Abstract
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.
Highlights
Targeted gene modification is a powerful tool for researchers interested in functional analysis of genes and potentially for gene therapy applications
The low frequency of gene targeting, which relies on homologous recombination (HR) is due in part to the much higher frequency of random integration via nonhomologous end joining (NHEJ), which occurs in about 1 cell for every 102–104 treated cells [1]
It was shown that a DNA double-strand break (DSB) at the target site increases the frequency of gene targeting several orders of magnitude in bacteria [2], yeast [3], plants [4], fruit flies [5], mice [6], human embryonic stem cells [7] and many other cell types
Summary
Targeted gene modification is a powerful tool for researchers interested in functional analysis of genes and potentially for gene therapy applications. It was shown that a DNA double-strand break (DSB) at the target site increases the frequency of gene targeting several orders of magnitude in bacteria [2], yeast [3], plants [4], fruit flies [5], mice [6], human embryonic stem cells [7] and many other cell types. Another strategy to increase gene targeting in mammalian cells has been achieved through the overexpression of key recombination proteins from HR proficient organisms. Knockdown of Ku70 and XRCC4 in human colon cancer cells led to a 30-fold increase in gene targeting [12]
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