We’re excited to present our newest paper, Protocol for editing fibroblasts with in vitro transcribed Cas9 mRNA and profile off-target editing by optimized GUIDE-seq, published in the STAR Protocols journal in December 2023.
Highlights and Summary
- A protocol for in vitro transcription of Cas9 mRNA with quality control step
- Electroporation transfection to edit fibroblast with Cas9 mRNA
- Optimization of dsODN concentration used for GUIDE-seq
- Profile off-target editing by optimized GUIDE-seq method
CRISPR-Cas9 gene editing is an efficient technique to modify specific sites/regions of DNA. Delivery of the Cas9 by mRNA is particularly promising in pre-clinical genome editing applications for its transient, nonintegrating feature.
However, the off-target of Cas9-gRNA still remains a concern and needs a specific monitor. Here, we present a revised protocol to edit fibroblasts by in vitro transcribed Cas9 mRNA and profile its off-target effect by the optimized GUIDE-seq method. This protocol can also be applied to other cell lines.