Abstract
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.
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Claims 6
Claim · model-written summary
Two dTALE-STAP systems activated reporter expression in stable transgenic rice.
- Taxon
- rice
Oryza sativae0 - Outcome
- STAP-driven reporter gene expressione0
- Comparator
- two different systems-dTALE1-STAP1 and dTALE2-STAP2e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“two different systems-dTALE1-STAP1 and dTALE2-STAP2-can activate STAP-driven reporter gene expression in stable transgenic rice lines”
Claim · model-written summary
Transcript levels depended on dTALE and STAP sequence identities.
- Taxon
- riceOryza sativat0
- Gene
- dTALE and STAP sequence identitiese0
- Outcome
- transgene transcript levelse0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“transgene transcript levels dependent on both dTALE and STAP sequence identities”
t0“rice”
Claim · model-written summary
Relative STAP strength was consistent between dTALE systems.
- Taxon
- riceOryza sativat0
- Outcome
- relative strength of individual STAP sequencese0
- Comparator
- dTALE1 and dTALE2 systemse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems”
t0“rice”
Claim · model-written summary
Relative STAP strength differed among cell types.
- Taxon
- riceOryza sativat0
- Organ / stage
- cell-typese0
- Outcome
- relative strength of individual STAP sequencese1
- Direction
- differse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“differs between cell-types”
e1“relative strength of individual STAP sequences”
t0“rice”
Claim · model-written summary
dTALE expression activated endogenous genes off target.
- Taxon
- riceOryza sativat0
- Treatment
- dTALE expressione0
- Outcome
- off-target activation of endogenous genese0
- Direction
- activatione0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“dTALE expression leads to off-target activation of endogenous genes”
t0“rice”
Claim · model-written summary
dTALE expression affected substantially fewer genes than somaclonal variation during regeneration.
- Taxon
- riceOryza sativat0
- Outcome
- number of genes affectede0
- Direction
- substantially lesse0
- Comparator
- somaclonal variatione0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants”
t0“rice”