PUBLICATION
Knock-in of a Large Reporter Gene via the High-Throughput Microinjection of the CRISPR/Cas9 System
- Authors
- Chen, S., Jiao, Y., Pan, F., Guan, Z., Cheng, S.H., Sun, D.
- ID
- ZDB-PUB-220209-13
- Date
- 2022
- Source
- IEEE transactions on bio-medical engineering 69(8): 2524-2532 (Journal)
- Registered Authors
- Cheng, Shuk Han
- Keywords
- none
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems*/genetics
- Gene Knock-In Techniques
- Genes, Reporter/genetics
- Microinjections
- Zebrafish*/genetics
- PubMed
- 35133958 Full text @ IEEE Trans. Biomed. Eng.
Citation
Chen, S., Jiao, Y., Pan, F., Guan, Z., Cheng, S.H., Sun, D. (2022) Knock-in of a Large Reporter Gene via the High-Throughput Microinjection of the CRISPR/Cas9 System. IEEE transactions on bio-medical engineering. 69(8):2524-2532.
Abstract
The non-viral delivery of the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) nuclease system provides promising solutions for gene therapy. However, traditional chemical and physical delivery approaches for gene knock-in are confronted by significant challenges to overcome the draw-backs of low efficiency and high toxicity. An alternative method for directly delivering CRISPR components into single cells is microinjection. Here, we present the high-throughput robotic microinjection of CRISPR machinery plasmids to produce gene insertions. We demonstrate that the microinjection of CRISPR/Cas9 with an enhanced green fluorescent protein (eGFP) donor template into single HepG2 cells can achieve re-porter gene knock-in targeting the adeno-associated virus site 1 locus. Homology-directed repair-mediated knock-in can be ob-served with an efficiency of 41%. Assessment via T7E1 assay indicates that the eGFP knock-in cells exhibit no detectable changes at potential off-target sites. A case study of injecting the eGFP knock-in cells into zebrafish (Danio rerio) embryos to form an in vivo tumor model is conducted. Results demonstrate the efficiency of combining microinjection with the CRISPR/Cas9 system in achieving gene knock-in.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping