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Photoactivatable CRISPR-Cas9 for optogenetic genome editing

Abstract

We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. paCas9 consists of split Cas9 fragments and photoinducible dimerization domains named Magnets. 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. We also demonstrate activation of paCas9 in spatial patterns determined by the sites of irradiation. Optogenetic control of targeted genome editing should facilitate improved understanding of complex gene networks and could prove useful in biomedical applications.

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Figure 1: Design and characterization of photoactivatable Cas9.
Figure 2: Optogenetic genome editing of mammalian endogenous genes by the photoactivatable Cas9.
Figure 3: Spatiotemporal control of Cas9 nuclease activity with optimized paCas9-2.
Figure 4: Optogenetic control of RNA-guided transcription interference with padCas9.

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Acknowledgements

We would like to thank J. Galipon for technical assistance. This work was supported by Platform for Dynamic Approaches to Living System from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) and by grants from Japan Society for the Promotion of Science (JSPS).

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Authors and Affiliations

Authors

Contributions

Y.N. and M.S. conceived the project. Y.N., F.K., T.N. and M.S. designed the experiments. Y.N. performed experiments and analyzed data. Y.N. and M.S. wrote the manuscript.

Corresponding author

Correspondence to Moritoshi Sato.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Construction of rapamycin-inducible split-Cas9.

(a) Candidate 18 split sites of Streptococcus pyogenes Cas9 protein. (b) Constructs of rapamycin-inducible Cas9. N- and C- fragments of Cas9 were fused with FRB and FKBP, respectively.

Supplementary Figure 2 Screening of split-Cas9 fragments.

(a) Ligand-induced Cas9 activity measured by luciferase reporter plasmid HDR assay. HEK293T cells were transfected with N-fragment of Cas9 fused with FRB, C-fragment of Cas9 fused with FKBP, stop codon-inserted luciferase reporter and promoter-less luciferase donor vector. (b) Second screening of Cas9 split sites in the vicinity of residues 713 and 714.

Supplementary Figure 3 Schematic of paCas9.

The schematic is based on the crystal structures of Cas9 (PDB ID: 4UN3) (ref. 17) and the light-state dimer of Vivid (PDB ID: 3RH8) (supplementary ref. 1), a precursor protein of Magnets. N713 and C714 fragments are shown in red and blue, respectively. Positive Magnet and negative Magnet are shown in gold and cyan, respectively. sgRNA and targeted DNA are shown in green and yellow, respectively. The positions connected between Cas9 fragments and Magnets are circled in black.

Supplementary Figure 4 Time-course analysis of paCas9-mediated indel mutation in EMX1 locus.

HEK293T cells were transfected with paCas9-1 targeting EMX1. After 20 h incubation, the cells were illuminated by 1.2 W/m2 blue light and genomic DNA was extracted at indicated time points. Indel mutation frequencies were evaluated by mismatch-sensitive T7E1 assay. Data are shown as the mean ± s.e.m. (n=4 from two individual experiments with biological duplicates). Indel frequencies at 0, 1, 3 and 6 h were below the detection limit (1%).

Supplementary Figure 5 paCas9-mediated indel mutation of human EMX1 locus in HeLa cells.

Indel mutation frequencies were evaluated by mismatch-sensitive T7E1 assay.

Supplementary Figure 6 paCas9 nickase can facilitate efficient genome editing using a pair of sgRNAs.

(a) Schematic of light-induced DNA double-strand breaks using a pair of sgRNAs with paCas9 D10A nickase. By introducing D10A mutation in N-terminal fragment of Cas9, paCas9 nuclease can be converted into paCas9 nickase. Using a pair of sgRNAs targeting opposite strands of targeted gene, paCas9 nickase can offer optical control of DNA double-stranded break in targeted locus. (b) Schematic of the human EMX1 locus presenting the target region of a paired sgRNAs indicated by blue lines. PAMs are also shown in red. Red allows indicate putative cleavage site. (c) Representative T7E1 assay result for calculating indel mutation rates induced by paCas9 nickase (mean, n=2 from independent experiments).

Supplementary Figure 7 Spatial surrogate reporter activation by paCas9.

(a) Schematic of surrogate EGFP reporter system (ref. 24, 25). The surrogate reporter consists of mCherry, target sequence of paCas9 (here EMX1 target site) and EGFP. In the dark, while mCherry is constitutively expressed from CMV promoter, EGFP fluorescence is not observed because EGFP gene is out of frame in the absence of Cas9 activity. Upon blue light irradiation, paCas9 is activated and a double-strand break is introduced into the EMX1 target site of the reporter. By NHEJ pathway, this break is repaired with frameshift mutations. This frameshift mutation makes EGFP gene in frame, causing the expression of mCherry-EGFP fusion protein. (b) Slit-patterned activation of paCas9. HEK293T cells were transfected with paCas9-2, surrogate EGFP reporter and sgRNA targeting the surrogate reporter. Twenty hours post transfection, samples were incubated under dark state, global blue light irradiation, and slit-patterned blue light using a photomask for 24 h.

Supplementary Figure 8 Full-length gel images.

Full-length gel images corresponding to Figs 2a, c, d, f, 3a, f and Supplementary Figs 5. and 6c. Unrelated lanes are marked with cross.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 2 and 3, and Supplementary Notes 1 and 2 (PDF 1694 kb)

Supplementary Table 1

Target sequences of sgRNAs and oligonucleotide sequences for constructing sgRNAs. (XLSX 24 kb)

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Nihongaki, Y., Kawano, F., Nakajima, T. et al. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat Biotechnol 33, 755–760 (2015). https://doi.org/10.1038/nbt.3245

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