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Light-inducible Cre/loxP System
The light-inducible Cre/loxP system allows researchers to control the timing and location of recombination by using a light-activated form of Cre, such as the Photorhabdus luminescens histidine kinase (Phot), which activates Cre in response to specific wavelengths of light.
What Is Light-inducible Cre/loxP System?
The Light-inducible Cre/loxP system refers to a technology that allows for the inducible expression of genes in response to light. It is a combination of site-specific recombination systems, such as Cre/lox, with a light-inducible system, such as a photoactivatable protein. Followings are some popular light-inducible Cre/loxP systems.
Caged Biomolecules-based Cre/loxP System
Caging groups can be installed at the key point of chemical inducers or the catalytic site of Cre recombinase to inhibit the activity of the biomolecules. The caged Cre reactivation following the caging group can be removed by UV light illumination. The gene expression can be switched on or off by UV light activated recombination in cell culture systems. Caging is a form of photoreversible chemical modification that has been used in the light-mediated activation of molecules. Generally, that caging groups can be divided into two types, caged Cre recombinase and pohotcaged chemical inducers.
Strategies of light-inducible site-specific recombinase systems (Tian X, et al. 2021)
CRY2-CIB1 System
The blue light–inducible dimerization modules CRY2 and CIB1 are one of the most commonly used optogenetics system, in which the CRY2 and CIB1 are fused to N-Cre and C-Cre respectively. Upon the illumination of blue light, the dimerization of CRY2 and CIBN leads to the reconstitution of split Cre recombinase activity. This system has been adapted in a variety of models and cell line to optically manufacture recombinase systems.
VVD System
In VVD system, two distinct VVD variants are designed as Magnets, positive and negative (PMag and nMag). The synergistic activation of two units prevents dimerization in the dark state. The blue light dependent heterodimerization of the Magnet system enables the reconstruction of split Cre fragments.
Light-Inducible Recombinases for Bacterial Optogenetics (Sheets, Michael B et al. 2020)
Why Light-inducible Recombinase System
Even though, the chemical-inducible system is very powerful tools for genomic manipulation, there are still some limitations of them:
- Ligands are not always neutral to the cell and therefore can disrupt the biological process under study;
- As the chemical ligands spread through the tissue, the control of activation is sometimes not sufficiently precise in space or time;
- The cost or side effects of chemical induction agents may discourage industrial or biomedical applications.
Therefore, here comes light-inducible systems, which can be induced by light instead of chemicals. Compared with chemical-induced system, this presents several advantages:
- Light can be used with great temporal and spatial precision and high reproducibility;
- Light is neutral for most cell types, when applied at low energy;
- It's very cost-effective and therefore scalable to industrial application.
This system is widely used in basic and applied research, including studies of gene function, tissue-specific gene expression, gene therapy, and conditional knockout of genes. It is particularly useful for studying developmental processes and for analyzing the role of genes in specific cell types or tissues. The light-inducible aspect of the system provides an additional level of control and precision, enabling researchers to carry out studies in a more timely and spatially restricted manner.
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References
- Tian X, Zhou B. Strategies for site-specific recombination with high efficiency and precise spatiotemporal resolution. J Biol Chem. 2021;296:100509.
- Sheets, Michael B et al. "Light-Inducible Recombinases for Bacterial Optogenetics." ACS synthetic biology vol. 9,2 (2020): 227-235.