Engineers at the University of Pennsylvania and Rice University have developed more precise DNA base-editing tools that could improve the safety of gene-editing approaches for diseases such as cystic fibrosis.
The researchers focused on cytosine base editors (CBEs) – CRISPR-derived tools that convert cytosine (C) to thymine (T) at targeted genomic sites without introducing double-stranded DNA breaks. Such single-base substitutions underlie many inherited disorders, but current editors can unintentionally modify nearby bases when multiple cytosines occur close together in a DNA sequence.
“More than a thousand different genetic mutations can cause cystic fibrosis,” says Xue “Sherry” Gao, Presidential Penn Compact Associate Professor in Chemical and Biomolecular Engineering and Bioengineering at Penn Engineering and co-senior author of the study. “The fact that different mutations require distinct corrective tools highlights the importance of precision medicine.”
To improve editing specificity, the team engineered variants of an APOBEC3G-based editor. The researchers modified the molecular “linker” connecting the targeting and editing components and introduced mutations designed to reduce the enzyme’s interaction with neighboring DNA bases.
“We essentially tightened the leash to ensure only our target was edited,” says Tyler Daniel, a Penn Engineering doctoral candidate and co-first author of the study. The redesigned system also incorporates Cas9 variants with relaxed protospacer-adjacent motif (PAM) constraints, expanding the range of genomic sites the editor can target.
In laboratory experiments in human cells, the engineered editors significantly reduced unintended bystander edits while maintaining efficient editing at the target site. At several cystic fibrosis–related sites, unintended edits fell from around 50–60 percent to less than 1 percent.
The team also demonstrated that the editors could both introduce and correct cystic fibrosis–causing mutations in human bronchial epithelial cells. The edits altered cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels, protein expression, and ion channel function.
“We were able to introduce specific cystic-fibrosis mutations into human epithelial cells relevant to the disease, generating cell models that will improve our understanding,” says Gang Bao, co-senior author. “We were also able to reverse those mutations and show improved cellular functions using the same editor.”
Although the work remains at a preclinical stage, the authors suggest the improved base editors could support both the development of gene therapies and the creation of more accurate cellular models for studying genetic diseases.
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