1. Cell Biology
  2. Neuroscience

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

  1. Haining Zhong  Is a corresponding author
  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
  1. Oregon Health and Science University, United States
https://doi.org/10.7554/eLife.64911
Share this article
Cite this article
  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
  2. Download BibTeX
  3. Download .RIS

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.

Metrics

  • 8,352
    views
  • 997
    downloads
  • 24
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Categories and tags

Research organisms

Further reading

    1. Cancer Biology
    2. Cell Biology

    A syngeneic spontaneous zebrafish model of tp53-deficient, EGFRvIII, and PI3KCAH1047R-driven glioblastoma reveals inhibitory roles for inflammation during tumor initiation and relapse in vivo

    Alex Weiss, Cassandra D'Amata ... Madeline N Hayes
    Research Article

    High-throughput vertebrate animal model systems for the study of patient-specific biology and new therapeutic approaches for aggressive brain tumors are currently lacking, and new approaches are urgently needed. Therefore, to build a patient-relevant in vivo model of human glioblastoma, we expressed common oncogenic variants including activated human EGFRvIII and PI3KCAH1047R under the control of the radial glial-specific promoter her4.1 in syngeneic tp53 loss-of-function mutant zebrafish. Robust tumor formation was observed prior to 45 days of life, and tumors had a gene expression signature similar to human glioblastoma of the mesenchymal subtype, with a strong inflammatory component. Within early stage tumor lesions, and in an in vivo and endogenous tumor microenvironment, we visualized infiltration of phagocytic cells, as well as internalization of tumor cells by mpeg1.1:EGFP+ microglia/macrophages, suggesting negative regulatory pressure by pro-inflammatory cell types on tumor growth at early stages of glioblastoma initiation. Furthermore, CRISPR/Cas9-mediated gene targeting of master inflammatory transcription factors irf7 or irf8 led to increased tumor formation in the primary context, while suppression of phagocyte activity led to enhanced tumor cell engraftment following transplantation into otherwise immune-competent zebrafish hosts. Altogether, we developed a genetically relevant model of aggressive human glioblastoma and harnessed the unique advantages of zebrafish including live imaging, high-throughput genetic and chemical manipulations to highlight important tumor-suppressive roles for the innate immune system on glioblastoma initiation, with important future opportunities for therapeutic discovery and optimizations.

    1. Cancer Biology
    2. Cell Biology

    Cancer: Modeling glioblastoma

    Ian Lorimer
    Insight

    Establishing a zebrafish model of a deadly type of brain tumor highlights the role of the immune system in the early stages of the disease.

    1. Cell Biology
    2. Neuroscience

    Presynaptic Rac1 in the hippocampus selectively regulates working memory

    Jaebin Kim, Edwin Bustamante ... Scott H Soderling
    Research Article

    One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation signaling networks. Previous studies have shown that Rac1-mediated signaling is associated with hippocampal-dependent working memory and longer-term forms of learning and memory and that Rac1 can modulate forms of both pre- and postsynaptic plasticity. How these different cognitive functions and forms of plasticity mediated by Rac1 are linked, however, is unclear. Here, we show that spatial working memory in mice is selectively impaired following the expression of a genetically encoded Rac1 inhibitor at presynaptic terminals, while longer-term cognitive processes are affected by Rac1 inhibition at postsynaptic sites. To investigate the regulatory mechanisms of this presynaptic process, we leveraged new advances in mass spectrometry to identify the proteomic and post-translational landscape of presynaptic Rac1 signaling. We identified serine/threonine kinases and phosphorylated cytoskeletal signaling and synaptic vesicle proteins enriched with active Rac1. The phosphorylated sites in these proteins are at positions likely to have regulatory effects on synaptic vesicles. Consistent with this, we also report changes in the distribution and morphology of synaptic vesicles and in postsynaptic ultrastructure following presynaptic Rac1 inhibition. Overall, this study reveals a previously unrecognized presynaptic role of Rac1 signaling in cognitive processes and provides insights into its potential regulatory mechanisms.