Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing

Matthew J Tarnowski,Thomas E Gorochowski

doi:10.1038/s41467-022-28074-5

Matthew J Tarnowski, Thomas E Gorochowski

Open Access

https://doi.org/10.1038/s41467-022-28074-5

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Journal: Nature Communications	Publication Date: Jan 21, 2022
Citations: 11	License type: open-access

Affiliation: University of Bristol

Abstract

Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. However, because termination is stochastic, two different forms of transcript could be produced: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs at the level of transcription. Here, we explore this possibility by repurposing terminators as ‘transcriptional valves’ that can tune the proportion of RNAP read-through. Using one-pot combinatorial DNA assembly, we iteratively construct 1780 transcriptional valves for T7 RNAP and show how nanopore-based direct RNA sequencing (dRNA-seq) can be used to characterize entire libraries of valves simultaneously at a nucleotide resolution in vitro and unravel genetic design principles to tune and insulate termination. Finally, we engineer valves for multiplexed regulation of CRISPR guide RNAs. This work provides new avenues for controlling transcription and demonstrates the benefits of long-read sequencing for exploring complex sequence-function landscapes.

Highlights

Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA
T7 RNAP was selected due to its broad use in synthetic biology, which stems from the fact that it is a single-subunit RNAP with high processivity, making it ideal for both in vitro use[25] as well as an orthogonal transcription system in vivo[26,27]
While diverse terminators are available for the native E. coli RNAP7,16, for T7 RNAP only a single terminator exists in the T7 phage genome[28] and only a few alternatives have been characterized[22,29,30]

Summary

Introduction

Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs at the level of transcription We explore this possibility by repurposing terminators as ‘transcriptional valves’ that can tune the proportion of RNAP read-through. An ability to design transcript isoforms in this way could open new avenues to control the stoichiometry of multi-gene expression purely at the level of transcription This regulatory approach could be more efficient and impose less burden[11,12,13] than other more commonly used methods, like operons, as not all RNA polymerases (RNAPs) would need to synthesize full-length multigene transcripts and could be freed more quickly for other tasks. By comparing the ratio of fluorescence for each reporter with and without the terminator present, it is possible to indirectly quantify the fraction of transcriptional read through and the termination efficiency of the terminator[7,16,17]

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