Journal: Science (New York, N.Y.)

Article Title: Very Fast CRISPR on Demand

doi: 10.1126/science.aay8204

Figure Lengend Snippet: (A) Schematic of Cas9 activation by modulating base-pairing between the PAM distal region of cgRNA and genomic DNA. (B) Without light, Cas9/cgRNA bound to target DNA without cleavage, causing a clear band shift. Proteinase K degraded Cas9, causing target DNA to shift back to the original position. (C, D) Fast and efficient in vitro cleavage kinetics of Cas9 after light activation. (E) Indels detected by high-throughput sequencing of PCR-amplified genomic DNA extracted from cells without RNP, with RNP but no light, and with RNP 48 h after light activation. (F) DSBs detected by DSB-ddPCR of genomic DNA extracted from cells without RNP, with RNP but no light, and with RNP 30 s after light activation. (G-H) DSB % over time using our method (red) compared to either RNP electroporation (G, target sequence at ACTB) or a chemically inducible system (H, target sequence at MYC). (I) DSBs and Normalized Indels (Materials and Methods) at ACTB over time after Cas9 activation.

Article Snippet: Competing interests: The authors and Johns Hopkins University have filed a provisional patent application on the method of spatiotemporal control of Cas9 activities via cgRNA.

Techniques: Activation Assay, Electrophoretic Mobility Shift Assay, In Vitro, Next-Generation Sequencing, Amplification, Electroporation, Sequencing