Site-Specific and Enzymatic Cross-Linking of sgRNA Enables Wavelength-Selectable Photoactivated Control of CRISPR Gene Editing

Journal of the American Chemical Society, Год журнала: 2022, Номер 144(10), С. 4487 - 4495

Опубликована: Март 8, 2022

Chemical cross-linking enables rapid identification of RNA-protein and RNA-nucleic acid inter- intramolecular interactions. However, no method exists to site-specifically covalently cross-link two user-defined sites within an RNA. Here, we develop RNA-CLAMP, which site-specific enzymatic (clamping) selected guanine residues Intramolecular clamping can disrupt normal RNA function, whereas subsequent photocleavage the cross-linker restores activity. We used RNA-CLAMP clamp stem loops single-guide (sgRNA) CRISPR-Cas9 gene editing system via a photocleavable cross-linker, completely inhibiting editing. Visible light irradiation cleaved restored with high spatiotemporal resolution. Design linkers responsive different wavelengths allowed multiplexed photoactivation in mammalian cells. This photoactivated platform benefits from undetectable background activity, provides choice activation wavelengths, has multiplexing capabilities.

Язык: Английский

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A Conformational Restriction Strategy for the Control of CRISPR/Cas Gene Editing with Photoactivatable Guide RNAs**

Angewandte Chemie International Edition, Год журнала: 2022, Номер 62(5)

Опубликована: Дек. 1, 2022

The CRISPR/Cas system is one of the most powerful tools for gene editing. However, approaches precise control genome editing and regulatory events are still desirable. Here, we report spatiotemporal efficient CRISPR/Cas9- Cas12a-mediated with conformationally restricted guide RNAs (gRNAs). This approach relied on only two or three pre-installed photo-labile substituents followed by an intramolecular cyclization, representing a robust synthetic method in comparison to heavily modified linear gRNAs that often require extensive screening time-consuming optimization. tactic could direct cleavage genes encoding green fluorescent protein (GFP) vascular endothelial growth factor A (VEGFA) within predefined cutting region without notable leakage live cells. We also achieved light-mediated myostatin (MSTN) embryos, wherein new bow-knot-type gRNA was constructed excellent OFF/ON switch efficiency. Overall, our work provides significant strategy circular precisely manipulate where when edited.

Язык: Английский

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Photoactivated DNA Assembly and Disassembly for On‐Demand Activation and Termination of cGAS‐STING Signaling

Angewandte Chemie International Edition, Год журнала: 2023, Номер 62(33)

Опубликована: Июнь 27, 2023

Abstract Despite significant progress in DNA self‐assembly for interfacing with biology, spatiotemporally controlled regulation of biological process via situ dynamic assembly remains an outstanding challenge. Here, we report optically triggered and disassembly strategy that enables on‐demand activation termination the cyclic GMP‐AMP synthase (cGAS)‐stimulator interferon genes (STING) signaling pathway. In design, activatable hairpin is engineered a photocleavable group at defined site to modulate its activity. Light induces configurational switching consequent hairpins form long linear double‐stranded structures, allowing stimulate cGAS protein synthesize 2′,3′‐cyclic‐GMP‐AMP (cGAMP) STING stimulation. Furthermore, by endowing pre‐assembled scaffold built‐in photolysis feature, demonstrate cGAS‐STING stimulation can be efficiently terminated through remote photo‐triggering, providing first time route control temporal “dose” such We envision this will benefit inspire both fundamental research therapeutic applications regarding

Язык: Английский

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Cascade dynamic assembly/disassembly of DNA nanoframework enabling the controlled delivery of CRISPR-Cas9 system

Science Advances, Год журнала: 2023, Номер 9(35)

Опубликована: Авг. 30, 2023

CRISPR-Cas9 has been explored as a therapeutic agent for down-regulating target genes; the controlled delivery of Cas9 ribonucleoprotein (RNP) is essential efficacy and remains challenge. Here, we report cascade dynamic assembly/disassembly DNA nanoframework (NF) that enables RNP. NF was prepared with acrylamide-modified initiated hybridization chain reaction (HCR). Through an HCR, single-guide RNA incorporated to NF; simultaneously, internal space expanded, facilitating loading protein. designed hydrophilic acylamino hydrophobic isopropyl, allowing swelling aggregation. The responsive release RNP realized by introducing disulfide bond-containing

Язык: Английский

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Regulation of the CRISPR-Cas12a system by methylation and demethylation of guide RNA

Chemical Science, Год журнала: 2023, Номер 14(22), С. 5945 - 5955

Опубликована: Янв. 1, 2023

Chemical modifications of CRISPR-Cas nucleases help decrease off-target editing and expand the biomedical applications CRISPR-based gene manipulation tools. Here, we found that epigenetic guide RNA, such as m6A m1A methylation, can effectively inhibit both cis- trans-DNA cleavage activities CRISPR-Cas12a. The underlying mechanism is methylations destabilize secondary tertiary structure gRNA which prevents assembly Cas12a-gRNA nuclease complex, leading to decreased DNA targeting ability. A minimum three adenine methylated nucleotides are required completely activity. We also demonstrate these effects reversible through demethylation by demethylases. This strategy has been used in regulation expression, demethylase imaging living cells controllable editing. results methylation-deactivated demethylase-activated a promising tool for CRISPR-Cas12a system.

