Photoswitchable gRNAs for Spatiotemporally Controlled CRISPR-Cas-Based Genomic Regulation

Abstract

The recently discovered CRISPR-Cas gene editing system and its derivatives have found numerous applications in fundamental biology research and pharmaceutical sciences. The need for precise external control over the gene editing and regulatory events has driven the development of inducible CRISPR-Cas systems. While most of the light-controllable CRISPR-Cas systems are based on protein engineering, we developed an alternative synthetic approach based on modification of crRNA/ tracrRNA duplex (guide RNA or gRNA) with photocaging groups, preventing the gRNA from recognizing its genome target sequence until its deprotection is induced within seconds of illumination. This approach relies on a straightforward solid-phase synthesis of the photocaged gRNAs, with simpler purification and characterization processes in comparison to engineering a lightresponsive protein. We have demonstrated the feasibility of photocaging of gRNAs and light-mediated DNA cleavage upon brief exposure to light in vitro. We have achieved light-mediated spatiotemporally resolved gene editing as well as gene activation in cells, whereas photocaged gRNAs showed virtually no detectable gene editing or activation in the absence of light irradiation. Finally, we have applied this system to spatiotemporally control gene editing in zebrafish embryos in vivo, enabling the use of this strategy for developmental biology and tissue engineering applications.

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

“…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.…”

Durable Control of HIV-1 Using aStaphylococcus aureusCas9-Expressing Lentivirus Co-Targeting Viral Latency and Host Susceptibility

“…These numbers are in accordance with previous mathematical This strategy resulted in improved editing efficiencies. This strategy could be further optimized and de-risked for clinical application by developing inducible Pol III promoters which restrict HIV-1 gRNAs expression to HIV-1-infected cells [44][45][46][47] .…”

Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein

“…For instance, photocleavage of NPOM-modified DNA/RNA has been used to control the activities of DNAzymes 29 , antisense DNA/RNA 13,30 , restriction endonucleases 31 , DNA-binding transcription factors 32 , polymerase chain reaction (PCR) rates 33 , as well as CRISPR-Cas gene editing 9,18,[34][35] . In these cases, NPOM-modifications prevent the DNA or RNA from hybridizing to the complementary strands, which could be reversed upon NPOM cleavage with light, converting the unhybridized 'inactive' molecules to hybridized 'active' molecules.…”