A novel CRISPR/RNA-aptamer-mediated base editing system with potential therapeutic value

Abstract:

Description Nuclease-dependent precise genome editing such as correction of point mutations requires introduction of targeted DNA double strand breaks (DSB) and activation of homology dependent repair (HDR), limiting its application to proliferating cells. To expand GE capabilities for therapeutic use in non-dividing somatic cells it is necessary to precisely modify nucleotides avoiding DSBs. Recently, Cas9-cytidine deaminase fusions, also known as based editors (BE), were shown to precisely modify target bases at certain genomic loci. To expand the base editing toolbox, we sought to engineer a novel base editing system based on RNA-aptamer mediated recruitment. To this end, we engineered a nuclease-deficient CRISPR/Cas9 system as a recruitment platform for non-nuclease DNA/RNA editing enzymes that catalyze C· G→ T· A conversions by cytidine deamination. Targeted nucleotide modification was achieved with high precision in prokaryotic and eukaryotic cells. In bacteria, we tested our system targeting the rifampicin resistance determining region of the rpoB gene. Survival in rifampicin reached over 1000-fold higher than untreated cells. To examine whether the system can correct loss of function mutations in human genome, we treated a stably integrated non-fluorescent EGFP gene containing an A· T→ G· C mutation on the chromophore sequence. Fluorescence was efficiently restored in treated cells, detecting around 10% of GFP positive cells after treatment. Next generation sequencing confirmed a G· C→ A· T conversion in 60% of reads at the target position, restoring the wild type sequence, with low by-stander effect. Exome …

Año de publicación:

2019

Keywords:

Fuente:

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