Investigation of A Novel Gene Editing System and Its Application in Uncovering Key Mutations in ASD

摘要

The CRISPR/Cas9 genome editing platform has revolutionized basic research and gene/cell therapy. In 2023, the approval of Casgevy by regulatory agencies in the UK and US as the first CRISPR-based therapy for sickle cell disease marked a new era in gene therapy. However, current approaches remain limited: most strategies rely on gene knockouts rather than precise repair, and conventional systems induce DNA double-strand breaks at specific genomic loci using Cas9 nucleases or nickases, which can lead to adverse outcomes such as chromosomal loss or translocations, thereby restricting broader therapeutic application. To overcome these challenges, we fused microbial single-strand annealing proteins(SSAPs) with catalytically inactive Cas9(dCas9) to develop a cleavage-free gene editing platform, dCas9–SSAP. This system enables efficient long-sequence knock-ins in mammalian cells with minimal on-target errors and negligible off-target effects, achieving efficiencies up to ～20% across diverse donor designs and cell types, including human stem cells. We further applied this optimized platform to identify key mutations associated with autism spectrum disorder(ASD) progression. Collectively, dCas9–SSAP provides a safer and more precise strategy for large-fragment genome engineering, offering a powerful tool for elucidating mechanisms and identifying therapeutic targets in mutation-associated diseases.