PC-Linker RNA-Caged CRISPR Systems For Diagnostics And Gene Editing
A PC-linker RNA-caged CRISPR system uses a photocleavable (PC) linker to block temporarily, or "cage," guide RNA activity until a specific wavelength of light unlocks it. [Jain et al. 2016, Hu et al. 2022, Moroz-Omori et al. 2020]
In this approach, a complementary "protector" or blocking oligonucleotide binds to the functional region of the CRISPR RNA, for example, the spacer domain or direct repeat, using a PC linker. In the inactive state, the linkage forms an inactive duplex that prevents the guide RNA from assembling with the Cas protein or recognizing its target sequence. Exposure to ultraviolet (UV) light, typically at 365 nm, cleaves the PC linker, releasing the blocking strand to activate CRISPR-Cas activity.
In one-pot diagnostics, target pre-amplification is decoupled from CRISPR-Cas12a detection to prevent premature reactions and greatly improve sensitivity.
PC-linker RNA-caged CRISPR systems enable spatiotemporal control by triggering gene editing, gene activation, or cellular responses in precise locations and at exact moments using light. More recently developed CRISPR systems use circular RNA designs incorporating PC linkers to increase resistance to degradation before light activation.
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Figure 1: Photoactivatable blockage of Cas9-mediated DNA targeting.
a) Light-activated “turn on” of CRISPR using a photocleavable protector or blocking oligonucleotides (Jain et al. 2016). The hybridization of single chimeric guide RNAs (sgRNA) with a complementary blocking oligonucleotide containing photocleavable groups creates an inactive, blocked CRISPR complex. A remote light trigger cleaves and fragments the protector oligonucleotides, activating CRISPR nuclease activity.
b) Structural schematics of blocking units. In the design by Hue et al. (2022), the CRISPR-Cas12a system is temporarily blocked so that the recombinase polymerase amplification (RPA) reaction is unaffected. After the RPA reaction is complete, the CRISPR-Cas12a system is rapidly activated using 365-nm light irradiation. Introducing photocaged RNA significantly improves the sensitivity of DNA and RNA detection.
In a different approach, Moroz-Omori et al. (2022) used photocaged gRNAs for light-induced CRISPR-Cas-mediated target DNA cleavage, enabling spatiotemporally resolved gene editing and gene activation in cells.
Figure 2: Photo-release of caged thymidine.
The photocaged thymidine (T) is placed in the sgRNA with 5- or 6-nucleotide spacing within the target-recognition region of the gRNA. Moroz-Omori et al. introduced three phosphorothioate linkages at each terminus of the variable part of the crRNA to improve duplex stability intracellularly using NPOM caged-dT-CE phosphoramidites (5'-Dimethoxytrityl-N3-[[1-(6-nitro-1,3-benzodioxol-5-yl)ethoxy]methyl]-2'-deoxy-thymidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidites). Two photocaging groups were positioned in the targeting region of crRNA to prevent Watson–Crick base pairing with the complementary genomic DNA. Incorporating two photocaging groups within the target-recognizing region of the gRNA proved to be sufficient to prevent CRISPR-Cas-mediated gene editing or transcription activation. Light activation reconstituted CRISPR activity.
The use of photo-controlled caged gRNA significantly improves spatiotemporal control of CRISPR-Cas-based genomic regulation. Further, solid-phase synthesis enables production of the photocaged gRNAs, which is simpler than engineering a light-responsive protein. This approach enables spatiotemporal control of gene editing in vivo, supporting developmental biology and tissue engineering applications.
Reference
Hu M, Qiu Z, Bi Z, Tian T, Jiang Y, Zhou X. Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics. Proc Natl Acad Sci U S A. 2022 Jun 28;119(26):e2202034119. [PMC]
Jain PK, Ramanan V, Schepers AG, Dalvie NS, Panda A, Fleming HE, Bhatia SN. Development of Light-Activated CRISPR Using Guide RNAs with Photocleavable Protectors. Angew Chem Int Ed Engl. 2016 Sep 26;55(40):12440-4. [PMC]
Moroz-Omori EV, Satyapertiwi D, Ramel MC, Høgset H, Sunyovszki IK, Liu Z, Wojciechowski JP, Zhang Y, Grigsby CL, Brito L, Bugeon L, Dallman MJ, Stevens MM. Photoswitchable gRNAs for Spatiotemporally Controlled CRISPR-Cas-Based Genomic Regulation. ACS Cent Sci. 2020 May 27;6(5):695-703. [Euro PMC]