Synthetic genetic circuits enable reprogramming of plant roots

Brophy, Jennifer A N; Magallon, Katie J.; Kniazev, Kiril; Dinneny, José R.

doi:10.1101/2022.02.02.478917

Cited by 5 publications

(7 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although a number of systems for the manipulation of transgene expression in plants have been reported, most have shortcomings. For example, artificial transcription factors that are based on fixed DNA-binding domains have been used for tissue-specific and multiplexed regulation of plant transgene expression but heritability was not investigated (Belcher et al, 2020;Brophy et al, 2022). Furthermore, off-target activation of endogenous genes was not considered, and if proven to be a problem, fixed DNA-binding domains cannot be adapted to prevent such activation.…”

Section: Discussionmentioning

confidence: 99%

A single promoter‐TALE system for tissue‐specific and tuneable expression of multiple genes in rice

Danila

Schreiber

Ermakova

et al. 2022

Plant Biotechnology Journal

View full text Add to dashboard Cite

In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems-dTALE1-STAP1 and dTALE2-STAP2-can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

show abstract

Section: Discussionmentioning

confidence: 99%

A single promoter‐TALE system for tissue‐specific and tuneable expression of multiple genes in rice

Danila

Schreiber

Ermakova

et al. 2022

Plant Biotechnology Journal

View full text Add to dashboard Cite

show abstract

“…Our CRISPRi circuit design has significant advantages over past systems that use different TF DNA binding domains to specify different binding events in a circuit 14,15,26 . Essentially, the circuit logic is programmed simply by changing the input sgRNAs and target sequences and linking the compact integrator modules (∼650 bp each) together, providing a very high level of flexibility.…”

Section: Discussionmentioning

confidence: 99%

“…Essentially, the circuit logic is programmed simply by changing the input sgRNAs and target sequences and linking the compact integrator modules (∼650 bp each) together, providing a very high level of flexibility. This has substantial advantages compared to circuit designs that require different recombinases due to the compactness of the system (recombinases can be ∼1.5 - 2kb in length) 16 or complex synthesis and delivery of many different DNA binding domains 15,26 , with potentially challenging and limited reprogrammability. Importantly, the integrator components of our circuits do not require the use of repurposed plant TFs, providing greater orthogonality from endogenous plant regulatory processes that may vary between cell types and conditions.…”

Section: Discussionmentioning

confidence: 99%

“…To build gene circuits, recombinases and transcriptional regulators obtained from other host organisms are often used, however refactored proteins may be limited in diversity or may not function as expected in plants, and thus require performance and cross-reactivity testing for each intended plant species. Nevertheless, gene circuits have been constructed using recombinase proteins or orthogonal transcription factors (TFs) in plants [13][14][15][16][17][18] . Alternatively, plant TF domains can be combined with orthogonal DNA binding domains and synthetic promoters to create logic 13,19 , however the use of plant TF domains requires endogenous regulatory factors, and therefore are difficult to program beyond their natural range of activity.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

CRISPRi-based circuits for genetic computation in plants

Khan

Herring

Oliva

et al. 2022

Preprint

View full text Add to dashboard Cite

Synthetic gene circuits can enable new cellular behaviours by integrating multiple input signals into customisable genetic programs. However, gene circuit development in plants has been limited by a lack of orthogonal and modular parts required for their construction. Here, we present a tool-kit of reversible CRISPRi-based gene circuits for use in plants. First, we created a range of engineered repressible promoters of different strengths and used them as integrators for the construction of NOT and NOR gates in Arabidopsis cells. Next, we determined the optimal processing system to express sgRNAs from RNA Pol II promoters to introduce NOR gate programmability and interface it with host regulatory sequences. Finally, we connected multiple NOR gates together in layered arrangements to create OR, NIMPLY, and AND logic functions. Our CRISPRi circuits are orthogonal, compact, reversible, programmable, and modular, providing a new platform for sophisticated and deliberate spatio-temporal control of gene expression in plants.

show abstract

“…With characterization of additional recombinases that function effectively in plants, including more split-recombinases, this system could easily be expanded to create highly complex single layer gene circuits, integrating multiple inputs and potentially allowing for a single plant to respond to multiple environmental cues. Our diverse set of logic gates operating in plants with the ability to record a form of memory of past events has the potential to greatly advance plant biotechnology and complement transcriptional-based systems for gene circuits 7 .…”

Section: Discussionmentioning

confidence: 99%

Synthetic memory circuits for programmable cell reconfiguration in plants

Lloyd

Gong

et al. 2022

Preprint

View full text Add to dashboard Cite

Plant biotechnology predominantly relies on a restricted set of genetic parts with limited capability to customize spatiotemporal and conditional expression patterns. Synthetic gene circuits have the ability to integrate multiple customizable input signals through a processing unit constructed from biological parts, to produce a predictable and programmable output. Here, we present a suite of functional recombinase-based gene circuits for use in plants. We first established a range of key gene circuit components compatible with plant cell functionality. We then used these to develop a range of operational logic gates using the identify function (activation) and negation function (repression) in Arabidopsis protoplasts and in vivo, demonstrating their utility for programmable manipulation of transcriptional activity in a complex multicellular organism. Through utilization of genetic recombination these circuits create stable long-term changes in expression and recording of past stimuli. This highly-compact programmable gene circuit platform provides new capabilities for engineering sophisticated transcriptional programs and previously unrealised traits into plants.

show abstract

Synthetic genetic circuits enable reprogramming of plant roots

Cited by 5 publications

References 46 publications

A single promoter‐TALE system for tissue‐specific and tuneable expression of multiple genes in rice

A single promoter‐TALE system for tissue‐specific and tuneable expression of multiple genes in rice

CRISPRi-based circuits for genetic computation in plants

Synthetic memory circuits for programmable cell reconfiguration in plants

Contact Info

Product

Resources

About