High-performance chemical- and light-inducible recombinases in mammalian cells and mice

Weinberg, Benjamin H.; Cho, Jang Hwan; Agarwal, Yash; Pham, Nam; Caraballo, Leidy D.; Walkosz, Maciej; Ortega, Charina; Trexler, Micaela; Tague, Nathan; Law, Billy; Benman, William; Letendre, Justin; Beal, Jacob; Wong, Wilson

doi:10.1038/s41467-019-12800-7

Cited by 59 publications

(89 citation statements)

References 34 publications

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“…In particular, light-induced heterodimerization provides a simple and versatile way to control the activity of split enzymes including Cre recombinase [ 109 ], CRISPR-Cas9 [ 18 ], T7 RNA polymerase [ 48 ] and intracellular antibodies (intrabodies) [ 110 ]. Notably, the VVD-based optogenetic tools have been reported to be temperature-sensitive [ 111 , 112 ] and stimulating cells with colder temperatures can also induce dimerization [ 113 ]. Accordingly, the pre-incubation of cells at the experimental temperature [ 47 ], and keeping the temperature constant during cell imaging, are important considerations for this system.…”

Section: Lov Domain-based Optogenetic Toolsmentioning

confidence: 99%

Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators

Shen

Campbell

2020

IJMS

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Optogenetic (photo-responsive) actuators engineered from photoreceptors are widely used in various applications to study cell biology and tissue physiology. In the toolkit of optogenetic actuators, the key building blocks are genetically encodable light-sensitive proteins. Currently, most optogenetic photosensory modules are engineered from naturally-occurring photoreceptor proteins from bacteria, fungi, and plants. There is a growing demand for novel photosensory domains with improved optical properties and light-induced responses to satisfy the needs of a wider variety of studies in biological sciences. In this review, we focus on progress towards engineering of non-opsin-based photosensory domains, and their representative applications in cell biology and physiology. We summarize current knowledge of engineering of light-sensitive proteins including light-oxygen-voltage-sensing domain (LOV), cryptochrome (CRY2), phytochrome (PhyB and BphP), and fluorescent protein (FP)-based photosensitive domains (Dronpa and PhoCl).

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Section: Lov Domain-based Optogenetic Toolsmentioning

confidence: 99%

Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators

Shen

Campbell

2020

IJMS

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“…Synthetic split proteins are useful tools for biologists 1 . Often, they are created to serve as sensors for protein-protein interaction 2 , detectors for biomolecules 3,4 , molecular switches 5,6 , and logic gates in synthetic circuits [7][8][9][10] . In other instances, proteins are split to be endowed with temporal-spatial 11 or userdefined controls 12 , or to reduce their sizes for viral delivery 13 .…”

Section: Introductionmentioning

confidence: 99%

“…Doing so could risk perturbations to the protein structure and function. By either approach, the split site design space is often sparsely sampled by educated guesses with split sites tested through trial-and-error 6,23,24 , which is inefficient. A better solution would be to predict split sites computationally from protein crystal structures.…”

Section: Introductionmentioning

confidence: 99%

Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering

Tyh

Shao

et al. 2020

Preprint

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Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augmented a mini-Mu transposon-based screening approach and devised the intein-assisted bisection mapping (IBM) method. IBM robustly revealed clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further showed that the use of inteins expands functional sequence space for splitting a protein. We also demonstrated the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins. Furthermore, the intein inserted at an identified site could be engineered by the transposon again to become partially chemically inducible, and to some extent enabled post-translational tuning on host protein function. Our work offers a generalizable and systematic route towards creating split protein-intein fusions and conditional inteins for protein activity control.

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“…However, this is limited by the dynamical complexity and increased burden on cellular resources caused by increasing the number of biological components [ 3 , 35 ]. Furthermore, there are a finite number of recombinases that have been characterised that can be used reliably as inputs [ 36 ], which limits scalability regardless of the difficulty associated with the number of components.…”

Section: Introductionmentioning

confidence: 99%

A mechanistic model of the BLADE platform predicts performance characteristics of 256 different synthetic DNA recombination circuits

et al. 2020

Self Cite

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Boolean logic and arithmetic through DNA excision (BLADE) is a recently developed platform for implementing inducible and logical control over gene expression in mammalian cells, which has the potential to revolutionise cell engineering for therapeutic applications. This 2-input 2-output platform can implement 256 different logical circuits that exploit the specificity and stability of DNA recombination. Here, we develop the first mechanistic mathematical model of the 2-input BLADE platform based on Cre- and Flp-mediated DNA excision. After calibrating the model on experimental data from two circuits, we demonstrate close agreement between model outputs and data on the other 111 circuits that have so far been experimentally constructed using the 2-input BLADE platform. Model simulations of the remaining 143 circuits that have yet to be tested experimentally predict excellent performance of the 2-input BLADE platform across the range of possible circuits. Circuits from both the tested and untested subsets that perform less well consist of a disproportionally high number of STOP sequences. Model predictions suggested that circuit performance declines with a decrease in recombinase expression and new experimental data was generated that confirms this relationship.

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High-performance chemical- and light-inducible recombinases in mammalian cells and mice

Cited by 59 publications

References 34 publications

Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators

Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators

Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering

A mechanistic model of the BLADE platform predicts performance characteristics of 256 different synthetic DNA recombination circuits

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