Search for: All records

The complexity of eukaryotic organisms is intricately tied to transcriptome‐level processes, notably alternative splicing and the precise modulation of gene expression through a sophisticated interplay involving RNA‐binding protein (RBP) networks and their RNA targets. Recent advances in our understanding of the molecular pathways responsible for this control have paved the way for the development of tools capable of steering and managing RNA regulation and gene expression. The fusion between a rapidly developing understanding of endogenous RNA regulation and the burgeoning capabilities of CRISPR‐Cas and other programmable RBP platforms has given rise to an exciting frontier in engineered RNA regulators. This review offers an overview of the existing toolkit for constructing synthetic RNA regulators using programmable RBPs and effector domains, capable of altering RNA sequence composition or fate, and explores their diverse applications in both basic research and therapeutic contexts.

 

Transcription factor (TF)–promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF–promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ⁵⁴-dependent, butanol-responsive TF–promoter pair, BmoR-P_BMO derived from Thauera butanivorans, at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized P_BMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in P_BMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ⁵⁴-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development. 

Transcription factor (TF)–promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF–promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ 54 -dependent, butanol-responsive TF–promoter pair, BmoR-P BMO derived from Thauera butanivorans , at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized P BMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in P BMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ 54 -dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development.