Abstract
Transcription activator-like effectors (TALEs) have been effectively used for targeted genome editing, transcriptional regulation, epigenetic modification, and locus-specific DNA imaging. However, with the advent of the clustered regularly interspaced short palindromic repeat/Cas9 system, an easy-to-use tool with the same function as TALEs, TALEs have recently been abandoned because of their complexity, time consumption, and difficult handling in common labs. Here, we described a degenerated codon-based TALE assembly system for simple, rapid, and efficient TALE assembly. TALE trimers with nonrepetitive DNA sequences were amplified by PCR and sequentially assembled via Gibson assembly. Our method is cost-effective, requires only commonly used basic molecular biology reagents, and takes only 2 h from target sequence analysis to completion. This simple, rapid, and lab-friendly TALE assembly method will restore the value of TALEs in DNA targeting.
Highlights
The recent emergence of programmable DNA-binding proteins that are built on transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 architectures has heralded a new era of efficient genome editing and manipulation
All TALE plasmid backbones were derived from pCAG-T7-TALE nucleases (TALENs) with heterodimeric (ELD, KKR) domains obtained from Addgene (Plasmids #37184 and #40131)
We know that the TALE repeat contains 34 amino acid (AA) with repeat variable diresidues (RVDs) at positions 12 and 13 (NI, NG, NN, HD, Figure 1A)
Summary
The recent emergence of programmable DNA-binding proteins that are built on transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas architectures has heralded a new era of efficient genome editing and manipulation. These tools provide new approaches to mastering and programming gene function and gene regulatory circuits and can be applied for the advancement of agriculture and medicine. The TALEN system has many advantages in gene editing and regulation, it is far less commonly applied than Cas technology, mainly because it is formed by multiple repetitive modules in series. DcTA will enable labs to master TALE assembly, which can re-emerge as the main tool for genetic modification and regulation
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