Date of Award
Fall 12-4-2019
Track
Biochemistry
Degree Type
Thesis
Degree Name
Master of Science in Chemical Sciences (MSCB)
Department
Chemistry
Committee Chair/First Advisor
Dr. Jonathan L. McMurry
Committee Member
Dr. Daniel P. Morris
Committee Member
Dr. Susan M.E. Smith
Abstract
Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) complexes are becoming the preferred technique for genomic editing because of their precision, efficiency and accuracy. Prokaryotes naturally use CRISPR/Cas complexes to detect and eliminate foreign nucleic acids after infections. Cas proteins form complexes with RNA to cause double-stranded breaks (DSBs) in the invader’s sequence. Researchers are trying to use CRISPR complexes to treat many human diseases caused by these mutations. Research using CRISPR/Cas systems in the treatments of many of the genetic diseases has resulted in misfortunes (such as toxicity to the patient or new diseases) because of the method of delivery such as viral delivery.
The current work attempted to delivery Cas proteins complexed with RNA to the nuclei of living eukaryotic cells using a novel cell-penetrating peptide (CPP)-adaptor system. Since the discovery of CPPs in the 1980s, researchers have used CPPs to deliver biomolecules such as proteins and nucleic acids into cells (the delivered biomolecules are called cargos). Researchers encountered the problem of endosomal entrapment: the CPP-cargos remained trapped in endosomes likely because of CPP-receptor interactions. To solve this problem, a novel CPP adaptor, TAT-CaM was developed. TAT-CaM fuses the CPP from HIV TAT to human calmodulin (CaM) in one polypeptide. When Ca2+ is present, TAT-CaM binds to an engineered or endogenous calmodulin-binding site (CBS) on cargo proteins. After endocytosis, TAT-CaM dissociates from cargos as Ca2+ concentrations drop during trafficking. Like other CPPs, TAT-CaM remains trapped in endosomes, but cargos escape efficiently.
Here, we use biolayer interferometry (BLI) and confocal microscopy to show Ca2+-dependent binding and delivery of CRISPR complexes to the nuclei of cells. Donor DNA sequences were also delivered to the nucleus of the cell. The donor DNA contained a biotin tag that bound to CBS-tamavidin, an avidin from the mushroom Pleurotus cornucopia with a high affinity for biotin. CBS-tamavidin bound to both TAT-CaM and biotinylated donor DNA during delivery to living cells. Ultimately, we aim to deliver CRISPR complexes and donor DNA simultaneously delivered to the cell nucleus, where the cell’s homologous recombination machinery can use the donor DNA sequences to repair the DSBs break.