The introduction of photocleavable molecules to nucleic acids has a long tradition. An early example is the conjugation of the photolabile 2-nitrobenzyl to ATP to produce caged ATP. This caged ATP allowed McCray et al. in 1980 to study the kinetics of ATP dissociation from actomyosin by measuring the amount and rate of ATP generation after laser flash photolysis of caged ATP.
Figure 1: Photolysis of caged ATP results in the formation of 2-nitrosoacetophenone and a proton, depending on the ionization state of ATP.
Photocleavable guide RNAs (pcRNAs) are engineered CRISPR guide RNAs containing photolabile linkers, such as 1-(2-nitrophenyl)-1,2-ethanediol, that break upon UV or visible light exposure. This light-triggered degradation deactivates the Cas9 complex, enabling precise spatiotemporal control of gene editing, drastically reducing off-target effects, and halting editing cycles on demand.
The RNA-programmed CRISPR/Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats) is now widely used for genome editing, gene expression regulation, and other tasks requiring manipulation of genetic material. Controlling this molecular tool more precisely by switching it on and off in time and space in response to specific signals will be an important enhancement.
1-(2-nitrophenyl)-1,2-ethanediol is an ortho-nitrobenzyl (oNB) derivative commonly used as a photolabile protecting group or "caged" linker in biology and chemistry. Upon UV or near-visible light irradiation, it undergoes photocleavage, releasing the appended functional groups and forming a 2-nitroso aromatic byproduct. oNB esters are widely used as photolabile protecting groups and linkers in diverse applications ranging from materials science to chemical biology.
The oNB moiety in a PC spacer phosphoramidite enables incorporation of the linker at various positions in oligonucleotides during automated solid-phase synthesis. When incorporated at the 5'- end, after photocleavage, a 5'-phosphate is generated on the DNA, rendering it suitable for further biological transformations, such as gene construction and cloning after ligation.
| 1-(2-Nitrophenyl)-1,2-ethanediol or 2-Nitrostyrene glycol | PC Linker Phosphoramidite 3-(4,4’-Diemthoxytrityl)-1-(2-nitrophenyl)-propan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite |
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The photolysis of the linker follows the classical pathway of ortho-nitrobenzyl compounds: During photoexcitation, the nitroaromatic group absorbs light, typically in the 300–365 nm UVA range, exciting the molecule to its triplet state.
Photochemical Decomposition Mechanism for oNB Esters via a Triplet State and an Intramolecular H-Abstraction (according to Fink et al. 2025).
During rapid hydrogen transfer, the excited nitro group abstracts a hydrogen atom from the adjacent benzylic carbon, resulting in an unstable aci-nitro Intermediate. Next, the intermediate cyclizes into a five-membered isoxazole derivative. The ring decomposes, cleaving the attached molecule and leaving behind 2-nitrosobenzaldehyde or a related ketone/aldehyde derivative.
By placing photocleavable linkers at strategic points in pcRNAs, the guide RNA remains functional for DNA targeting until light irradiation cleaves the linker. Protein-binding sites or hairpin regions are optimal positions for incorporating the linker. Once cleaved, the guide RNA degrades, completely abolishing Cas9 nuclease activity. pcRNA systems can completely inactivate Cas9 function in less than a minute post-irradiation.
In CRISPR-based gene-editing experiments, light-controlled systems can prevent prolonged, uncontrolled DNA-cutting and repair cycles, thereby significantly reducing off-target mutations. Correctly designed pcRNAs enable turning gene-editing activity "off" in specific regions of cells or organisms at precise moments, enabling highly targeted developmental studies. pcRNA systems utilize both UV and visible light, allowing for non-invasive manipulation of Cas9 and base editors in mammalian cells.
Automated solid-phase oligonucleotide synthesis allows the manufacturing of synthetic crRNAs or sgRNAs as well as the incorporation of photolabile linkers into their backbone. In the absence of light, the pcRNA binds to the Cas9 protein and guides it to the complementary genomic target for cleavage. When mild UV or visible light is applied to the target area, photons cleave the synthetic linkers, fragmenting the guide RNA. Without the intact guide RNA, the Cas9 nuclease dissociates or remains inactive, halting all downstream genome editing.
