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Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs
Published on: June 20, 2016
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One-Day TALEN Assembly Protocol and a Dual-Tagging System for Genome Editing
Shuyan Zhang1, Jun Wang1, Jinke Wang1
1State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
ACS Omega
|August 18, 2020
Summary
A new one-day protocol for preparing transcription activator-like effector nucleases (TALENs) offers faster gene editing than CRISPR. This method efficiently generates custom TALENs and demonstrates high editing efficiency for various genes and cell lines.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
- Protein Engineering
Background:
- Transcription activator-like effector nucleases (TALENs) are powerful gene editing tools.
- Existing TALEN preparation methods can be time-consuming and complex.
- CRISPR technology offers rapid gene editing but TALENs provide distinct advantages in specificity and design.
Purpose of the Study:
- To develop a rapid and efficient protocol for preparing custom transcription activator-like effector nucleases (TALENs).
- To compare the gene editing efficiency of the new TALEN protocol with CRISPR.
- To establish a dual-tagging system for protein labeling using TALENs.
Main Methods:
- Redesigned the Golden Gate TALEN and TAL Effector Kit 2.0 for faster assembly.
- Utilized linear monomers amplified via high-fidelity PCR and a TALE-FokI backbone.
- Developed a pipeline for assembling ready-to-use TALEN expression plasmids.
- Targeted five NF-κB genes (RELA, RELB, CREL, NFKB1, NFKB2) in 293T, HepG2, and PANC1 cell lines.
- Implemented a dual-tagging system for protein tagging (SBP, AviTag) via homology-directed repair.
Main Results:
- The new protocol enables the preparation of custom 18-bp binding TALENs in approximately 12 hours.
- Achieved high efficiency (over 80%) in obtaining positive colonies for TALEN construction.
- Demonstrated high efficiency, reproducibility, reliability, and applicability of the TALEN protocol across different genes and cell lines.
- The fabricated TALENs exhibited significantly higher gene editing efficiency compared to CRISPR.
- Successfully edited cells with SBP or AviTag using the dual-tagging system.
Conclusions:
- The developed rapid TALEN preparation protocol significantly reduces turnaround time for gene editing.
- This new method offers a highly efficient and reliable alternative to existing TALEN construction techniques.
- The TALEN system demonstrates superior editing efficiency over CRISPR in specific applications.
- The dual-tagging system provides a versatile tool for protein studies and applications in immunoprecipitation and ChIP assays.
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