Iterative SCRaMbLE for engineering synthetic genome modules and chromosomes

Abstract

Saccharomyces cerevisiae is closing-in on the first synthetic eukaryotic genome with genome-wide redesigns, including LoxPsym site insertions that enable inducible genomic rearrangements in vivo via Cre recombinase through SCRaMbLE (Synthetic Chromosome Recombination and Modification by LoxPsym-mediated Evolution). Combined with selection, SCRaMbLE quickly generates phenotype-enhanced strains by diversifying gene arrangement and content. Here, we demonstrate how iterative cycles of SCRaMbLE reorganises synthetic genome modules and chromosomes to improve functions. We introduce SCOUT (SCRaMbLE Continuous Output and Universal Tracker), a reporter system that allows sorting of SCRaMbLEd cells into high-diversity pools. Paired with long-read sequencing, SCOUT enables high-throughput mapping of genotype abundance and genotype-phenotype relationships. Iterative SCRaMbLE is applied here to yeast strains with a full synthetic chromosome and histidine biosynthesis modules. Five HIS module designs are tested, and SCRaMbLE is used to optimise the poorest performer. Our results highlight iterative SCRaMbLE as a powerful tool for data driven modular genome design.

Fig. 1. Design and construction of histidine genome modules.

A Metabolic pathway of L-histidine biosynthesis from 5-phospho-α-D-ribose 1-diphosphate (PRPP) in S. cerevisiae. Phosphoribosyl-ATP, phosphoribosyl-AMP, phosphoribosylformiminoAICAR-phosphate, phosphoribulosylformiminoAICAR-phosphate, imidazole glycerol phosphate, imidazole acetol-phosphate are shown as their abbreviations “PR-ATP”, “PR-AMP”, “Pro-FAR”, “PR-FAR”, “IGP”, “IAP”, respectively. B Schematic overview of the relocation of HIS genes into a synthetic module by defragmentation and refactoring. Seven HIS genes, catalysing 10 steps of reactions in the histidine biosynthesis pathway, namely HIS1 to HIS7, including their native regulatory sequences, were partially removed from their native genomic loci and relocated as a module at the URA3 locus. Genes are labelled in distinct colours. White squares labelled on chromosomes represent deletion of seven HIS genes from their native loci and replacement with a 23 bp ‘landing pad’ which contains a unique CRISPR/Cas9 targeting sequence. C Schematic process of the defragmented and refactored HIS module assembly. For defragmentation, gene fragments containing the native promoter (1 kb region upstream of the gene), CDS and 3′UTR (500 bp region downstream of the gene) of each HIS gene were amplified from the BY4741 genomic DNA and then constructed into entry-level plasmids by Gibson assembly. The linker plasmids were constructed by inserting a loxPsym sequence into the synthetic connectors (ConL and ConR) from the YTK. For refactoring, the CDS of each HIS gene was firstly constructed into an entry-level plasmid as a synthetic ‘part’ that is compatible for YTK assembly. Next, the YTK promoter, terminator along with the CDS of HIS genes were constructed as gene cassettes into the vectors containing linkers embedded with a 34 bp loxPsym sequence. Gene fragments and linkers for defragmentation, and gene cassettes with their linkers for refactoring, were linearised from the plasmids and then integrated into the URA3 locus as a synthetic module by yeast homologous directed repair (HDR)-based assembly, respectively. D Heatmap representing the range of native (purple) and YTK promoter (blue) strengths. Each promoter was assigned for a value from 0-1000 based on the RNA-seq data and YTK characterisation data. Different combinations of promoters were selected for HIS gene expression cassettes. E Growth curves of the WT control strain (blue) and the strain harbouring the defragmented HIS module (purple) in SC-His. Mean OD₆₀₀ from 3 biological replicates are shown as squares, with error bars representing standard deviation. F Growth curves of the WT control strain (purple, n  =  4 biologically independent samples) and the strain harbouring the refactored HIS modules (HIS_refactor-1, light blue, HIS_refactor-2, cyan, HIS_refactor-3, light green, HIS_refactor-4, yellow, n  =  3 biologically independent samples) in SC-His. Mean values are plotted and error bars indicate standard deviation. G Microscopy images of the WT control strain and strains harbouring the defragmented and refactored synthetic HIS module (HIS_refactor-4) from the overnight culture in SC-His. Source data are provided as a Source Data file.

Fig. 2. SCOUT for SCRaMbLE screening.

A Schematic of SCRaMbLE inducing rearrangements, such as gene deletion, inversion and duplication between loxPsym sites in synthetic DNA. Genes are labelled in distinct colours. B Schematic of the SCOUT reporter design and its application in iterative SCRaMbLE. The SCOUT cassette is an antisense-orientated mGFPmut2 gene lacking ATG start codon flanked by two pairs of loxP variant sites, loxP2272 and loxP5171. The reporter also encodes a CreEBD expression cassette, where Cre recombinase is fused to the oestrogen binding domain (EBD) so that its function can be induced by β-estradiol. Cre-mediated inversion and excision occur at each pair of loxP variant sites, respectively, resulting in the stable inversion of the mGFPmut2 gene and deletion of one of each pair of lox sites. The flipped mGFPmut2 orientation turns on GFP fluorescence. The reporter plasmid can be removed from SCRaMbLEd strains and reintroduced for a new cycle of SCRaMbLE. C Assessment of SCRaMbLE reporter (SCOUT) performance. SCRaMbLE was induced by adding 1 μM β-estradiol to a strain containing a defragmented HIS module transformed with either of the reporter plasmids. After 4 h of induction, cells were analysed by flow cytometry to determine GFP fluorescence. Percentages of GFP⁺ cells are shown in the left table. Remaining cells were washed twice and plated on SC-Leu and SC-His-Leu plates at a 10⁻⁴ dilution rate. Plates were incubated at 30 °C for 3 days and then imaged under blue light. GFP colony numbers, highlighted in green, were counted and shown in the right table. D PCR of a FACS-sorted GFP⁺ cell library to examine the gene rearrangements by SCRaMbLE in a synthetic HIS module. Cells were grown in synthetic complete media without histidine (SC-His) and with histidine (SC). PCR primers are designed to target the URA3 locus, within which the synthetic module is located. Lanes and marker were run on the same gel but were rearranged as indicated. The experiment was independently repeated three times. Source data are provided as a Source Data file.

