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

Abstract

The function of CRISPR/Cas9 can be conditionally controlled by the rational engineering of guide RNA (gRNA) to target the gene of choice for precise manipulation of genome. Particularly, chemically modified...

Smart Strategies for Precise Delivery of CRISPR/Cas9 in Genome Editing

“…To allow photocontrol of CRISPR/Cas9 activity without disturbing the Watson−Crick base pairing while still maintaining control over the gene expression process in cells, a photolabile protecting group (6-nitropiperonyloxymethylene) can be employed to mask and protect gRNA using site-specific chemical modification. 48 In one recent study, light was used to control CRISPR (crRNA) by incorporating vitamin E (which can inhibit the binding of ribonucleoprotein to the targeted DNA) and a photolabile linker at the 5′ terminus to switch the CRISPR-Cas9 system off. The vitamin E-caged crRNA was successfully activated under light exposure, allowing for controlled gene editing with minimal off-target effects.…”

Optical control of CRISPR-Cas editing with cyclically caged guide RNAs

“…This strategy has already been applied in many examples where the photolabile functionalities were indiscriminately distributed among the spacer and scaffold regions of short crRNAs (36-43 nt). 19,[22][23][24][25]27 However, methodology that allows for the preparation of longer gRNAs of high-purity with over 100 nucleotides containing dozens of site-specifically incorporated photocleavable functionalities has been documented as a limitation. We believe that the anchoring of two modified nucleotides at the 5'-and 3'-end respectively, followed by an intramolecular covalent cyclization similar to the head-to tail splice junction of its natural counterpart, the circRNAs, 51 would form a closed loop with a rigid conformation but still could be fragmented in the presence of light (Fig.…”

“…The standard phosphoramidite RNA chemistry has enabled the incorporation of versatile modified nucleotide motif in a site-specific manner, which can improve the stability of gRNAs towards ribonucleases and the specificity with Cas endonuclease. [9][10][11][12][13][14][15][16] Among these approaches, photolabile groups, [17][18][19][20][21][22][23][24][25][26][27] small-moleculeinduced cleavable linkers, 26,[28][29][30] oligo-binding sites, [31][32][33][34][35][36] and thermo-reversible substituents 37 could be deliberately introduced which, in addition to magnify the duration of oligonucleotides, eliminated the genome editing activity of synthetic gRNAs that can later be switched on by external stimuli hence triggering the editing process. However, a high number of modifying sites, usually one in every 5~6 nucleotides, 22,29 were required to hinder the formation of gRNA-DNA heteroduplex during R-loop expansion and destabilize the conformational flexibility of gRNA:DNA:Cas9 complex.…”

Photoswitchable architecture transformation of a DNA-hybrid assembly at the microscopic and macroscopic scale

“…35,[47][48][49][50][51] Within these stimuli, light stands out due the traceless nature of the photon as a reactant, its spatiotemporal precise dosing and the high tunability of its energy and intensity. 51 In this way, the structure [52][53][54][55][56][57][58] and function 57,[59][60][61][62][63] of DNA and ribonucleic acid (RNA) could be controlled by light. Moreover, controlling these architectures at the molecular level, will allow control of the characteristics of DNA-based supramolecular selfassemblies over various length scales from nano-dimensions up to the macroscopic world (Fig.…”