Zou et al. 2021 developed photocleavable guide RNAs (pcRNAs) to enable light-mediated, rapid, and complete deactivation of CRISPR-Cas9 nucleases and base editors. A photocleavable moiety is chemically incorporated into the guide RNA (gRNA) that directs Cas9 to its target DNA. When exposed to light of a specific wavelength, this linker molecule undergoes photochemical cleavage, destroying the gRNA and preventing Cas9 from binding to its target site. This built-in mechanism avoids the need for separate inhibitors, which can be slow, incomplete, or difficult to deliver. Deactivation occurs within seconds to less than 1 minute after light exposure. Also, residual activity is extremely low, with approximately <1% residual indels, ensuring near-total shutdown. The pcRNA modification enhances specificity of wild-type Cas9.
This approach works with both Cas9 nucleases and base editors.Time-resolved experiments revealed the following time course: For CAS9, ~12–36 hours of activity is sufficient for high editing efficiency. For base editors, ~2–4 hours of activity is sufficient for high editing efficiency.This pcRNA design enables on-demand editing without prolonged Cas9 activity, reducing off-target effects. By synchronizing the termination of DNA damage, pcRNA allows researchers to study cellular responses to DNA damage and repair without the confounding effects of sustained cycles of damage and repair. pcRNAs improve the safety profile of CRISPR applications by allowing rapid shutdown when needed.
Wang et al. in 2023 utilized a similar approach for the production of a molecular glue.
Sakovina et al. in 2024 reported the design of a series of photocleavable guide CRISPR RNAs (crRNA) and their analogs modified with 2′-fluoro and locked nucleic acid (LNA) containing one or two 1-(2-nitrophenyl)-1,2-ethanediol photolabile linkers. The research group demonstrated that the crRNAs can be destroyed by relatively mild UVA irradiation with the rate constants 0.24–0.77 min−1 and that the photocleavage markedly slows down the action of Cas9 nuclease in the model in vitro system.
The incorporation of two photolabile linkers provided more rapid crRNA destruction than a single linker. Photolabile linkers incorporated in the crRNA structure improved the specificity of DNA cleavage by Cas9 nuclease for the fully complementary target. The use of photocleavable crRNA in CRISPR/Cas9 genome editing permits the system to be switched off in a spatiotemporally controlled manner, thus alleviating its off-target effects.
References
Blanc A, Bochet CG. Bis(o-nitrophenyl)ethanediol: A practical photolabile protecting group for ketones and aldehydes. J Org Chem. 2003 Feb 7;68(3):1138-41. [PubMed]
Fink AL, Groß AG, Puch F, Geitner R. Photolysis of ortho-Nitrobenzyl Esters: Kinetics and Substituent Effects. ACS Omega. 2025 Nov 20;10(47):57560-57567. [PMC]
McCray JA, Herbette L, Kihara T, Trentham DR. A new approach to time-resolved studies of ATP-requiring biological systems; laser flash photolysis of caged ATP. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7237-41.
Sakovina L, Vokhtantsev I, Akhmetova E, Vorobyeva M, Vorobjev P, Zharkov DO, Novopashina D. Photocleavable Guide crRNAs for a Light-Controllable CRISPR/Cas9 System. Int J Mol Sci. 2024 Nov 19;25(22):12392. [PubMed, PMC]
Wang J, Peled TS, Klajn R. Photocleavable Anionic Glues for Light-Responsive Nanoparticle Aggregates. J Am Chem Soc. 2023 Feb 9;145(7):4098–108. [PMC]
Zou RS, Liu Y, Ha T. CRISPR deactivation in mammalian cells using photocleavable guide RNAs. STAR Protoc. 2021 Oct 20;2(4):100909. [PMC]