Characterization and Engineering of Transposons for Genome Editing

Abstract

Since the human genome was sequenced in 2000, we have rapidly elucidated previously unappreciated associations between our genetic code and human disease. Driven by the rapid development of new molecular technologies, there has been increased desire to treat these genetically rooted diseases by directly manipulating DNA. Using genome editing tools, primarily based on bacterial CRISPR systems, scientists can treat the genome like a text file, adding, deleting, or changing small stretches of DNA. However, the ability to cut-and-paste large fragments of DNA into a defined location in the genome has remained elusive. In the first part of this thesis, we characterize and engineer multiple genome editing systems to address the problem of DNA insertion and, more broadly, problems in human health.

First, we functionally characterize a system of unknown function, a type V-K CRISPR-associated transposase from the cyanobacteria Scytonema hofmanni (ShCAST). We demonstrate that ShCAST performs self-sufficient targeted DNA insertion and can be reconstituted in bacteria for genome editing with efficiency of up to 80% without selection. We then go on to characterize transposon homing, a key mechanism in the natural lifecycle of CAST systems, which enables these mobile elements to navigate to the sites in which they are found. We show that type V-K systems use a non-canonical CRISPR RNA (crRNA) to perform this task. Surprisingly, the distinct type I-B CAST uses a dedicated sequence-specific DNA binding protein for homing to an attachment site.

Next, we engineer a non-long terminal repeat (LTR) retrotransposon, R2 from the silkworm Bombyx mori (R2bm), as a site-specific DNA insertion tool in human cells. We show that R2bm can be used to perform DNA insertion into human 28S ribosomal DNA (rDNA) repeats and validate this strategy for delivering a functional transgene. We also demonstrate that R2bm’s target site can be changed through association with a reprogrammable DNA-binding protein like SpCas9, enabling functional correction of a truncated protein in the human genome.

In early 2020, while we were harnessing R2 for DNA insertion, the COVID-19 pandemic broke out and general laboratory activities were shut down. In the second part of this thesis, we develop a new chemistry for detection of SARS-CoV-2 called STOPCovid to address the urgent need for diagnostics in the COVID-19 pandemic. STOPCovid aims to simplify liquid handling for sensitive nucleic acid detection in low-complexity and point-of-care settings. We demonstrate that STOPCovid has a sensitivity of 93.1% and specificity of 98.5% using over 400 patient samples.