An artificial triazole backbone linkage provides a split-and-click strategy to bioactive chemically modified CRISPR sgRNA

Abstract

As the applications of CRISPR-Cas9 technology diversify and spread beyond the laboratory to diagnostic and therapeutic use, the demands of gRNA synthesis have increased and access to tailored gRNAs is now restrictive. Enzymatic routes are time-consuming, difficult to scale-up and suffer from polymerase-bias while existing chemical routes are inefficient. Here, we describe a split-and-click convergent chemical route to individual or pools of sgRNAs. The synthetic burden is reduced by splitting the sgRNA into a variable DNA/genome-targeting 20-mer, produced on-demand and in high purity, and a fixed Cas9-binding chemically-modified 79-mer, produced cost-effectively on large-scale, a strategy that provides access to site-specific modifications that enhance sgRNA activity and in vivo stability. Click ligation of the two components generates an artificial triazole linkage that is tolerated in functionally critical regions of the sgRNA and allows efficient DNA cleavage in vitro as well as gene-editing in cells with no unexpected off-target effects.

Versatile 3′ Functionalization of CRISPR Single Guide RNA

“…sgRNAs are typically 90–115‐nt in length and challenging to produce in high yield and purity via solid‐phase synthesis alone, hence we aimed to synthesize the sgRNA in two parts and use splint ligation to form our desired sgRNA . To accomplish this, we chemically synthesized a 30‐nt fragment of the sgRNA modified at the 3′‐end with a primary amine (Figure A and Table S1 in the Supporting Information).…”

“…For this purpose, we used an sgRNA bearing an RNA scaffold that had been shown previously to support cleavage and was used for structural studies of Cas9 complexes . We chose to study the impact of modification at this location using the in vitro DNA cleavage assay, which is known to show a strong correlation with cleavage in a cellular context . None of the chemically modified sgRNAs disabled Cas9′s ability to cleave the target DNA in vitro, suggesting this location is suitable for a wide variety of modifications.…”

“…While chemically modified gRNAs have been explored in some detail, most studies investigate modifications to the sugar or use the dual guide RNA system (e.g., crRNA+trRNA) . Generally, these modifications (e.g., 2′‐ O ‐methyl) are intended for increasing or maintaining on‐target potency while reducing off‐target gene editing or sgRNA stability to nucleases and less envisioned for application‐based experiments although some examples exist .…”

Biomedical applications of copper-free click chemistry: in vitro, in vivo, and ex vivo

“…Antibody-modied TCO/Tz-modied magnetic nanoparticles iEDDA Amplication of signal for diagnosis purpose 61 Antibody-modied TCO/Tz-modied magneto-uorescent nanoparticles iEDDA Study of protein dynamics in the neuronal system 62 Azidohomoalanine/DIFO-biotin SPACC Study of the conformational change of protein by FRET 63 Unnatural amino acid containing tetrazine or norbornene/BODIPY-FL tetrazine or BODIPY-TMR-X bicyclononyne iEDDA, SPACC Nucleotide ligation for the CRISPR Cas9 system 64 RNAs modied with alkyne or DBCO/RNAs modied with azide CuAAC, SPACC regarding click chemistry for biomedical purposes and the prospect of this technique in the future. Particularly, we will focus on copper-free click reactions occurring on a cell surface, in cell cytosol, in a body, or at least with proteins or nucleotides, emphasizing the advantage of copper-free click chemistry considering the environment in which the reaction occurs.…”

Engineering nucleic acid chemistry for precise and controllable CRISPR/Cas9 genome editing

“…It has a comparable function to the in vitro transcribed dual RNA. Taenaitree et al [45] adopted a similar split-and-click strategy (Fig. 3b) to generate gRNA for genome editing.…”