Browsing by Author "Rivera-Torres, Natalia"
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Item CRISPR directed targeted gene alteration: mechanism to application(University of Delaware, 2019) Rivera-Torres, NataliaSingle base mutations can be repaired by introducing single stranded DNA oligonucleotides (ssODN) into a target cell. The frequency at which this occurs is dependent on several of factors: the length of ssODN, the position of the cell in its proliferative cycle, and the presence of double-stranded DNA breaks in the host genome. Genome editing offers a promising strategy for gene repair and correction by overcoming difficulties associated with lack of precision. CRISPR/Cas has increased the pace and lowered the cost of research, allowing the genetic manipulation even in organisms that have historically been difficult to modify. Furthermore, the combinatorial approach uniting ssODNs and CRISPR/Cas9 has emerged as a feasible therapeutic approach. In the work presented in this dissertation I focused on the mechanism and application of gene editing utilizing CRISPR systems. I tested combinatorial approach of utilizing CRISPR/Cas9 system along with ssODN to promote single base pair correction and demonstrate it is now possible to direct single nucleotide exchange in efficient manner. We find that both insertions and deletions accompany single base repair as result from allelic analysis of clonally expanded cell populations. CRISPR/Cas9 and single-stranded oligonucleotide donor DNA molecules working in tandem can lead to the precise repair of the point mutation in the eGFP gene, and led to propose a new model for the repair of point mutations, a process we have termed ExACT. The relationship between transfection efficiency and gene editing activity was tested and analyzed based on experimental and visual data and found that there is no direct correlation between efficient cellular uptake and genome modification directed by an RNP. By understanding the mechanisms by which CRISPR/Cas executes gene editing in human cells, a more efficacious and potential approach to drug development could be undertaken. The application of the CRISPR gene editing system in two different approaches to study pediatric Leukemia was explored. (1) pediatric patient specific ALL chromosomal translocation (4:11)(q21:q23) was re-created by utilizing the CRISPR/Cas9 system in HEK293 cells. This led to the development of a convenient platform for rapid modeling of cancer-related genetic mutations in vitro. (2) Implemented the use of a novel gene editing approach to create expression vectors that harbor patient specific mutations that were tested against TKI. We have developed a diagnostic system to monitor the impact of mutant FLT3 ITDs on the progression of oncogenesis and to evaluate the efficacy of novel AML drugs.Item Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides(Public Library of Science, 2017-01-04) Rivera-Torres, Natalia; Banas, Kelly; Bialk, Pawel; Bloh, Kevin M.; Kmiec, Eric B.; Kelly Banas; Pawel Bialk; Kevin M. Bloh; Eric B. Kmiec; Natalia Rivera-Torres; Kmiec, EricCRISPR/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