Incorporating programmable DNA-binding domains enhances the efficiency and flexibility of miniature CRISPR base editors

The therapeutic application of base editors is currently limited by their large sizes, which are often beyond the packaging capabilities of adeno-associated viral (AAV) vectors. Despite recent progress in mega genome mining that has identified a diverse array of compact CRISPR proteins (e.g. Cas12f, TnpB, and IscB), the resulting miniature base editors are often exhibited reduced activities and a limited targeting scope, leaving a broad spectrum of disease-relevant genetic variants inaccessible. In this study, we have developed a platform, designated as Zinc Finger Proteins (ZFP)-enhanced miniature base editor (zmBE), which integrates programmable DNA-binding domains to enhance the efficiency and expand targeting scope of miniature base editors, including those based on Un1Cas12f1 and OgeuIscB. Utilizing protein language model (PLM) for the design of ZFPs further simplified development and optimization of zmBEs tailored to a specific target. Leveraging these methodologies, we engineered a zmBE that effectively induced the SMN2 exon 7 A6>G conversion and restored SMN2 exon 7 inclusion. Our study thus provides a versatile platform for developing miniature base editors for in vivo therapeutic applications. Overall design: Using RNA-seq to detect RNA off-target effect induced by dCas12f-ABE and dCas12f-ABE-ZFP, and unedited cells were included as mock.