High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion.

ABSTRACT: Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CR

SUBMITTER: Zhong H 

PROVIDER: S-EPMC8211447 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature