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Summary: 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.For complete details on the use and execution of this protocol, please refer to Ganna Reint et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.