High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion
- Oregon Health and Science University, United States
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.
Data availability
All data are included in the manuscript and supporting source data files. Source data files have been provided for Figures 1, 2, 3, 5, and 6.
Article and author information
Author details
Funding
NIH/NIMH (RF1MH120119)
- Haining Zhong
- Tianyi Mao
NINDS (R01NS081071)
- Tianyi Mao
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Animal handling and experimental protocols were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee (IACUC) of the Oregon Health & Science University (#IS00002792).
Reviewing Editor
- Kang Shen, Howard Hughes Medical Institute, Stanford University, United States
Publication history
- Received: November 14, 2020
- Accepted: June 7, 2021
- Accepted Manuscript published: June 8, 2021 (version 1)
- Version of Record published: June 17, 2021 (version 2)
- Version of Record updated: June 22, 2021 (version 3)
- Version of Record updated: January 27, 2022 (version 4)
Copyright
© 2021, Zhong et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion
eLife 10:e64911.
https://doi.org/10.7554/eLife.64911
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