Aptamer-guided gene targeting in yeast and human cells.
Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location via homologous recombination (HR) and is useful both for functional analysis and gene therapy applications. HR is inefficient in most organisms and cell types, including mammalian cells, often limiting the effectiveness of gene targeting. Therefore, increasing HR efficiency remains a major challenge to DNA editing. Here, we present a new concept for gene correction based on the development of DNA aptamers capable of binding to a site-specific DNA binding protein to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting). We selected DNA aptamers to the I-SceI endonuclease. Bifunctional oligonucleotides containing an I-SceI aptamer sequence were designed as part of a longer single-stranded DNA molecule that contained a region with homology to repair an I-SceI generated double-strand break and correct a disrupted gene. The I-SceI aptamer-containing oligonucleotides stimulated gene targeting up to 32-fold in yeast Saccharomyces cerevisiae and up to 16-fold in human cells. This work provides a novel concept and research direction to increase gene targeting efficiency and lays the groundwork for future studies using aptamers for gene targeting.
School of Biology, Georgia Institute of Technology, Atlanta, GA, 30332, USA.
Patrick
P
Ruff
Kyung Duk
KD
Koh
Havva
H
Keskin
Rekha B
RB
Pai
Francesca
F
Storici
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D018390:Gene Targeting; D057809:HEK293 Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D051721:Rad52 DNA Repair and Recombination Protein / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid
D052157:Aptamers, Nucleotide; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific