Simple new strategy improves safety and accuracy of CRISPR gene editing

CRISPR genome editing

A breakthrough new strategy strengthens CRISPR editing, minimizing large deletions and increasing safety and precision in gene editing. Credit: 2024 KAUST

KAUST researchers have improved the safety of CRISPR gene editing by reducing harmful DNA deletions and improving repair mechanisms, advancing safer gene treatments.

A simple and powerful strategy developed by KAUST scientists can help improve safety and ACCURATELY of CRISPR gene editing, a tool that has already been approved for clinical use to treat inherited blood disorders.

This approach addresses a critical issue with CRISPR technology: the act of cutting the genome at specific points and then putting it back together, which inherently risks damaging DNA in a way that can cause large-scale and unpredictable disruption.

Hoping to alleviate this problem, a team led by Mo Li, a stem cell biologist at KAUST, investigated the DNA repair pathways that lead to large genomic deletions after CRISPR editing in human stem cells.

Their analysis led them to a process known as microhomology-mediated end-joining (MMEJ), an error-prone mechanism that, while capable of repairing breaks in DNA, often leaves large deletions in its wake.

Key genetic findings

The scientists interrogated various genes implicated in this MMEJ process and found two that played central—but opposite—roles in these unwanted deletion events.

A gene, called POLK, turned out to exacerbate the risk of large deletions after CRISPR editing. The other, called PRAemerged as a genomic chaperone with protective effects.

By manipulating these genes, either with drugs that inhibit them POLK or through genetic techniques that promote the expression of PRAthe KAUST team was then able to reduce the occurrence of large deleterious deletions without compromising the efficiency of genome editing and, in doing so, preserve the genomic integrity of the edited stem cells.

“This easy-to-use approach can reduce the chances of these large, damaging DNA deletions occurring,” says Baolei Yuan, a former Ph.D. student in Li’s lab and one of the architects of the study, along with Chongwei Bi and Yeteng Tian from Li’s lab.

Improving Repair Mechanisms

Moreover, the same interventions were found to increase the efficiency of homology-directed repair, a mechanism known for its ability to enable precise genome editing without adding unwanted mutations.

This was evident in experiments involving stem cells carrying mutations in two genes associated with sickle cell disease and Wiskott-Aldrich syndrome, both inherited blood disorders. Modulating POLK OR PRAthe researchers achieved very precise and reliable gene editing in these cells.

The findings mark an important step forward in refining CRISPR technology, Li claims. “It’s really exciting because it means we’re getting closer to safer and more effective treatments for genetic diseases,” he says.

With a provisional patent application filed for this innovative strategy, the team continues to explore the mechanisms behind a broader set of unwanted mutations and refine its techniques to make CRISPR safer and more efficient.

“Achieving high efficiency and safety remains a challenge that requires further development,” says Li, “and our lab remains at the forefront, seeking new solutions.”

Reference: “Modulation of the microhomology-mediated end-joining pathway suppresses large deletions and enhances homology-directed repair after CRISPR-Cas9-induced DNA breaks” by Baolei Yuan, Chongwei Bi, Yeteng Tian, ​​Jincheng Wang, Yiqing Jin, Khaled Alsayegh, Muham Tehseen, Gang Yi, Xuan Zhou, Yanjiao Shao, Fernanda Vargas Romero, Wolfgang Fischle, Juan Carlos Izpisua Belmonte, Samir Hamdan, Yanyi Huang, and Mo Li, April 29, 2024, BMC Biology.
DOI: 10.1186/s12915-024-01896-z

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