Invention and development of programmable base-to-kilobase DNA writing and integration technologies
Name
tou-ctou-phd-be-2025-thesis.pdf
Size
22.46 MB
Format
Adobe PDF
Checksum (MD5)
68cbe8ed1a2614311f7397ae3fb26e5e
Author(s)
Tou, Connor J.
Advisor(s)
Collins, James J.
Kleinstiver, Benjamin P.
Date Issued
May 2025
Publisher
Massachusetts Institute of Technology
Abstract
Programmable genome editing technologies are revolutionizing biological applications across medicine, agriculture, and industry. In this thesis, we leverage diverse enzymatic functions and mechanisms inherent to distinct single-stranded DNA (ssDNA) viruses and mobile genetic elements (MGEs) to invent three molecular approaches which enable precise and programmable modification of genomes in bacteria, mammalian cells, and animals.
First, we developed click editing, a new genome editing method that enables genome writing of nearly any nucleotide-scale change into a genome at user-defined target sites. Click editors (CEs) couple the unique capability of HUH endonucleases to perform protein-ssDNA attachment with the advantageous properties of CRISPR-guided nickases and DNA-dependent DNA polymerases.
While existing technologies capable of performing small DNA changes are well suited to correct the root cause of many genetic diseases, most genetic diseases are caused by heterogeneous genetic mutations spread across a gene. This imposes a major therapeutic bottleneck because the optimization of bespoke genome editors for hundreds or thousands of mutations is impractical and cannot be scaled to all patients. As a tantalizing alternative, targeted DNA integration could deliver a curative, mutation-agnostic strategy that benefits from endogenous gene regulation.
To realize this potential, we developed three systems for targeted DNA integration: (1) Second-generation click editors (CE2) that more efficiently write longer sequences into the genome, facilitating their use with site-specific recombinases for programmable DNA integration; (2) Engineered CRISPR-associated transposase systems (termed HELIX) which perform RNA-programmable DNA integration with high product purity and specificity in bacteria and human cells; and (3) a new genome editing method termed INSTALL, which combines immune-evasive DNA donors and recombinases to enable non-viral DNA insertion in human cells and animals.
Together, CEs, HELIX, and INSTALL are emergent approaches for programmable genome editing from the single DNA base to the multi-kilobase scale, establishing proof-of-concept platforms for personalized and generalized genetic medicine that overcome the limitations of current approaches.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Terms of Use
In Copyright - Educational Use Permitted
Copyright retained by author(s)
Persistent DSpace Link
