Engineering Synthetic Chromosomes by Sequential Loading of Multiple Genomic Payloads over 100 Kilobase Pairs in Size

Abstract

Gene delivery vehicles currently in the clinic for treatment of monogenic disorders lack sufficient carrying capacity to efficiently address complex polygenic diseases. Thus, to engineer multifaceted genetic circuits for bioengineering human cells as a therapeutic option for polygenic diseases, we require new tools that are currently in their infancy. Mammalian artificial chromosomes, or synthetic chromosomes, represent a viable approach for delivery of large genetic payloads that are mitotically stable and remain independent of the host genome. Previously, we described a mammalian synthetic chromosome platform, termed the ACE system, that requires a single unidirectional integrase for the introduction of multiple genes onto the ACE platform chromosome. In this report, we provide a proof of concept that the ACE synthetic chromosome bioengineering platform is amenable to sequential delivery of off-the-shelf large genomic fragments. Specifically, large genomic clones spanning the human solute carrier family 2, facilitated glucose transporter member 1 ( SLC2A1 or GLUT1 , 169 kbp), and human monocarboxylate transporter 1 ( SLC16A1 or MCT1 , 144 kbp) genetic loci were engineered onto the ACE platform and demonstrated to express and correctly splice both gene transcripts. Thus, the ACE system provides a facile and tractable engineering platform for the development of gene-based therapeutic agents targeting polygenic diseases.

Building a community to engineer synthetic cells and organelles from the bottom-up

“…A minimal synthetic cell consisting of only 473 genes, capable of metabolizing and reproducing, has been constructed using a top-down approach ( Hutchison et al, 2016 ). With the objective of engineering a minimal synthetic cell synthetic chromosomes have been designed to generate artificial genetic blueprints for operating synthetic cell systems ( Greene et al, 2019 ). In addition, droplet-based synthetic cells with an artificial photosynthetic metabolism that can bind CO 2 have been created, and synthetic cell systems for the scalable bio-production of natural plant products have also been built ( Miller et al, 2020 ).…”

Synthetic biology tools for engineering Goodwin oscillation in Trypanosoma brucei brucei

“…Most synthetic biology research to date utilises the three archetypal workhorse cells of biotechnology: Escherichia coli ( E. coli ), Saccharomyces cerevisiae and immortalised mammalian cells ( Saito and Yokobayashi 2019 ), in anticipation of rapid industrial application. While alternative bacterial ( Hoff et al., 2020 ) and budding yeast host cells ( Henríquez et al., 2020 ) continue to be exploited by synthetic biologists, longer term goals include the eventual de novo design of genomes to control bacterial ( Robertson et al., 2021 ), yeast ( Ong et al., 2021 ) and even mammalian ( Greene et al., 2019 ) cellular ‘chassis’. Against this background the protozoa present an interesting opportunity for synthetic biologists.…”