Язык: Английский

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Modular Weaving DNAzyme in Skeleton of DNA Nanocages for Photoactivatable Catalytic Activity Regulation

Angewandte Chemie, Год журнала: 2024, Номер 136(18)

Опубликована: Март 22, 2024

Abstract DNAzymes exhibit tremendous application potentials in the field of biosensing and gene regulation due to its unique catalytic function. However, spatiotemporally controlled DNAzyme activity remains a daunting challenge, which may cause nonspecific signal leakage or silencing systems. Here, we report photochemical approach via modular weaving active into skeleton tetrahedral DNA nanocages (TDN) for light‐triggered on‐demand liberation thus conditional control activity. We demonstrate that direct encoding TDN could improve biostability ensure delivery efficiency, comparing with conventional surface anchoring strategy. Furthermore, molecular nanostructures allows remote DNAzyme‐mediated high spatiotemporal precision light. In addition, is applicable editing functions other functional nucleic acids.

Язык: Английский

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Small Molecule-Inducible RNA-Targeting Systems for Temporal Control of RNA Regulation

ACS Central Science, Год журнала: 2020, Номер 6(11), С. 1987 - 1996

Опубликована: Окт. 28, 2020

All aspects of mRNA lifetime and function, including its stability, translation into protein, trafficking through the cell, are tightly regulated coordinated post-transcriptional modifications interactions with a multitude RNA effector proteins. Despite increasing recognition regulation as critical layer mammalian gene expression control excitement therapeutic target, tools to study regulatory mechanisms temporal precision in their endogenous environment lacking. Here, we present small molecule-inducible RNA-targeting effectors based on our previously developed CRISPR/Cas-inspired targeting system (CIRTS). The CIRTS biosensor platform is guide (gRNA)-dependent binding domains that interact target transcript using Watson–Crick–Franklin base pair interactions. Addition molecule recruits an transcript, thereby eliciting local effect transcript. In this work, showcase these biosensors can trigger inducible editing, degradation, or transcripts molecule-dependent manner. We further go show editor induce editing molecule-controllable manner vivo. Collectively work provides new set probe dynamics systems at level.

Язык: Английский

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CRISPRoff enables spatio-temporal control of CRISPR editing

Nature Communications, Год журнала: 2020, Номер 11(1)

Опубликована: Окт. 7, 2020

Abstract Following introduction of CRISPR-Cas9 components into a cell, genome editing occurs unabated until degradation its component nucleic acids and proteins by cellular processes. This uncontrolled reaction can lead to unintended consequences including off-target chromosomal translocations. To address this, we develop method for light-induced sgRNA termed CRISPRoff. Here show that inactivation ribonucleoprotein attenuates within cells allows titratable levels efficiency spatial patterning via selective illumination.

Язык: Английский

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Photochemical modifications for DNA/RNA oligonucleotides

RSC Advances, Год журнала: 2022, Номер 12(11), С. 6484 - 6507

Опубликована: Янв. 1, 2022

Photoconvertible groups (PG) can be incorporated into an oligonucleotide to undergo various irreversible and reversible light-induced reactions such as cleavage, crosslinking, isomerization, intramolecular cyclization reactions.

Язык: Английский

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G-quadruplex-based CRISPR photoswitch for spatiotemporal control of genomic modulation

Nucleic Acids Research, Год журнала: 2023, Номер 51(8), С. 4064 - 4077

Опубликована: Март 13, 2023

Abstract CRISPR (clustered regularly interspaced short palindromic repeats) technology holds tremendous promise for gene regulation and editing. However, precise control of editing is essential to overcome its uncontrollable reaction process excessive activity that leads off-target To this problem, we engineered a photoswitch on G-quadruplex gRNA (GqRNA) precisely controlled expression by embedding dicationic azobenzene derivatives (AZD++). Our results demonstrated rational design the onto crRNA conferred higher stability sequence recognition specificity than unmodified single guide (sgRNA). Light-induced isomerization AZD++ quickly transformed state GqRNA, which facilitated rapid activation ribonucleoprotein genome on-target sites in cells with excellent efficiency. In turn, AZD++–GqRNA promptly refolded an off inhibit genomic cleavage, limited generation effects by-products. Therefore, proposed strategy photo-reversible modality presents new opportunity CRISPR-Cas9 modulation improve safety applicability.

Язык: Английский

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