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theses

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-wide sequence analysis revealed no detectable off-target effects. Targeting of endogenous loci also resulted in highly efficient nucleotide conversion at the desired C positions. We also show that our system can destroy the 3’ splice acceptor site of intron 50 in human DMD gene, potentially inducing exon 51 skipping, providing evidence of a therapeutic application to treat Duchenne muscular dystrophy. Taken together, the data show that our GE system represents a safe and promising technology for editing specific nucleotides, correcting genetic mutations or other clinically relevant applications, independent of DSB and HDR, with potential therapeutic value in non-dividing cells.

ETD_9363

Ph.D.

Includes bibliographical references

by Juan Carlos Collantes

doi:10.7282/t3-s2dw-xk36

ETD doctoral

The author owns the copyright to this work.