Photoactivatable CRISPR-Cas9 for optogenetic genome editing

@article{Nihongaki2015PhotoactivatableCF,
  title={Photoactivatable CRISPR-Cas9 for optogenetic genome editing},
  author={Yuta Nihongaki and Fuun Kawano and Takahiro Nakajima and Moritoshi Sato},
  journal={Nature Biotechnology},
  year={2015},
  volume={33},
  pages={755-760}
}
We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. [] Key Result In response to blue light irradiation, paCas9 expressed in human embryonic kidney 293T cells induces targeted genome sequence modifications through both nonhomologous end joining and homology-directed repair pathways. Genome editing activity can be switched off simply by extinguishing the light.

Optical Control of Genome Editing by Photoactivatable Cas9.

A detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9 is provided, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.

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Recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems are reviewed.

Optogenetics and CRISPR: A New Relationship Built to Last.

An overview of the state-of-the-art methods for light-control of CRISPR effectors and the plethora of exciting applications enabled by these systems, including spatially confined genome editing, timed activation of endogenous genes, as well as remote control of chromatin-chromatin interactions are detailed.

Optical control of CRISPR-Cas editing with cyclically caged guide RNAs

A spatiotemporal and efficient CRISPR/Cas9 and Cpf1-mediated editing with photo-sensitive circular gRNAs that achieves light-mediated MSTN gene editing in embryos and a new bow-knot-type gRNA showed no background editing in the absence of light irradiation.

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein

This work reports the first optogenetic tool to control NmeCas9 activity in mammalian cells via an engineered, light-dependent anti-CRISPR (Acr) protein and demonstrates optogenetics regulation of a type II-C CRISPR effector and might suggest a new route for the design of optogenetically Acrs.

Precise and efficient scarless genome editing in stem cells using CORRECT

This protocol outlines in detail how to implement either the re-Guide or re-Cas variants of CORRECT to generate scarlessly edited isogenic stem cell lines with intended monoallelic and biallelic sequence changes in ∼3 months.

Programmable Live‐Cell CRISPR Imaging with Toehold‐Switch‐Mediated Strand Displacement

A toehold-switch-based approach to engineer the conformation of single guide RNA (sgRNA) for programmable activation of Cas9 and provides a new route for programing CRISPR in living cells for genome imaging and engineering.

Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light-Activated Guide RNA

A new method for conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos via photochemically activated, caged guide RNAs, enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.

Programmable live-cell CRISPR imaging with toehold switch-mediated strand displacement reactions.

A toehold switch-based approach to engineer the conformation of single guide RNA (sgRNA) for programmable activation of Cas9 with orthogonal DNA inputs is reported, which provides a new route to program CRISPR in living cells for genome imaging and engineering.

CRISPR/Cas9 for Human Genome Engineering and Disease Research.

The broad application of this system to the study of functional genomics, especially genome-wide genetic screening, and to therapeutics development, including establishing disease models, correcting defective genetic mutations, and treating diseases are discussed.
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