Inducible CRISPR-TR system for the conditional regulation of gene expression

CRIPSR-based transcription regulators (CRISPR-TRs) can be used to control the expression of any gene of interest by simply reprogramming its associated single guide RNA and tethering various effector domains to the dCas9 protein. Current inducible CRISPR-TR systems require complex protein engineering and cannot be scaled up to create transcriptional programmes involving multiple genes.

Oxford researchers have developed a versatile inducible CRISPR-TR platform by engineering the single guide RNA and devised a system of inducers to regulate the activity of CRISPR-TR. This system enables the regulation of target gene expression in any space and time and the assembly of complex gene circuits. It has wide applications in synthetic biology and the development of cell-based therapeutic strategies.

Current CRISPR-TR systems and their limitations

The CRISPR-based transcription regulator (CRISPR-TR) system was designed to control the output expression of any gene of interest. The system relies on a nuclease-deficient Cas9 fused with various effector domains, directed to specific genes by the single guide RNA (sgRNA). Chemically inducible or photo-activated CRISPR/Cas9 solutions are available but they require complex protein engineering and are difficult to scale up to implement synthetic networks across multiple genes.

The Oxford inducible CRISPR-TR system

To address these limitations, Oxford researchers have developed an inducible CRISPR-TR system based on minimal engineering of the sgRNA. In this system, a spacer-blocking hairpin (SBH) structure is appended at the 5’ end of the sgRNA to temporally block CRISPR-TR activity. To conditionally enable the activity of Cas9, a range of inducible SBH (iSBH) modules were further developed, including proteins, small molecules as aptazyme and single-stranded DNA oligonucleotides. With the action of these molecules, the full repression of CRISPR-TF activity is annulled and can be controlled.

Compared to current methods, the system confers the following benefits:

  • Simple, rapid and highly versatile
  • Compatible with all Cas9 based applications
  • Possible to encode a complete transcriptional programme in a single RNA molecule
  • Facilitates the assembly of more complex gene circuits

As potential applications, the system can be used to:

  • Answer fundamental biological questions involving precise spatiotemporal regulation of gene products
  • Rewire cellular behaviour in basic research
  • Develop smart therapeutics
  • Create scalable gene circuits of interest such as orthogonal and parallel transcriptional programmes

As a proof of principle, the system has been used to assemble gene regulatory modules in human cells and the results are published in Nature Communications.

Commercialisation

This technology is subject to a patent application. Oxford University Innovation is interested in hearing from companies that would like to license this technology.

Ferry et al, Nature Communications 8, Article number: 14633 (2017)
doi:10.1038/ncomms14633
https://www.nature.com/articles/ncomms14633

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