DNA Replication, cell cycle progression and the targeted gene repair reaction

Engstrom, Julia U.; Kmiec, Eric B.

doi:10.4161/cc.7.10.5826

Cited by 38 publications

(51 citation statements)

References 44 publications

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“…The three NT-ssODNs are of identical sequence through 40 bases but vary in length from 40 to 72 to 100 nucleotides respectively. This system is well established as a model for analyzing the mechanism of gene editing in human cells [9] [29]–[30]. In most applications, 72NT ssODN has been used in optimization studies for delivery and the response of cell and genomic DNA in the gene editing reaction [20].…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…Over the course of the last 15 years we and others have established the mechanism and regulation of short oligo induced gene editing [2–4,10,11,34]. More recent studies have shown that oligonucleotides of length between 49 and 72 bases respectively can direct single base repair as the sole agent of the gene editing reaction or in combination with both TALENs and CRISPR/Cas9 [33,35].…”

Section: Discussionmentioning

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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“…Studies on oligonucleotide ssODNs and cell cycle indicated that gene modification frequencies varied considerably between the various cell cycle phases, in particular late S-phase was shown to be the most amenable for gene repair [20], probably due to increased activity of replication forks and to a more favorable chromatin conformation. Transfecting SDFs into cells synchronized in different cell cycle phases, we noticed that replacement efficiencies were highest in vinblastine-enriched G 2 /M-phase cells.…”

Section: Discussionmentioning

confidence: 99%

Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System

et al. 2012

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Homologous Replacement is used to modify specific gene sequences of chromosomal DNA in a process referred to as “Small Fragment Homologous Replacement”, where DNA fragments replace genomic target resulting in specific sequence changes. To optimize the efficiency of this process, we developed a reporter based assay system where the replacement frequency is quantified by cytofluorimetric analysis following restoration of a stably integrated mutated eGFP gene in the genome of SV-40 immortalized mouse embryonic fibroblasts (MEF-SV-40). To obtain the highest correction frequency with this system, several parameters were considered: fragment synthesis and concentration, cell cycle phase and methylation status of both fragment and recipient genome. In addition, different drugs were employed to test their ability to improve technique efficiency. SFHR-mediated genomic modification resulted to be stably transmitted for several cell generations and confirmed at transcript and genomic levels. Modification efficiency was estimated in a range of 0.01–0.5%, further increasing when PARP-1 repair pathway was inhibited. In this study, for the first time SFHR efficiency issue was systematically approached and in part addressed, therefore opening new potential therapeutic ex-vivo applications.

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DNA Replication, cell cycle progression and the targeted gene repair reaction

Cited by 38 publications

References 44 publications

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

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

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

Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System

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