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

Rivera-Torres, Natalia; Strouse, Bryan; Bialk, Pawel; Niamat, Rohina A.; Kmiec, Eric B.

doi:10.1371/journal.pone.0096483

Cited by 19 publications

(26 citation statements)

References 45 publications

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“…This specific combination of CRISPR/Cas9 and ssODN has been shown previously to be optimal for RGEN-directed correction [14]. In the experiments reported herein, we utilize the 72NT oligonucleotide since previous data have established that targeting the non-transcribed strand at this ssODN length leads to a higher level of gene editing [31]. In addition, the use of the complementary oligonucleotide, targeting the transcribed strand, leads to artefactual annealing to the sgRNA component of the CRISPR/Cas9 complex, reducing overall activity [14].…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…The efficiency of the corrective change depends on several of factors (i.e., the length of the ssODN, the proliferative cell cycle, and the presence of DNA breaks in the host genome) [29, 30]. Recently, a single base change on the genome has been achieved through a combined use of the TALEN and ssODN templates [16, 17]. In the present study, we applied the TALEN protein delivery system for a specific base change in two systems (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…However, a significant challenge of this method is the low efficiency, requiring the use of a selection marker or screening a large number of subclones [15]. Recently, precise genome editing was achieved through a combined use of the TALEN and single‐stranded oligonucleotide DNA (ssODN) templates [16, 17]. In the present study, we combined the method of bacterial T3SS‐mediated TALEN protein injection and ssODN template transfection, providing an effective alternative method for precise DNA modification in stem cells.…”

Section: Introductionmentioning

confidence: 99%

Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins

Jia

Bai

Jin

et al. 2015

Stem Cells Translational Medicine

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The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies.

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“…A number of laboratories reported that treatment of cells with anticancer drugs such as camptothecin, which induces double‐strand breaks in chromosomal DNA, elevates the frequency of gene editing directed by ssODNs (Ferrara and Kmiec, ; Ferrara et al ., ; Olsen et al ., ; Storici et al ., ; Wang et al ., ). In effect, these drugs were used as adjuvants, pretreating cells prior to the addition of oligonucleotides and thereby elevating the number of gene repair events in a particular cell.…”

Section: Dna Damage and Dna Replication In Gene Editingmentioning

confidence: 97%

Genetic spell‐checking: gene editing using single‐stranded DNA oligonucleotides

Rivera-Torres

Kmiec

2015

Plant Biotechnology Journal

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SummarySingle-stranded oligonucleotides (ssODNs) can be used to direct the exchange of a single nucleotide or the repair of a single base within the coding region of a gene in a process that is known, generically, as gene editing. These molecules are composed of either all DNA residues or a mixture of RNA and DNA bases and utilize inherent metabolic functions to execute the genetic alteration within the context of a chromosome. The mechanism of action of gene editing is now being elucidated as well as an understanding of its regulatory circuitry, work that has been particularly important in establishing a foundation for designing effective gene editing strategies in plants. Double-strand DNA breakage and the activation of the DNA damage response pathway play key roles in determining the frequency with which gene editing activity takes place. Cellular regulators respond to such damage and their action impacts the success or failure of a particular nucleotide exchange reaction. A consequence of such activation is the natural slowing of replication fork progression, which naturally creates a more open chromatin configuration, thereby increasing access of the oligonucleotide to the DNA template. Herein, how critical reaction parameters influence the effectiveness of gene editing is discussed. Functional interrelationships between DNA damage, the activation of DNA response pathways and the stalling of replication forks are presented in detail as potential targets for increasing the frequency of gene editing by ssODNs in plants and plant cells.

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

Cited by 19 publications

References 45 publications

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

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

Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins

Genetic spell‐checking: gene editing using single‐stranded DNA oligonucleotides

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