Education & Certifications
B.S., University of California, San Diego, Biochemistry and Cell Biology (2019)
dCas9-based gene editing for cleavage-free genomic knock-in of long sequences.
Nature cell biology
Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR-Cas, gene editing could cause DNA damage and trigger DNA repair processes that are often error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here we couple microbial single-strand annealing proteins (SSAPs) with catalytically inactive dCas9 for gene editing. This cleavage-free gene editor, dCas9-SSAP, promotes the knock-in of long sequences in mammalian cells. The dCas9-SSAP editor has low on-target errors and minimal off-target effects, showing higher accuracy than canonical Cas9 methods. It is effective for inserting kilobase-scale sequences, with an efficiency of up to approximately 20% and robust performance across donor designs and cell types, including human stem cells. We show that dCas9-SSAP is less sensitive to inhibition of DNA repair enzymes than Cas9 references. We further performed truncation and aptamer engineering to minimize its size to fit into a single adeno-associated-virus vector for future application. Together, this tool opens opportunities towards safer long-sequence genome engineering.
View details for DOI 10.1038/s41556-021-00836-1
View details for PubMedID 35145221
Cleavage-Free dCas9 Knock-In Gene-Editing Tool Leveraging RNA-Guided Targeting of Recombineering Proteins
CELL PRESS. 2021: 107
View details for Web of Science ID 000645188700204