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Wednesday April 06, 4:00 PM - 7:00 PM

Efficient allelic conversion by homologous chromosome-templated repair in Drosophila somatic tissues


Authors:
Annabel Guichard; Sitara Roy; Sara Juste; Marketta Sneider; Ankush Araudkar; Zhiqian Li; Ehan Bier

Affiliation: Univ California, San Diego, CA

Keywords:
g. CRISPR/Cas9; j. pairing/transvection

Recent advances in Cas9-based genome editing offer broad applications in research, agriculture and medicine. These techniques typically entail insertion of exogenous gene sequences provided by a donor template into specific genomic sites following Cas9-mediated DNA cleavage. Here, we demonstrate efficient somatic allelic conversion wherein cleavage-resistant sequences present on one chromosome correct a targeted cleavage-sensitive allele at the corresponding site on the homologous chromosome. Using a set of allelic combinations of the white locus of Drosophila, we show successful allelic conversion as revealed by quantifiable red eye clones produced by Cas9-dependent cleavage, or by its nickase variants Cas9D10A and Cas9H840A. Repair phenotypes elicited by Cas9 versus nickases differ substantially in several respects, including efficiency, production of cleavage-resistant alleles through the NHEJ mutagenic pathway and developmental timing. We show that allelic correction does not strictly require long-range chromosomal pairing, and that sequence homology alone can promote such repair from a non-homologous location in the genome. If generalized to human cells, this simple approach could be adapted to correct a wide array of dominant or trans-heterozygous genetic conditions offering novel gene therapy strategies.