Design and Construction of Bacterial Genomes at the Megabase-Scale

Author: Sanfiorenzo, Charles John

Year: 2024

Degree: Dissertation (Ph.D.)

Advisor: Wang, Kaihang

Committee Members: Goentoro, Lea A.; Newman, Dianne K.; Hay, Bruce A.; Wang, Kaihang

Option: Biology

Abstract

Building genomic chimeras would enable melding of the diverse functions and properties of life. However, prior arts in genome synthesis are limited to reconstituting functions within singular genomes rather than combining diverse genomic functions across multiple distinct genomes. Existing methods are also prohibitively expensive, laborious, time-consuming, and not scalable for creating large genomes. Addressing these limitations, the author invented Additive Conjugative-CAST Engineering (ACE) combining conjugation with CRISPR-associated transposition (CAST) to deliver and integrate up to half a genome per step from a donor into a precisely defined position in the recipient’s genome. This work demonstrates ACE’s engineering capacities integrating a 2-megabase donor genomic segment in a single step and at least three megabase in two steps. Importantly, ACE’s generality is confirmed through the creation of genomic chimeras across species, genus, order, and class barriers. With ACE, this work further showcases that such chimeric organisms, denoted genome expanded organisms (GEOs), can be forged from at least three starting bacterial strains, and are stably maintained to express all acquired genomic parts. Principles confounding ACE are expanded onto genome editing technologies, such as Prime Editing, and further explored for the megabase-scale transfer of DNA into eukaryotes. ACE and derivative technologies thereof offer a new paradigm of creating artificial lifeforms to combine and potentially create novel functions beyond the constraints of nature, while probing and elucidating genome plasticity, architecture, and expression patterns of GEOs.