Guidescan Software For Improved Single And Paired Crispr Guide Rna Design Coupled With Computational Studies In Leukemia

CRISPR technology has revolutionized the field of genome engineering. CRISPR allows for the easy and efficient manipulation of virtually any genetic locus through a two-component system: a CRISPR endonuclease and guide RNA (sgRNA). These components form a complex that enacts double strand breaks in target DNA. The repair of the double strand break is the main mechanism by which genetic editing of a locus takes place. While the endonuclease cleaves target DNA, it is the sgRNA that specifies targets through complementary binding to a target site. Determining the specificity of sgRNAs to their target site represented a crucial challenge to the genome-engineering field. To facilitate the design of CRISPR libraries, we developed Guidescan, a software package that allowed for the customizable production of sgRNA databases that were guaranteed to match user-defined requirements for sgRNA uniqueness. Furthermore, several computational studies of leukemia are described in this thesis that illustrate different molecular actors and mechanisms through which a leukemic like disease, Myelodysplastic Syndrome, can progress towards leukemia, how leukemia hijacks a splicing protein to maintain its pathology, and finally, how a leukemia can develop resistance to a targeted therapy

Targeting the non-coding genome and temozolomide signature enables CRISPR-mediated glioma oncolysis

Summary: Glioblastoma (GBM) is the most common lethal primary brain cancer in adults. Despite treatment regimens including surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy, growth of residual tumor leads to therapy resistance and death. At recurrence, a quarter to a third of all gliomas have hypermutated genomes, with mutational burdens orders of magnitude greater than in normal tissue. Here, we quantified the mutational landscape progression in a patient’s primary and recurrent GBM, and we uncovered Cas9-targetable repeat elements. We show that CRISPR-mediated targeting of highly repetitive loci enables rapid elimination of GBM cells, an approach we term “genome shredding.” Importantly, in the patient’s recurrent GBM, we identified unique repeat sequences with TMZ mutational signature and demonstrated that their CRISPR targeting enables cancer-specific cell ablation. “Cancer shredding” leverages the non-coding genome and therapy-induced mutational signatures for targeted GBM cell depletion and provides an innovative paradigm to develop treatments for hypermutated glioma