The involvement of replication in single stranded oligonucleotide-mediated gene repair

Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.

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Faculty Opinions recommendation of High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.737073806.793579888 ◽

2020 ◽

Author(s):

Yousin Suh

Keyword(s):

Cystic Fibrosis ◽

Stem Cells ◽

High Efficiency ◽

Gene Repair ◽

Free Gene

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Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity

Molecular and Cellular Biology ◽

10.1128/mcb.22.11.3852-3863.2002 ◽

2002 ◽

Vol 22 (11) ◽

pp. 3852-3863 ◽

Cited By ~ 59

Author(s):

Li Liu ◽

Michael C. Rice ◽

Miya Drury ◽

Shuqiu Cheng ◽

Howard Gamper ◽

...

Keyword(s):

Transcriptional Activity ◽

Fluorescent Protein ◽

Fusion Gene ◽

Strand Bias ◽

Nucleotide Exchange ◽

Gene Repair ◽

Protein Fusion ◽

Template Strand ◽

Targeted Gene Repair ◽

Targeting Vector

ABSTRACT Modified single-stranded DNA oligonucleotides can direct nucleotide exchange in Saccharomyces cerevisiae. Point and frameshift mutations are corrected in a reaction catalyzed by cellular enzymes involved in various DNA repair processes. The present model centers on the annealing of the vector to one strand of the helix, followed by the correction of the designated base. The choice of which strand to target is a reaction parameter that can be controlled, so here we investigate the properties of strand bias in targeted gene repair. An in vivo system has been established in which a plasmid containing an actively transcribed, but mutated, hygromycin-enhanced green fluorescent protein fusion gene is targeted for repair and upon conversion will confer hygromycin resistance on the cell. Overall transcriptional activity has a positive influence on the reaction, elevating the frequency. If the targeting vector is synthesized so that it directs nucleotide repair on the nontranscribed strand, the level of gene repair is higher than if the template strand is targeted. We provide data showing that the targeting vector can be displaced from the template strand by an active T7 phage RNA polymerase. The strand bias is not influenced by which strand serves as the leading or lagging strand during DNA synthesis. These results may provide an explanation for the enhancement of gene repair observed when the nontemplate strand is targeted.

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Construction of Adoxophyes honmai nucleopolyhedrovirus bacmid DNA by using a gene-repair BAC cassette

Applied Entomology and Zoology ◽

10.1007/s13355-014-0313-8 ◽

2015 ◽

Vol 50 (1) ◽

pp. 143-146

Author(s):

Yasumasa Saito ◽

Yasuhisa Kunimi ◽

Madoka Nakai

Keyword(s):

Gene Repair ◽

Adoxophyes Honmai

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Gene repair tool for stem cells

Nature ◽

10.1038/474008c ◽

2011 ◽

Vol 474 (7349) ◽

pp. 8-8

Keyword(s):

Stem Cells ◽

Gene Repair

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Sickle Cell Disease Hematopoietic Stem Cell gene therapy with globin gene addition is promising

10.31219/osf.io/j5fkb ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Gene Therapy ◽

Stem Cell ◽

Sickle Cell Disease ◽

Hematopoietic Stem Cell ◽

Sickle Cell ◽

Globin Gene ◽

Autologous Transplantation ◽

Gene Repair ◽

Cell Disease ◽

Hematopoietic Stem

Autologous transplantation of gene-modified HSCs might be used to treat Sickle Cell Disease (SCD) once and for all. Hematopoietic Stem Cell (HSC) gene therapy with lentiviral-globin gene addition was optimized by HSC collection, vector constructs, lentiviral transduction, and conditioning in the current gene therapy experiment for SCD, resulting in higher gene marking and phenotypic correction. Further advancements over the next decade should allow for a widely approved gene-addition therapy. Long-term engraftment is crucial for gene-corrected CD34+ HSCs, which might be addressed in the coming years, and gene repair of the SCD mutation in the-globin gene can be achieved in vitro using genome editing in CD34+ cells. Because of breakthroughs in efficacy, safety, and delivery strategies, in vivo gene addition and gene correction in BM HSCs is advancing. Overall, further research is needed, but HSC-targeted gene addition/gene editing therapy is a promising SCD therapy with curative potential that might be widely available soon.

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