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Improving transgene expression and CRISPR-Cas9 efficiency with molecular engineering-based molecules.
Clinical and Translational Medicine 2020 October
As a novel and robust gene-editing tool, the Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-associated protein 9 (CRISPR-Cas9) system has revolutionized gene therapy. Plasmid vector delivery is the most commonly used method for integrating the CRISPR-Cas9 system into cells. However, such foreign cytosolic DNAs trigger an innate immune response (IIR) within cells, which can hinder gene editing by inhibiting transgene expression. Although some small molecules have been shown to avoid the action of IIR on plasmids, they only work on a single target and may also affect cell viability. A genetic approach that works at a comprehensive level for manipulating IIR is still lacking. Here, we designed and constructed several artificial nucleic acid molecules (ANAMs), which are combinations of aptamers binding to two key players of IIR (β-catenin and NF-κB). ANAMs strongly inhibited the IIR in cells, thus improving transgene expression. We also used ANAMs to improve the gene-editing efficiency of the CRISPR-Cas9 system and its derivatives, thus enhancing the apoptosis of cancer cells induced by CRISPR-Cas9. ANAMs can be valuable tools for improving transgene expression and gene editing in mammalian cells.
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