Protocol for editing fibroblasts with in vitro transcribed Cas9 mRNA and profile off-target editing by optimized GUIDE-seq

Zhuokun Li; Ganna Reint; Emma Maria Haapaniemi

doi:10.1016/j.xpro.2023.102662

Protocol for editing fibroblasts with in vitro transcribed Cas9 mRNA and profile off-target editing by optimized GUIDE-seq

STAR Protoc. 2023 Dec 15;4(4):102662. doi: 10.1016/j.xpro.2023.102662. Epub 2023 Oct 27.

Authors

Zhuokun Li¹, Ganna Reint², Emma Maria Haapaniemi³

Affiliations

¹ Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway. Electronic address: zhuokun.li@ncmm.uio.no.
² Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway.
³ Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway. Electronic address: e.m.haapaniemi@ncmm.uio.no.

Abstract

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.

Keywords: CRISPR; Cell Culture; Sequencing.

MeSH terms

CRISPR-Cas Systems* / genetics
DNA / genetics
Gene Editing / methods
RNA, Guide, CRISPR-Cas Systems*
RNA, Messenger / genetics

Substances

RNA, Guide, CRISPR-Cas Systems
RNA, Messenger
DNA