Fig. 3. SCRaMbLE rescues function by duplication of HIS5.

A Schematic of SCRaMbLE workflow for synthetic modules. Strain yXL216 that harbours the synthetic HIS module (HIS_refactor-4) was transformed with a SCRaMbLE reporter plasmid (pXL005), generating strain yXL219, which can induce Cre expression and indicate SCRaMbLEd cells by GFP fluorescence. Cells showing GFP expression were collected through FACS and subsequently grown in SC-His to enrich improved phenotypes. Genotyping of the post-SCRaMbLE library was performed through POLAR-seq. Top candidates with improved phenotypes were subjected to iterative rounds of SCRaMbLE after curing and re-introducing the SCRaMbLE reporter plasmid (pXL005). B Frequency of reads with different numbers of HIS genes, identified from the post-SCRaMbLE library yXL219_pool. C Bar chart showing the duplication frequency of each gene in the detected reads from yXL219_pool. D Schematic of representative post-SCRaMbLE genotypes identified in more than 10 reads from yXL219_pool. Frequency in total reads is labelled on the right in percentage. Rearranged genes are highlighted with dashed lines. Gene duplications are highlighted with black squares. E Maximum growth rates of the 14 strains isolated from yXL219_pool. Bars in grey, purple, and black represents the defective parental strain yXL219, the strains isolated from yXL219_pool and the WT control strain (yXL014), respectively. Cultures were grown at 30 °C and performed in 3 biological replicates (WT, n = 2 biologically independent samples). Mean values are plotted and error bars represent standard deviation. Culture medium is stated above each graph. F Bar chart showing the numbers of HIS genes in each distinct genotype identified from yXL219_pool (purple), yXL327_pool (blue) and yXL397_pool (light blue) and their frequency in total reads. G Frequency of reads detected with HIS5 gene triplication (green) or quadruplication (light green) from yXL219_pool, yXL327_pool and yXL397_pool. Source data are provided as a Source Data file.

Fig. 4. Increasing sfGFP expression by iterative SCRaMbLE of a synthetic chromosome.

A SCRaMbLE was induced in strain synV-pBAB016-pSCW11-creEBD (R0) for four hours to create a diversified SCRaMbLE library. The top 36 strains were selected, and fluorescence characterised using a plate reader. The top performing strain (R1) was stocked and subsequently subject to a second round of SCRaMbLE, as before, to produce strain R2, this was repeated for strains R3, R4, and R5. B Strains were selected and picked by eye under illumination of blue light (‘blue box’). Endpoint 520 nm fluorescence and OD₆₀₀ was measured after 48-hour growth. Mean is shown with error bars that indicate standard deviation for n  =  7 biologically independent samples. Pairwise comparisons were assessed using one-sided unpaired t-tests. R0 vs R1, p = 9.27 × 10⁻⁶; R1 vs R2, p = 0.0073; R2 vs R3, p = 1.24 × 10⁻⁵; R3 vs R4, p = 0.519; R4 vs R5, p = 0.140. ****p < 0.0001; **p < 0.01; ns not significant. C Colonies were counted for induced and uninduced cultures after each round of SCRaMbLE (R1-R5). Population survival is shown as the percentage of induced colonies to uninduced colonies. For clarity values for each bar are shown as numbers. D A simplified two-dimensional depiction of a phenotype and fitness landscape. Transition of a cell (circle) upwards indicates an improvement in fitness and/or phenotype. Arrows indicate individual rounds of SCRaMbLE. During independent SCRaMbLE experiments many local maxima are sampled but not necessarily reaching the optimum fitness/phenotype within each maximum. Iterative-SCRaMbLE commits a cell lineage to a single local maximum. Once at the optimum fitness/phenotype (depicted here as occurring in R3) no further fitness or phenotype improvements can be seen with subsequent rounds of SCRaMbLE (R4 and R5, inset). Any further SCRaMbLE events, in combination with those already existing result in decreased fitness and/or phenotype. E Rearrangement events at each round of iterative SCRaMbLE determined by multiplexed long-read sequencing. Inversions are shown in blue. Grey arrows below each chromosome indicates the new orientation of the region. Deletions are shown in red with the affected region subsequently marked with a dotted vertical line. The centromere is depicted as a grey circle. F Summary of SCRaMbLE events in strain R3 (and R4-R5). Deletions are shown in red, inversions are shown in blue, and translocations are shown in green. Pink genes indicate those with a disrupted 3’ UTR, red genes indicate those that are deleted, and green genes indicate those that have undergone a copy number change through a duplication event. LoxPsym sites are indicated by white circles on sticks. Source data are provided as a Source Data file.