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

eLife

Stefanie Kaech Petrie¹  Tianyi Mao²  Cesar C Ceballos²  Michael A Muniak²  Maozhen Qin²  Haining Zhong²  Crystian I Massengill²  Lei Ma² 

[1] Department of Neurology, Oregon Health & Science University, Portland, United States;Vollum Institute, Oregon Health & Science University, Portland, United States;

关键词: CRISPR/Cas9;    endogenous fluorescent protein labeling;    precise protein sequence insertion;    cytoskeleton labeling;    synaptic protein labeling;    in vivo imaging;    of endogenous proteins;    Human;    Mouse;    Rat;   

DOI  :  10.7554/eLife.64911

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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.

【 授权许可】

CC BY