanding technological risks and limitations and steps to address them. "
Scientists say their next steps are to determine how they could reduce their chances of inadvertent editing. They plan to use their new system to further investigate the accuracy of CRISPR and, ideally, develop out-of-target prediction algorithms based on their findings.
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