Sc3.0: revamping and minimizing the yeast genome

Abstract

Recent improvements in DNA synthesis and editing techniques enable engineering the entire genome of an organism, offering new tools to directly probe relationships between genotype and phenotype. Genome synthesis potentially allows the researchers to gain a much greater degree of control of an organism, and it also leads to a completely new way to understand the biology of genomes. In 2008, the first mega-size bacteria genome was built from oligonucleotides [1]. Next, the 4-Mb genome of E. coli was redesigned and engineered [2, 3]. More recently, the synthesis of first eukaryotic genome, the 12-Mb Saccharomyces cerevisiae genome, is nearing completion as the goal of the Sc2.0 initiative [4] and Genome Project-Write (GP-Write) has been proposed to engineer higher eukaryotes with gigabase-sized genomes [5]. As genome sizes increase, the design principles of synthetic genomes are becoming more sophisticated and complex. In the first synthetic genome, the Mycoplasma genome, only few watermarks were introduced [1]. The nearly completed Sc2.0 project involves building a genome that is heavily modified [4]. These modifications include the removal of all retrotransposons, subtelomeric repeats, and introns; eliminating and relocating all tRNA genes; swapping all TAG stop codon to TAA; and introducing numerous PCRTags (a type of watermark) by synonymous recoding of coding sequences. More importantly, over 4000 LoxPSym sites need to be inserted in the 3′ UTR of all non-essential genes, as well as at synthetic "landmarks," a system designated as "synthetic chromosome rearrangement and modification by loxP-mediated evolution" (SCRaMbLE [6]). Overall, the native genome will be reduced in size by about 8% with an aim to reduce genomic contents and stabilize the genome, while still maintaining similar 3D structures and functions as wild-type chromosomes. One thing we learned while constructing new chromosomes is that despite the variety of changes introduced, cells are quite tolerant to these perturbations. For example, the relocation of the megabase-size, highly repetitive ribosomal gene cluster on chrXII to a much smaller chromosome, chrIII, conferred only very minor, if any, effects on cell growth [7]. These results lead us to propose a new hypothesis that the yeast genome contains a larger variety of redundant elements. Therefore, more radical changes might be introduced to generate a much more compact genome. Here, we present a proposal to design and synthesize the next version of the synthetic yeast genome, dubbed Sc3.0.

Addressing Evolutionary Questions with Synthetic Biology

“…So far, the synthesis of six (out of 16) synthetic Sc2.0 chromosomes has been published and we expect soon the publication of the whole genome (Richardson et al 2017;Kannan & Gibson 2017). The next version of a synthetic yeast genome (Sc3.0) is also already in planning (Dai et al 2020) with the aim for further compacting the synthetic chromosomes (Z. Luo et al 2020).…”

SCRaMbLE: A Study of Its Robustness and Challenges through Enhancement of Hygromycin B Resistance in a Semi-Synthetic Yeast

“…Rearrangements in the NGMLR mapping data were called using Sniffles (v1.0.11) [ 46 ] with a threshold of ≥10 reads confirming the rearrangement. De novo genome assembly of each strain was performed using Canu (v1.8) [ 47 ]. SCRaMbLE rearrangements in synII were evaluated and confirmed by comparing the mapping and variant calling data from Sniffles with the de novo assembly obtained by Canu [ 47 ].…”

“…De novo genome assembly of each strain was performed using Canu (v1.8) [ 47 ]. SCRaMbLE rearrangements in synII were evaluated and confirmed by comparing the mapping and variant calling data from Sniffles with the de novo assembly obtained by Canu [ 47 ].…”

Genetic Engineering and Synthetic Genomics in Yeast to Understand Life and Boost Biotechnology

“…This may result in the possibility that the optimal genotype in a certain SCRaMbLE experiment cannot be obtained. However, there is a new approach to circumvent this limitation by concatenating the essential genes of the synthetic chromosome on a “non-SCRaMbLEable” centromeric plasmid [ 105 ]. This additional copy of essential genes now allows for the loss of the essential rafts of the synthetic chromosomes and increases the number of combinatorial possibilities which can be obtained by SCRaMbLE.…”

“…Intermediate versions, where the synthetic genome is reduced, altered or extended according to the Sc2.0 rules or with new, additional rules, have great potential for future application. How Sc3.0 would look remains unclear, however, recently, a perspective article by Junbiao Dai and others [ 105 ] pointed out a minimised version of yeast with major changes in the genetic code might be the Sc3.0 project. One should keep in mind the intermediate versions from Sc2.0 to Sc3.0 have the potential to answer questions in basic and applied research.…”