TY - JOUR
T1 - High-fidelity, efficient, and reversible labeling of endogenous proteins using crispr-based designer exon insertion
AU - Zhong, Haining
AU - Ceballos, Cesar C.
AU - Massengill, Crystian I.
AU - Muniak, Michael
AU - Ma, Lei
AU - Qin, Maozhen
AU - Petrie, Stefanie Kaech
AU - Mao, Tianyi
N1 - Funding Information:
We thank Dr Zefeng Wang for discussions regarding exon-intron junctions; Drs Paul Meraner and Marc Freeman for the SpCas9 mouse line; Dr Kevin Wright for the Neuro 2A cell line; Dr Bart Jong-bloets for help with imaging, Dr James Frank for help with experimental attempts that are not included, Mr Matthew Schleisman at the OHSU flow cytometry core, and Ms Brittany Daughtry at the OHSU Gene Profiling Shared Resource for technical assistance on FACS and mRNA purification experiments, respectively. We thank Dr Gail Mandel for critical comments on the manuscript. The work was supported by an NIH/BRAIN Initiative grant (RF1MH120119) to HZ and TM, and an NIH/ NINDS R01 (R01NS081071) to TM.
Publisher Copyright:
© Zhong et al.
PY - 2021/6
Y1 - 2021/6
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85108741510&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85108741510&partnerID=8YFLogxK
U2 - 10.7554/eLife.64911
DO - 10.7554/eLife.64911
M3 - Article
C2 - 34100715
AN - SCOPUS:85108741510
VL - 10
JO - eLife
JF - eLife
SN - 2050-084X
M1 - e64911
ER -