Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biochemistry

Supervisor

Edgell, David R.

Abstract

We created a novel dual endonuclease called TevCas12a. TevCas12a is a fusion of the I-TevI nuclease and linker domains to Cas12a. The goal was to create a dual endonuclease that made precise double-strand breaks leaving non-compatible ends for exogenous DNA insertion and specific deletions. TevCas12a was expected to make two double-strand breaks, one by I-TevI and the other from Cas12a. However, when tested in vitro, TevCas12a cut the DNA substrate multiple times, rapidly degrading the substrate. We call this activity permissive cleavage. TevCas12a activity is guide RNA dependent, but not guide RNA sequence specific, it can degrade any DNA substrate with a non-targeted guide RNA. Sequencing of the TevCas12a cleavage products with Oxford Nanopore revealed that most of the non-guide directed cuts were within I-TevI’s cleavage motif, indicating that I-TevI is responsible for the cuts. A mechanism is proposed for how I-TevI and Cas12a work together to permissively cleave DNA.

Summary for Lay Audience

CRISPR is a bacterial immune system which has been adapted to be used as a genomic editing tool. CRISPR has many different Cas proteins which act as molecular scissors to cut DNA and make a double-strand break. However, mammalian cells are good at repairing breaks in their DNA which allows the Cas protein to cut again and begins a cycle of cleavage and repair. I made a new protein called TevCas12a which is the fusion of the CRISPR protein Cas12a to another protein I-TevI which also acts as molecular scissors. Together, TevCas12a was predicted to cut the DNA twice (once from Cas12a and once from I-TevI) to create two non-compatible double-strand breaks, since they each cut DNA differently. This would stop the cell from repairing the single cut made with Cas and with the two different ends from TevCas12a, new DNA could be inserted into the cell.

When I tested TevCas12a against a DNA substrate, I found that it did not cut twice as I expected, instead it cut the DNA multiple times degrading it into smaller and smaller pieces. Neither Cas12a nor I-TevI on their own are able to repeatedly cut DNA like TevCas12a can. My thesis sought to understand the mechanism of cleavage activity for TevCas12a that allows it to degrade DNA. TevCas12a does not randomly cut the DNA, it cleaves at very specific sequences. I-TevI cleaves at a specific pattern in the DNA and by sequencing TevCas12a cleavage products, I found that most of them were cut in I-TevI’s cleavage site. This indicates that I-TevI is responsible for making these specific cuts which degrade the DNA. I also proposed a mechanism for how I-TevI and Cas12a work together to be able to degrade DNA.

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