Genomic rearrangements are an important driving force for the evolution of organisms and a key factor leading to complex diseases such as cancer. The forms of genomic rearrangements include deletion, duplication, insertion, inversion and translocation that can be achieved by changes in gene copy number, perturbation of protein coding sequences as well as cis-regulatory network. Previous studies have shown that genome rearrangements are prevalent in all living systems, from prokaryotes to humans, and that species can evolve several orders of magnitude faster than base mutations. The implantation of the SCRaMbLE system is an important design in the Yeast Genome Synthesis Program (Sc 2.0)1, which has been proved as an effective method for achieving synthetic genome group rearrangements in yeast, and has been applied in many research fields such as conditional tolerance strain development, natural product production and genome streamlining2. However, because yeast containing all synthetic chromosomes have not yet been constructed, SCRaMbLE rearrangements on a genome-wide scale can not be achieved. Moreover, previous studies have shown that rearrangement events within a single chromosome and between multiple chromosomes show large differences in SCRaMbLE rearrangements in strains containing single or multiple synthetic chromosomes3,4. However, there is no good model for studying interchromosomal rearrangements.
On January 26, 2024, the research team led by Dai Junbiao with Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, published an online research paper entitled “Large-scale genomic rearrangements boost SCRaMbLE in Saccharomyces cerevisiae” in the journal Nature Communications. Dozens of loxPsym sites were introduced in the yeast genome-wide by the CRISPR system to construct a yeast system available for whole-chromosome rearrangements, which laid a foundation for studying the impact of genome structural variation on environmental adaptability and evolution of the strains. Studies revealed that structural variation in large segments could cause changes in gene transcription level and 3D genome structure, creating phenotypes that were more tolerant to stress. Further studies revealed that after hybridization with strains containing synthetic chromosomes, the heterozygous diploid strains after introduction of many loxPsym sites showed faster adaptive evolution under acetate conditions than diploid strains containing only synthetic chromosomes.
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Link: https: / / www.nature.com/articles/s41467-023-44511-5
In the research, the researchers first used the CRISPR / Cas9 system to insert 83 loxPsym sites in a scattered manner across 16 chromosomes in S. cerevisiae, with each chromosome containing at least 2 sites. They named this engineered strain as SparLox 83 and confirmed that the insertion of the 83 loxPsym sites did not affect the growth and genome stability of the strains.
To improve the screening efficiency of the rearranged strains, the researchers introduced the previously reported rearranged strain screening system - ReSCuES system in the SparLox 83 strain5, and obtained the engineered strain, SparLox 83R. At the same time, given the presence of essential genes between every two loxPsym sites in SparLox83R, the deletion would cause strain lethality, so the researchers hybridized SparLox83R and wild-type strain BY 4742 to form heterozygous diploids. In SparLox83R and its heterozygous diploid with wild type after five rounds of SCRaMbLE, the researchers found that haploid cell rearrangement rate (about 30%) was significantly higher than that of heterozygous diploid (about 3%). In the mixture of two genotypes, there were more interchromosomal rearrangement events than chromosome rearrangement events. This result was different from the previous studies with synthetic chromosome as the object of SCRaMbLE. Meanwhile, the researchers discovered that the frequency of rearrangements varied greatly between different loxPsym sites. Through the quantitative analysis of the changes, it was finally revealed that the distance between the two sites and the openness of chromatin had an impact on the frequency of rearrangements.
Large segment rearrangements can drive phenotypic diversification and environmental fitness, but it remains challenging to distinguish the impacts of large segment rearrangements and other types of mutations on adaptability in naturally evolved strains. Taking SparLox83R as the object, the researchers induced its rearrangements, and screened for strains with increased tolerance under multiple drug stress conditions. Later, the researchers sequenced a strain with enhanced tolerance to microtubule depolymerization drugs and analyzed the transcriptome and spatial three-dimensional genome (Hi-C), to interpret the mechanisms associated with structural variation and drug resistance in chromosomes 4, 19 and 14.
Further, to explore whether large segment rearrangements mediated by scattered loxPsym sites could increase the rate of directed evolution of synthetic chromosomes, SparLox83R was hybridized with strains containing synthetic chromosomes to induce SCRaMbLE. Sequencing revealed that scattered loxPsym sites in SparLox83R can produce multiple types of rearrangements together with loxPsym sites on synthetic chromosomes, including structural variants from synthetic chromosomes and SparLox83R, and resulting in loss of heterozygosity and aneuploidy with a high frequency.
To confirm that these rearrangements can provide driving force for strain evolution, the researchers compared two different heterozygous diploid strains: the diploid (JDY544) formed from hybridization between SparLox83R and synthetic chromosome 3 strains and the diploid (JDY546) formed from hybridization between wild type haploid strains and synthetic chromosome 3 under acetic acid stress. After induced rearrangement of 48 independent populations and growth detection on acetic acid plates, it was revealed that after three rounds of evolution, all 48 populations of JDY544 could grow under high concentrations of acetic acid (0.7%), while only a few populations of JDY546 could grow under such conditions. This result indicated that the population from JDY544 was able to produce strains with acetate tolerance faster than JDY546, implying that the rearrangements mediated by loxPsym sites scattered in SparLox83R may be associated with a rapid enhancement of acetate tolerance.
In conclusion, this study has constructed an engineered yeast strain containing 83 loxPsym sites distributed on 16 chromosomes. Taking them as the research object, through high-throughput sequencing and chromosome structure analysis of the strains obtained after SCRaMbLE, it proved that the yeast strain with genome-wide distribution loxPsym sites can be used as a powerful tool for studying genome rearrangements, and can drive the rapid evolution of strains and achieve environmental stress tolerance. The methods provided by this study also provide ideas for applying the SCRaMbLE system to other engineered strains or species, thus greatly expanding the application scope of the SCRaMbLE system.
Cheng Li, an assistant researcher with Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, PhD candidate Zhao Shijun, post-doctoral fellow Li Tianyi and PhD graduate Hou Sha are first authors of the paper. Professor Junbiao Dai is the corresponding author of the paper. The research was supported by programs such as the National Key Research and Development Program, National Natural Science Foundation of China, Guangdong Basic and Applied Research Foundation, Great Science Program of Chinese Academy of Sciences, Shenzhen Science and Technology Program, Innovation Program of Chinese Academy of Agricultural Sciences and Shenzhen Institute of Synthetic Biology.
References:
1. Dymond, J.S.et al.Synthetic chromosome arms function in yeast and generate phenotypic diversity by design.Nature 477, 471–476 (2011).
2. Steensels, J., Gorkovskiy, A.& Verstrepen, K.J.SCRaMbLEing to understand and exploit structural variation in genomes.Nat.Commun.9, 9–11 (2018).
3. Zhang, H.et al.Systematic dissection of key factors governing recombination outcomes by GCE-SCRaMbLE.Nat.Commun.13, 5836 (2022).
4. Zhou, S.et al.Dynamics of synthetic yeast chromosome evolution shaped by hierarchical chromatin organization.Natl.Sci.Rev.10, (2023).
5. Luo, Z.et al.Identifying and characterizing SCRaMbLEd synthetic yeast using ReSCuES.Nat.Commun.9, 1–10 (2018).
