Benchmarking of TALE- and CRISPR/dCas9-Based Transcriptional Regulators in Mammalian Cells for the Construction of Synthetic Genetic Circuits

Abstract

Transcriptional activator-like effector (TALE)- and CRISPR/Cas9-based designable recognition domains represent a technological breakthrough not only for genome editing but also for building designed genetic circuits. Both platforms are able to target rarely occurring DNA segments, even within complex genomes. TALE and dCas9 domains, genetically fused to transcriptional regulatory domains, can be used for the construction of engineered logic circuits. Here we benchmarked the performance of the two platforms, targeting the same DNA sequences, to compare their advantages for the construction of designed circuits in mammalian cells. Optimal targeting strands for repression and activation of dCas9-based designed transcription factors were identified; both platforms exhibited good orthogonality and were used to construct functionally complete NOR gates. Although the CRISPR/dCas9 system is clearly easier to construct, TALE-based activators were significantly stronger, and the TALE-based platform performed better, especially for the construction of layered circuits.

Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae

“…For BS13, a clear elevation of yEGFP fluorescence can be observed upon increasing the copy number from two to four, or eight copies. This is consistent with previous observations (Maeder et al, 2013 ; Lebar and Jerala, 2016 ). However, a sharp decrease occurs for 16 BSs.…”

“…In most cases, the yEGFP fluorescence for dCas9-based activation reaches roughly 10% of the corresponding synTALE-mediated expression. The weaker transactivation capacity of dCas9-based TFs compared to synTALEs targeting the same BS has just recently been reported (Lebar and Jerala, 2016 ). Interestingly, increasing the copy number of BSs for dCas9 has variable effects.…”

“…The two types of synTFs described above have been successfully used to modify the expression output of target genes in various organisms, including mammalian cell lines or S. cerevisiae (Farzadfard et al, 2013 ; Maeder et al, 2013 ; Gao et al, 2014 ; Lebar and Jerala, 2016 ). In most cases, the synTFs are programmed to modulate endogenous gene expression by specifically binding sequences within native promoters.…”

Synthetic Gene Expression Circuits for Designing Precision Tools in Oncology

“…TALEs are more straightforward to design than ZFs even though they pose challenges to cloning and delivery into host genomes. The CRISPR-based regulators are easier to construct than TALEs which, nonetheless, perform better for the construction of layered circuits (Lebar and Jerala, 2016 ). The plasmid pT181 antisense-RNA-mediated transcription attenuation platform is well established to control transcription through RNA–RNA interactions (Lucks et al, 2011 ).…”

On three genetic repressilator topologies

“…[2,16,43]. With the developments in the field of synthetic biology in the recent years, the use of designable repressors has become more and more frequent [36,26,31,20,8,19,29,30,34]. Such repressors can be designed to bind any DNA sequence due to their modular structure, which can be exploited to eliminate interactions with the cells' genome.…”