dCas9-based gene editing for cleavage-free genomic knock-in of long sequences

Authors:
Wang C, Qu Y, Cheng JKW, Hughes NW, Zhang Q, Wang M, Cong L.
In:
Source: Nat Cell Biol
Publication Date: (2022)
Issue: 24(2): 268-278
Research Area:
Immunotherapy / Hematology
Cells used in publication:
H9 ( human ES cell line) 
Species: human
Tissue Origin: embryo
Platform:
4D-Nucleofector® X-Unit
Experiment

For the transfection of hESC (H9) cells, a P3 primary cell 4D-NucleofectorTM X kit S (Lonza) was used following the manufacturer’s protocol. Briefly, the hESC (H9) cells were resuspended in Accutase (Innovative Cell Technology) and washed twice with PBS before electroporation. For each reaction, 3×10^5 cells were nucleofected with 4 µg total DNA mixed in 20 µl electroporation buffer using the DC100 Nucleofector program. For 4µg DNA, we used 2.6 µg of the dCas9–SSAP gRNA plasmids, 1 µg of pMCP-RecT or GFP control plasmid and 0.4 µg of PCR template DNA (the primer sequences are listed in Supplementary Table 4 and the template sequence are listed in Supplementary Notes). After electroporation, the cells were seeded into 12-well plates with 1ml mTeSR1 medium containing 10 µM Y27632. The culture medium was changed every 24h. 

Abstract

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