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

Abstract

We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5′‐protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA‐target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off‐to‐on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.

Photocontrol of CRISPR/Cas9 function by site-specific chemical modification of guide RNA

“…Besides, the single modication site at 2 0 -OH also made the synthesis of modied RNA much easier than introducing multiple modication sites. In addition, compared with previously reported multiple modications of gRNA by using UV labile groups, [37][38][39][40] our single-site modication made the optical response highly efficient with a relatively low energy dosage, which would be practically benecial for prompt and harmless temporal control. Notably, given the fact that optical control by light with a longer wavelength is more suitable for applications in biomedical samples, we believe introduction of visible or even near-infrared (NIR) light activated moieties 55 for visible or NIR light control could also be feasible through this convenient strategy.…”

“…Chemical modications have enriched the versatility of conditional control of RNA-based technology, such as siRNA for silencing of mRNA [52][53][54] and gRNA for CRISPR function. [36][37][38][39][40][41][42] However, previously reported RNA caging strategies were generally based on structural variation of nucleic acids through disrupting base pairing or signicant alteration of molecular identities, but they neglected the interactive impacts between RNA and RNA binding proteins. Our current study indicates that interruption of the interactive sites between 2 0 -OH of ribose in the seed region of gRNA and the Cas9 protein would significantly disturb the Cas9 activity, and these sites can be utilized to introduce photolabile groups for optical control of CRISPR function.…”

“…Several recent reports have shed some new light on this approach. These studies include utilization of photocleavable antisense DNA to regulate the activity of gRNA, 36 photoactivation of directly caged gRNA, [37][38][39][40] and ligand-induced deprotection of gRNA. 41,42 The general principle for these designs relies on signicant structural alteration of gRNA either by blocking the base pairing [36][37][38][39] or by changing the overall chemical identity through unspecic multiple modications.…”

“…These studies include utilization of photocleavable antisense DNA to regulate the activity of gRNA, 36 photoactivation of directly caged gRNA, [37][38][39][40] and ligand-induced deprotection of gRNA. 41,42 The general principle for these designs relies on signicant structural alteration of gRNA either by blocking the base pairing [36][37][38][39] or by changing the overall chemical identity through unspecic multiple modications. [40][41][42] The former approach needs an individual sequence design for insertion of multiple modied nucleotides with relative difficulties for sample preparation; the latter one utilizes chemical reactive agents to achieve unspecic and heavy modications of gRNA that may potentially encounter incomplete deprotection and result in reduced activation efficiency.…”

Multimerized self-assembled cagedtwo-in-onesiRNA nanoparticles for photomodulation of RNAi-induced gene silencing

“…A photolabile linker was used as a promising photochemical switch, such as DMNPE, NPE, NPP, NPOM and CD-DMNPE. 16 Photocaging strategies have been demonstrated to be successful and effective for temporal and spatial control of the gene expression in vitro and in vivo, including caged plasmids, caged antisense oligonucleotides, caged sgRNAs, [17][18][19][20][21][22] caged miR-NAs [23][24][25] , caged siRNAs etc. 26,27 These caging siRNA strategies usually apply one or more photolabile moieties to randomly or precisely insert into the sequences of siRNA and sterically block the association of siRNA with the RNA-induced silencing complex (RISC), or prevent mRNA cleavage.…”