Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression

Abstract

Engineered DNA-binding proteins that can be targeted to specific sites in the genome to manipulate gene expression have enabled many advances in biomedical research. This includes generating tools to study fundamental aspects of gene regulation and the development of a new class of gene therapies that alter the expression of endogenous genes. Designed transcription factors have entered clinical trials for the treatment of human diseases and others are in preclinical development. High-throughput and user-friendly platforms for designing synthetic DNA-binding proteins present innovative methods for deciphering cell biology and designing custom synthetic gene circuits. We review two platforms for designing synthetic transcription factors for manipulating gene expression: Transcription Activator-Like Effectors (TALEs) and the RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. We present an overview of each technology and a guide for designing and assembling custom TALE- and CRISPR/Cas9-based transcription factors. We also discuss characteristics of each platform that are best suited for different applications.

Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds

“…First, they are easily programmable using Watson-Crick base pairing to target desired endogenous loci in the genome. TAL effectors can be used to generate complex programs, and these effectors can produce larger effects on gene expression than CRISPR-based approaches, but this requires the custom design of many distinct TAL specificities (Kabadi and Gersbach, 2014). Second, scRNA programs allow for distinct regulatory actions to take place at each target locus.…”

Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation

“…In addition to RNAi, customized DNA-binding proteins such as zinc-finger proteins or transcription activator-like effectors (TALEs) have been used as tools for sequence-specific DNA targeting and gene regulation 3 . These proteins robustly target DNA through programmable DNA-binding domains and can recruit effectors for transcription repression or activation in a modular way 4–9 . However, because each DNA-binding protein needs to be individually designed, their construction and delivery for the purpose of simultaneously regulating multiple loci is technically challenging 10 .…”

Gene Therapy of Inherited Retinal Degenerations: Prospects and Challenges

“…Alternatively, zinc-finger transcriptional repressors have been designed to silence RHO at the level of its locus, 101 a strategy that may theoretically overcome the challenge of silencing this very abundant protein that accounts for > 70% of the total rod PR outer segments protein content. 102 New-generation customized DNA-binding modules, including TALE and CRISPR-inactive CAS9, 103 provide new tools to design effective RHO transcriptional repressors. While RHO expression has been successfully suppressed to therapeutically relevant levels in animal models by several groups, 94,97,98,101 its replacement to sufficient levels (at least 50% of endogenous) appears challenging given RHO's high expression levels in PR.…”