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 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

  1. Haining Zhong

    Vollum Institute, Oregon Health and Science University, Portland, United States
    For correspondence
    zhong@ohsu.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7109-4724
  2. Cesar C Ceballos

    Vollum Institute, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
  3. Crystian I Massengill

    Vollum Institute, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
  4. Michael A Muniak

    Vollum Institute, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8047-5871
  5. Lei Ma

    Vollum Institute, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
  6. Maozhen Qin

    Vollum Institute, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
  7. Stefanie Kaech Petrie

    Jungers Center for Neuroscience Research, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
  8. Tianyi Mao

    Vollum Institute, Oregon Health and Science University, Portland, United States
    Competing interests
    Tianyi Mao, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3532-8319

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.

Reviewing Editor

  1. Kang Shen, Howard Hughes Medical Institute, Stanford University, United States

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).

Version history

  1. Received: November 14, 2020
  2. Accepted: June 7, 2021
  3. Accepted Manuscript published: June 8, 2021 (version 1)
  4. Version of Record published: June 17, 2021 (version 2)
  5. Version of Record updated: June 22, 2021 (version 3)
  6. 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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  1. Haining Zhong
  2. Cesar C Ceballos
  3. Crystian I Massengill
  4. Michael A Muniak
  5. Lei Ma
  6. Maozhen Qin
  7. Stefanie Kaech Petrie
  8. Tianyi Mao
(2021)
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

Share this article

https://doi.org/10.7554/eLife.64911

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