Cutting-edge platform significantly reduces time required for genome engineer and selection of optimal editors for specific applications

A research team from the LKS Faculty of Medicine at the University of Hong Kong (HKUMed) has developed a groundbreaking method to optimize precise genome editors at scale. This new approach allows for the parallel engineering and testing of hundreds or more base editor variants, eliminating the need for time-consuming one-at-a-time testing. By leveraging this method, users can easily identify the most suitable precise genome editors for therapeutic genome editing. The team’s findings have been published in Cell Systems, and a patent application has been filed based on their work.

Base editing, a newer CRISPR-based genome editing technology, presents a safer option for addressing genetic diseases with single-base mutations. This technique works by correcting the mutated DNA to its normal form. However, the outcome of base editing depends on factors such as the type and version of the base editor used, the composition of the target DNA sequence, and the position of the DNA bases to be converted. Selecting an unsuitable base editor can lead to incorrect edits and additional mutations, resulting in undesired effects.

Currently, optimizing the use of existing base editors requires painstaking one-at-a-time testing to determine their editing performance for each therapeutic locus. Moreover, many therapeutic loci lack an optimized base editor, which means creating a new one with conventional methods can take months or even years, despite global efforts.

HKUMed’s research team has developed a platform that combines a base editor reporter system with CombiSEAL technology. CombiSEAL allows for the rapid engineering of hundreds or more base editor variants in parallel by combining varying enzymatic deaminase domains and CRISPR/Cas9-based DNA-recognition domains. The team used this platform to quantify each variant’s editing efficiency, purity, sequence motif preference, and bias in generating single and multiple base conversions in human cells. This comprehensive analysis enables the selection of the most suitable base editors for therapeutic targets, maximizing efficiency and minimizing undesired edits.

The team also expanded the platform’s applicability to enhance the current base editor system. By focusing on engineering the stem-loop-2 region of the sgRNA scaffold used in the base editor system, they identified two novel sgRNA scaffold variants, SV48 and SV240, that achieved significantly higher base editing efficiency compared to the wild-type scaffold.

Furthermore, the platform is not limited to base editors but is also compatible with other precise genome editor systems, including prime editors. This expands the scope of potential genome editors for correcting genetic mutations in therapeutic targets where base editors may not be applicable.

This platform revolutionizes the engineering of next-generation precise genome editors and their adaptation for future therapeutic applications. Dr. Alan Wong Siu-lun, Associate Professor of the School of Biomedical Sciences at HKUMed, describes it as an “accelerated checkout process in stores,” where barcode-labeled products (base editor variants) are quickly scanned in bulk, eliminating the need for individual testing.

Dr. Alan Wong Siu-lun led the research team, with John Fong Hoi-chun, a Ph.D. student, as the first author. The team also received assistance from Dr. Chu Hoi-yee and Dr. Zhou Peng, postdoctoral fellows at the School of Biomedical Sciences, HKUMed.

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