Researchers develop platform to accelerate engineering of precise genome editors

Researchers from the University of Hong Kong’s LKS Faculty of Medicine (HKUMed) has made a breakthrough in the field of genome editing. They have developed a novel method to overcome the current limitations in optimizing precise genome editors at scale. This new approach allows them to engineer hundreds of base editor variants simultaneously, rather than through time-consuming one-at-a-time testing. The team’s findings have been published in the scientific journal Cell Systems, and they have also filed a patent application based on their work.

Base editing is a relatively new genome editing technology based on CRISPR, which shows promise in safely addressing genetic diseases caused by single-base mutations. By correcting these mutations to their normal form, base editing offers a potentially safer approach for treating diseases like sickle cell disease and familial hypercholesterolemia.

However, the outcome of base editing can vary depending on factors such as the type and version of the base editor used, the sequence composition of the target DNA, and the position of the DNA bases to be modified. Choosing an inappropriate base editor can lead to incorrect edits and the introduction of additional mutations around the target site, potentially causing unintended effects.

Currently, the process of characterizing the editing performance of the available base editors requires laborious and exhaustive testing of each editor individually. Furthermore, many therapeutic targets lack an optimized base editor, and developing a new one using conventional methods can take months or even years.

The HKUMed research team has developed a platform that combines a base editor reporter system with a technology called CombiSEAL. CombiSEAL allows for the rapid engineering of hundreds of base editor variants in parallel by combining different enzymatic deaminase domains and CRISPR/Cas9-based DNA-recognition domains. The team used this platform to evaluate the editing efficiency, purity, sequence motif preference, and bias of each variant in human cells. This information helps identify the most suitable base editors for therapeutic targets, selecting those that achieve the desired type of base conversion with maximum efficiency and minimal undesired edits.

The platform was also extended to improve the efficiency of the base editor system. The researchers focused on engineering a region of the sgRNA scaffold used in the base editor system, known as the stem-loop-2 region. Through a screening process, they identified two novel sgRNA scaffold variants, SV48 and SV240, which outperformed the wild-type scaffold, resulting in up to 2.2-fold higher base editing efficiency.

Additionally, the team demonstrated that the platform is compatible not only with base editors but also with other precise genome editor systems, such as prime editors. This expands the scope of the search for suitable editors to correct genetic mutations at therapeutic targets where a base editor may not be applicable.

The newly developed platform accelerates the engineering of next-generation precise genome editors and facilitates their adaptation for future therapeutic applications. Dr. Alan Wong Siu-lun, Associate Professor of the School of Biomedical Sciences at HKUMed, likened it to an accelerated checkout process in stores. Each base editor variant is tagged with a barcode, and the platform enables the researchers to quickly analyze the editing performance of all variants simultaneously, without the need for individual testing.

The research was led by Dr. Alan Wong Siu-lun, with John Fong Hoi-chun as the first author, and assistance from Dr. Chu Hoi-yee and Dr. Zhou Peng, all affiliated with the School of Biomedical Sciences at HKUMed.

Source: The University of Hong Kong

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