High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion
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...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
eLife Sciences Publications Ltd
2021-06-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/64911 |
| _version_ | 1797998453251375104 |
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| author | Haining Zhong Cesar C Ceballos Crystian I Massengill Michael A Muniak Lei Ma Maozhen Qin Stefanie Kaech Petrie Tianyi Mao |
| author_facet | Haining Zhong Cesar C Ceballos Crystian I Massengill Michael A Muniak Lei Ma Maozhen Qin Stefanie Kaech Petrie Tianyi Mao |
| author_sort | Haining Zhong |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T10:48:59Z |
| format | Article |
| id | doaj.art-8ae934b12a2342f6aefbc97b9fbe8fff |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-11T10:48:59Z |
| publishDate | 2021-06-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-8ae934b12a2342f6aefbc97b9fbe8fff2022-12-22T04:28:58ZengeLife Sciences Publications LtdeLife2050-084X2021-06-011010.7554/eLife.64911High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertionHaining Zhong0https://orcid.org/0000-0002-7109-4724Cesar C Ceballos1Crystian I Massengill2Michael A Muniak3https://orcid.org/0000-0001-8047-5871Lei Ma4Maozhen Qin5Stefanie Kaech Petrie6Tianyi Mao7https://orcid.org/0000-0002-3532-8319Vollum Institute, Oregon Health & Science University, Portland, United StatesVollum Institute, Oregon Health & Science University, Portland, United StatesVollum Institute, Oregon Health & Science University, Portland, United StatesVollum Institute, Oregon Health & Science University, Portland, United StatesVollum Institute, Oregon Health & Science University, Portland, United StatesVollum Institute, Oregon Health & Science University, Portland, United StatesDepartment of Neurology, Oregon Health & Science University, Portland, United StatesVollum Institute, Oregon Health & Science University, Portland, United StatesPrecise 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.https://elifesciences.org/articles/64911CRISPR/Cas9endogenous fluorescent protein labelingprecise protein sequence insertioncytoskeleton labelingsynaptic protein labelingin vivo imaging |
| spellingShingle | Haining Zhong Cesar C Ceballos Crystian I Massengill Michael A Muniak Lei Ma Maozhen Qin Stefanie Kaech Petrie Tianyi Mao High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion eLife CRISPR/Cas9 endogenous fluorescent protein labeling precise protein sequence insertion cytoskeleton labeling synaptic protein labeling in vivo imaging |
| title | High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion |
| title_full | High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion |
| title_fullStr | High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion |
| title_full_unstemmed | High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion |
| title_short | High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion |
| title_sort | high fidelity efficient and reversible labeling of endogenous proteins using crispr based designer exon insertion |
| topic | CRISPR/Cas9 endogenous fluorescent protein labeling precise protein sequence insertion cytoskeleton labeling synaptic protein labeling in vivo imaging |
| url | https://elifesciences.org/articles/64911 |
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