Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

Aarts, Marieke; Riele, Hein te

doi:10.1038/gt.2010.161

Cited by 46 publications

(59 citation statements)

References 75 publications

(112 reference statements)

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“…Upon annealing to the single-stranded region of the laggingstrand template, ODNs are proposed to be incorporated into the genome as pseudo-Okazaki fragments (25,26). These results suggest that ODNs stabilize expanded TNRs by removal of genomic hairpins during replication; however, a direct demonstration of hairpin inhibition by ODNs in vivo is still wanting.…”

mentioning

confidence: 56%

Oligodeoxynucleotide Binding to (CTG) · (CAG) Microsatellite Repeats Inhibits Replication Fork Stalling, Hairpin Formation, and Genome Instability

Liu

Chen

Leffak

2013

Molecular and Cellular Biology

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(CTG) n · (CAG) n trinucleotide repeat (TNR) expansion in the 3= untranslated region of the dystrophia myotonica protein kinase (DMPK) gene causes myotonic dystrophy type 1. However, a direct link between TNR instability, the formation of noncanonical (CTG) n · (CAG) n structures, and replication stress has not been demonstrated. In a human cell model, we found that (CTG) 45 · (CAG) 45 causes local replication fork stalling, DNA hairpin formation, and TNR instability. Oligodeoxynucleotides (ODNs) complementary to the (CTG) 45 · (CAG) 45 lagging-strand template eliminated DNA hairpin formation on leading-and laggingstrand templates and relieved fork stalling. Prolonged cell culture, emetine inhibition of lagging-strand synthesis, or slowing of DNA synthesis by low-dose aphidicolin induced (CTG) 45 · (CAG) 45 expansions and contractions. ODNs targeting the laggingstrand template blocked the time-dependent or emetine-induced instability but did not eliminate aphidicolin-induced instability. These results show directly that TNR replication stalling, replication stress, hairpin formation, and instability are mechanistically linked in vivo.

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mentioning

confidence: 56%

Oligodeoxynucleotide Binding to (CTG) · (CAG) Microsatellite Repeats Inhibits Replication Fork Stalling, Hairpin Formation, and Genome Instability

Liu

Chen

Leffak

2013

Molecular and Cellular Biology

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“…These molecules direct nucleotide exchange at precise positions and without detectable off target effects [1,2]. While there is great utility in single agent gene editing, the frequency with which single base repair takes place has been consistently lower than needed for long-term development.…”

Section: Introductionmentioning

confidence: 99%

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

Rivera-Torres

Banas

Bialk³

et al. 2017

PLoS ONE

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CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex.

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“…Single base mutations can be repaired by the introduction of DNA oligonucleotides (ssODN) into a target cell [1]–[3]. The frequency of this corrective activity depends on a number of factors including the length of ssODN, the position of the cell in its proliferative cycle [4]–[5] and the presence of double-stranded DNA breaks in the host genome [6]–[7].…”

Section: Introductionmentioning

confidence: 99%

The Position of DNA Cleavage by TALENs and Cell Synchronization Influences the Frequency of Gene Editing Directed by Single-Stranded Oligonucleotides

et al. 2014

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With recent technological advances that enable DNA cleavage at specific sites in the human genome, it may now be possible to reverse inborn errors, thereby correcting a mutation, at levels that could have an impact in a clinical setting. We have been developing gene editing, using single-stranded DNA oligonucleotides (ssODNs), as a tool to direct site specific single base changes. Successful application of this technique has been demonstrated in many systems ranging from bacteria to human (ES and somatic) cells. While the frequency of gene editing can vary widely, it is often at a level that does not enable clinical application. As such, a number of stimulatory factors such as double-stranded breaks are known to elevate the frequency significantly. The majority of these results have been discovered using a validated HCT116 mammalian cell model system where credible genetic and biochemical readouts are available. Here, we couple TAL-Effector Nucleases (TALENs) that execute specific ds DNA breaks with ssODNs, designed specifically to repair a missense mutation, in an integrated single copy eGFP gene. We find that proximal cleavage, relative to the mutant base, is key for enabling high frequencies of editing. A directionality of correction is also observed with TALEN activity upstream from the target base being more effective in promoting gene editing than activity downstream. We also find that cells progressing through S phase are more amenable to combinatorial gene editing activity. Thus, we identify novel aspects of gene editing that will help in the design of more effective protocols for genome modification and gene therapy in natural genes.

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Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

Cited by 46 publications

References 75 publications

Oligodeoxynucleotide Binding to (CTG) · (CAG) Microsatellite Repeats Inhibits Replication Fork Stalling, Hairpin Formation, and Genome Instability

Oligodeoxynucleotide Binding to (CTG) · (CAG) Microsatellite Repeats Inhibits Replication Fork Stalling, Hairpin Formation, and Genome Instability

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

The Position of DNA Cleavage by TALENs and Cell Synchronization Influences the Frequency of Gene Editing Directed by Single-Stranded Oligonucleotides

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