CRISPR Directed Targeted Gene Alteration: Mechanism to Application

Single 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