Jef Boeke, PhD, DSc; Writing: Genomic Innovation and Precision Medicine Conference: April 5 2022

Jef D. Boeke, PhD, DSc

Professor, Department of Biochemistry and Molecular Pharmacology, NYU Grossman School of Medicine; Sol and Judith Bergstein Director, Institute of System Genetics

Tormenting Genes and Genomes

ABSTRACT

Rapid advances in DNA synthesis techniques have made it possible to engineer diverse genomic elements, pathways, and whole genomes, providing new insights into design and analysis of systems. The synthetic yeast genome project, Sc2.0, is well on its way with the 16 synthetic Saccharomyces cerevisiae chromosomes now completed by a global team. The synthetic genome features several systemic modifications, including TAG/TAA stop-codon swaps, deletion of subtelomeric regions, introns, tRNA genes, transposons, and silent mating loci. Strategically placed loxPsym sites enable genome restructuring using an inducible evolution system termed SCRaMbLE, which can generate millions of derived variant genomes with predictable structures leading to complex genotypes and phenotypes. The fully synthetic yeast genome provides a new kind of combinatorial genetics based on variations in gene content and copy number. Synthetic chromosome IV is the largest in the genome in terms of bp synthesized at over 1.4Mb. We have created an “inside out” version of this chromosome. Remarkably, the 3D structures of synthetic and native chromosomes are very similar despite the substantial number of changes introduced.

Chromosome I is the smallest S. cerevisiae chromosome, and anticipating issues of instability related to its small size, we decided to fuse it to other chromosomes–and were surprised by how easy it was to do this. This led to larger questions about whether it would be possibly to radically reduce chromosome number by continuing to fuse chromosomes. We completely reengineered the yeast karyotype, by systematically fusing pairs of telomeres and deleting single centromeres, generating an isogenic series of yeast ranging from n=16 to n=2. These strains show reproductive isolation and a massively altered 3D genome structure but are surprisingly “normal” and show high fitness. We have also developed a method that allows us to move megabase segments to distant locations in the genome in a single step, again, with surprisingly little impact on fitness.

Yet another form of genome torment is switching up the protein packaging of DNA. The substitution of human for yeast nucleosomes leads to a number of unexpected transcriptional and other phenotypes.

Last but not least, we reconfigured yeast as an efficient platform for the assembly of 100kb to megabase native mammalian genes or gene clusters. We are now enabled to rapidly deliver these genes and their many variants to embryonic stem cells and mice. This opens many avenues for tormenting human genes . . .