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

Abstract

The ability to remotely trigger CRISPR/Cas9 activity would enable new strategies to study cellular events with greater precision and complexity. We developed a method to photocage the activity of the guide RNA called ‘CRISPR-plus’ (CRISPR-precise light-mediated unveiling of sgRNAs). The photoactivatable capability of our CRISPR-plus method is compatible with simultaneous targeting of multiple DNA sequences and supports numerous modifications that can enable guide RNA labeling for use in imaging and mechanistic inquiries.

Optochemical Control of Biological Processes in Cells and Animals

“…[84] Moreover, optical control of gRNA function has been achieved, as discussed below (Figure 27). [85] …”

“…In an approach that is similar to the optical activation of antisense oligonucleotide function discussed above, [137,138,181] the inhibitory DNA strand binds to the target region of the gRNA and prevents Cas9 function until it is fragmented through UV-induced photolysis (Figure 27a,b). [85] As a proof of concept, this system was delivered into HeLa cells stably expressing a destabilized GFP reporter, and UVactivation led to reporter DNA cleavage and subsequent reduction in GFP expression. It was determined that longer protectors (24 nt) with three photolabile groups spaced six bases apart are optimal for inhibiting Cas9-mediated DNA cleavage (Figure 27c).…”

Light‐Triggered Genome Editing: Cre Recombinase Mediated Gene Editing with Near‐Infrared Light

“…[1,15] Alternatively, Jain et al avoided extensive protein engineering by photocaging key components of the genome editing system such as the guide RNA for the CRISPR/Cas9 technology. [16] A near-infrared (NIR) light responsive system still remains of keen interest; a technology that does not rely on the construction of complex fusion proteins, nor on transgenes in insertional mutagenesis has yet to be developed. [17] Here, we report a modular approach toward the delivery of genome editing enzymes based on plasmonic hollow gold nanoshells (HGNs).…”

Optimal Destabilization of DNA Double Strands by Single‐Nucleobase Caging

“…A wide scope of in vitro and in vivo applications has been reported for caged oligonucleotides. Antisense strategies, siRNA, miRNA and aptamers have been employed successfully for the photo‐regulation of gene expression and protein interaction. Caged oligonucleotides have also been used in transcriptomics, studies of RNA and DNA folding, RNA‐protein interactions and RNA therapeutics exist.…”