Rapid characterization of anti-CRISPR proteins and optogenetically engineered variants using a versatile plasmid interference system

Song, Guoxu; Tian, Chunhong; Li, Jiahui; Zhang, Fei; Peng, Yuxin; Gao, Xing; Tian, Yong

doi:10.1093/nar/gkad995

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2024

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Cited by 4 publications

References 51 publications

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Versatile plant genome engineering using anti-CRISPR-Cas12a systems

He,

Liu,

Chen

et al. 2024

Sci. China Life Sci.

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Versatile plant genome engineering using anti-CRISPR-Cas12a systems

He,

Liu,

Chen

et al. 2024

Sci. China Life Sci.

View full text Add to dashboard Cite

CRISPR/Cpf1–FOKI-induced gene editing in Gluconobacter oxydans

Wang,

Li,

Qin

et al. 2024

Synthetic and Systems Biotechnology

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Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Gligorovski,

Labagnara,

Rahi

2024

Preprint

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Directed evolution is a powerful method in biological engineering. Current approaches were devised for evolving steady-state properties such as enzymatic activity or fluorescence intensity. A fundamental problem remains how to evolve dynamic, multi-state, or computational functionalities, e.g., folding times, on-off kinetics, state-specific activity, stimulus-responsiveness, or switching and logic capabilities. These require applying selection pressure on all of the states of a protein of interest (POI) and the transitions between them. We realized that optogenetics and cell cycle oscillations could be leveraged for a novel directed evolution paradigm (‘optovolution’) that is germane for this need: We designed a signaling cascade in budding yeast where optogenetic input switches the POI between off (0) and on (1) states. In turn, the POI controls a Cdk1 cyclin, which in the re-engineered cell cycle system is essential for one cell cycle stage but poisonous for another. Thus, the cyclin must oscillate (1-0-1-0…) for cell proliferation. In this system, evolution can act efficiently on the dynamics, transient states, and input-output relations of the POI in every cell cycle. Further, controlling the pacemaker, light, directs and tunes selection pressures. Optovolution isin vivo, continuous, self-selecting, and genetically robust. We first evolved two optogenetic systems, which relay 0/1 input to 0/1 output: We obtained 25 new variants of the widely used LOV transcription factor El222. These mutants were stronger, less leaky, or green- and red-responsive. The latter was conjectured to be impossible for LOV domains but is needed for multiplexing and lowering phototoxicity. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss ofYOR1makes supplementing the expensive and unstable chromophore phycocyanobilin (PCB) unnecessary. Finally, we demonstrate the generality of the method by creating and evolving a destabilized rtTA transcription factor, which performs an AND operation between transcriptional and doxycycline input. Optovolution makes coveted, difficult-to-change protein functionalities evolvable.

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Rapid characterization of anti-CRISPR proteins and optogenetically engineered variants using a versatile plasmid interference system

Cited by 4 publications

References 51 publications

Versatile plant genome engineering using anti-CRISPR-Cas12a systems

Versatile plant genome engineering using anti-CRISPR-Cas12a systems

CRISPR/Cpf1–FOKI-induced gene editing in Gluconobacter oxydans

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

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