An artificial triazole backbone linkage provides a split-and-click strategy to bioactive chemically modified CRISPR sgRNA.
Lapatrada TaemaitreeArun ShivalingamAfaf H El-SagheerTom BrownPublished in: Nature communications (2019)
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.
Keyphrases
- crispr cas
- genome editing
- genome wide
- circulating tumor
- induced apoptosis
- cell free
- single molecule
- hiv testing
- dna binding
- dna methylation
- cell cycle arrest
- risk factors
- cancer therapy
- men who have sex with men
- endoplasmic reticulum stress
- nitric oxide
- cell death
- cell proliferation
- human immunodeficiency virus
- oxidative stress
- gene expression
- signaling pathway
- drug delivery
- circulating tumor cells