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

Haining Zhong, Cesar C. Ceballos, Crystian I. Massengill, Michael A. Muniak, Lei Ma, Maozhen Qin, Stefanie Kaech Petrie, Tianyi Mao

Research output: Contribution to journalArticlepeer-review

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 CRISPIE to fluorescently label endogenous proteins in mammalian neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.

Original languageEnglish (US)
Article numbere64911
JournaleLife
Volume10
DOIs
StatePublished - Jun 2021

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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