Targeted chromosomal gene modification in human cells by single-stranded oligodeoxynucleotides in the presence of a
Frank Radecke1, Ingrid Peter, Sarah Radecke
1Department of Transfusion Medicine, University Hospital Ulm, and Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, 89081 Ulm, Germany.
Abstract:
A DNA double-strand break (DSB) cannot be tolerated by a cell and is dealt with by several pathways. Here, it was hypothesized that DSB induction close to a targeted mutation in the genome of a mammalian cell might attract oligodeoxynucleotide (ODN)-directed gene repair. A HEK-293-derived cell line had been engineered harboring a single target locus with open reading frames encoding the living-cell reporter proteins LacZ and EGFP, the latter translationally decoupled by a DNA spacer with a unique I-SceI recognition site for defined DSB induction. To enable expression of a fluorescent LacZ-EGFP fusion protein, single-stranded (ss) ODNs (80 or 96 nucleotides long) spanning the DSB were designed to fuse both reading frames by altering a few base-pair positions, deleting 59 bp or introducing a 10-bp fragment. The ssODNs alone generated few EGFP-positive cells. With I-SceI transiently expressed, more than 0.3% of cells revealed EGFP expression 7 days after transfection, with up to 96% of the loci faithfully corrected, depending on the ssODN used. During these correction events, the ssODN did not become physically incorporated into the chromosome, but served only as information template. Unwanted insertional mutagenesis also occurred. Both observations have important implications for gene therapy.
Insights
Oligodeoxynucleotides (ODNs) can facilitate gene repair at DNA double-strand breaks (DSBs) in mammalian cells. This study shows ODNs act as templates for precise gene correction, with implications for gene therapy.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- DNA double-strand breaks (DSBs) are critical DNA lesions that cells repair through various pathways.
- Targeted induction of DSBs near genomic mutations could potentially enhance gene repair efficiency.
Purpose of the Study:
- To investigate if inducing a DNA double-strand break (DSB) near a targeted mutation attracts oligodeoxynucleotide (ODN)-directed gene repair in mammalian cells.
- To assess the efficiency and fidelity of gene correction mediated by single-stranded ODNs (ssODNs) in response to a targeted DSB.
Main Methods:
- Engineered a HEK-293 cell line with a target locus for LacZ and EGFP reporter genes, separated by an I-SceI recognition site.
- Designed and transfected cells with single-stranded oligodeoxynucleotides (ssODNs) of 80 or 96 nucleotides to bridge the DSB and fuse reporter genes.
- Induced DSBs using transient expression of I-SceI endonuclease.
Main Results:
- Transfection with ssODNs alone resulted in minimal EGFP expression.
- Transient expression of I-SceI led to EGFP expression in over 0.3% of cells.
- Faithful gene correction occurred in up to 96% of loci, depending on the specific ssODN used.
- ssODNs served as informational templates for repair and were not incorporated into the chromosome.
- Unwanted insertional mutagenesis was also observed during the correction process.
Conclusions:
- DSB induction proximal to a mutation can facilitate ODN-templated gene repair in mammalian cells.
- The efficiency of gene correction is dependent on the specific ssODN design.
- While promising for gene therapy, the potential for insertional mutagenesis requires careful consideration.
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