Development of Light-Activated CRISPR Using Guide RNAs with Photocleavable Protectors

• Published in: Angewandte Chemie (International ed.) Vol. 55; no. 40; pp. 12440 - 12444
• Main Authors: Jain, Piyush K., Ramanan, Vyas, Schepers, Arnout G., Dalvie, Nisha S., Panda, Apekshya, Fleming, Heather E., Bhatia, Sangeeta N.
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 26.09.2016; Wiley Subscription Services, Inc
• Edition: International ed. in English
• Subjects: Base Sequence; Compatibility; Complexity; CRISPR; CRISPR-Cas Systems - genetics; Deoxyribonucleic acid; DNA; DNA cleavage; Gene sequencing; gene technology; Green Fluorescent Proteins - genetics; HeLa Cells; Humans; Imaging; Labels; Light; Light effects; Marking; Nucleic Acid Hybridization; nucleic acids; Nucleotide sequence; Photoactivation; photochemistry; Photolysis - radiation effects; Protectors; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems - chemistry; RNA, Guide, CRISPR-Cas Systems - metabolism; CRISPR; DNA cleavage; nucleic acids; photochemistry; gene technology
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201606123

The ability to remotely trigger CRISPR/Cas9 activity would enable new strategies to study cellular events with greater precision and complexity. In this work, we have developed a method to photocage the activity of the guide RNA called “CRISPR‐plus” (CRISPR‐precise light‐mediated unveiling of sgRNAs). The photoactivation capability of our CRISPR‐plus method is compatible with the simultaneous targeting of multiple DNA sequences and supports numerous modifications that can enable guide RNA labeling for use in imaging and mechanistic investigations. Turn “ON” CRISPR with light: CRISPR can be brought under the control of light simply by hybridizing a single chimeric guide RNA (sgRNA) with a complementary oligonucleotide containing photocleavable groups (protector oligonucleotide). The protected sgRNA (p‐sgRNA) remains inactive, blocking CRISPR activity, until the protector oligonucleotide is cleaved with a remote light trigger.