Large-scale genomic rearrangements boost SCRaMbLE in Saccharomyces cerevisiae

Domain: earth_science
Published: 2024-01-26
Editorial outcome: accept

Abstract
Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. However, the potential of SCRaMbLE to study genomic rearrangements is currently hindered, because a strain containing all 16 synthetic chromosomes is not yet available. Here, we construct SparLox83R, a yeast strain containing 83 loxPsym sites distributed across all 16 chromosomes. SCRaMbLE of SparLox83R produces versatile genome-wide genomic rearrangements, including inter-chromosomal events. Moreover, when combined with synthetic chromosomes, SCRaMbLE of hetero-diploids with SparLox83R leads to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to hetero-diploids only with wild-type chromosomes. Analysis of the SCRaMbLEd strain with increased tolerance to nocodazole demonstrates that genomic rearrangements can perturb the transcriptome and 3D genome structure and consequently impact phenotypes. In summary, a genome with sparsely distributed loxPsym sites can serve as a powerful tool for studying the consequence of genomic rearrangements and accelerating strain engineering in Saccharomyces cerevisiae.

Introduction
Genomic rearrangements alter genetic linkage of discrete chromosomal regions and are an important mutational force in evolution1,2, underlying the pathology of various germline and somatic diseases such as nervous system disorders3, congenital heart diseases4, and cancers5. The profound effects of these genomic rearrangements, which include deletions, duplications, insertions, inversions, and translocations, are attributed to alterations in gene copy numbers, disruption of protein-coding sequences, and perturbation of cis-regulatory networks6–10. Previous studies indicate that the rate of genomic rearrangements is several orders of magnitude higher than the rate of base substitutions11, and genomic rearrangements are pervasive in all domains of life, from prokaryotes to humans12.

Genomic rearrangements likely occur randomly and sporadically, and discriminating their impacts from the confounding effects of other mutation types remains a challenge13,14. To quantify the impact of genomic rearrangement independently, several methods have been developed to construct targeted long-range rearrangements in model organisms, including Cre/loxP recombination13, I-SceI-induced15 and CRISPR/Cas9-induced14 DSB repair. However, only limited inter-chromosomal rearrangements can be generated and analyzed by these methods. A controlled system allowing genome-wide induction of a wide array of chromosomal rearrangements has not yet been established.

In the synthetic yeast genome project (Sc2.0), thousands of loxPsym sequences are incorporated in a designer genome to create a system known as Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE)16. Through SCRaMbLE, massive rearrangements can be introduced into the synthetic chromosomes, including intra-chromosomal deletions, inversions, duplications, and inter-chromosomal translocations17,18. SCRaMbLE therefore provides a strategy to investigate the potential mechanisms of genomic rearrangements and their functional consequences and facilitates delineation of the relationships between large-scale genomic rearrangements and diseases. However, to date, the power of SCRaMbLE has mostly been used in single synthetic chromosome contexts18–30. Moreover, intra-chromosomal rearrangements dominate, with inter-chromosomal rearrangements rarely detected in individual SCRaMbLEd strains despite the utilization of a strain with four synthetic chromosomes31. By sequencing SCRaMbLEd pools derived from inducing a strain harboring 5.5 synthetic chromosomes, comparable frequencies of intra- and inter-chromosomal events were detected at 47.24% and 52.67%, respectively32. However, only 9.87% of the total reads accounted for inter-chromosomal recombination, indicating that the occurrence of inter-chromosomal recombination was significantly lower than that of intra-chromosomal recombination in cells containing multiple synthetic chromosomes. Considering that the spatial proximity between loxPsym sites plays a crucial role in chromosome interactions, it can be inferred that translocation events would be more frequent when loxPsym sites are distributed throughout the entire genome.

Here, CRISPR/Cas9 was used to simultaneously integrate loxPsym sequences into multiple genomic loci. The resultant extensively engineered yeast strain, SparLox83R, possessed 83 Sparsely distributed LoxPsym sites across the genome with ReSCuES system. After inducing expression of Cre recombinase, the resultant SCRaMbLEd yeast population underwent diverse large-scale genomic rearrangements, dominated by inter-chromosomal events. By quantifying rearrangement frequencies at loxPsym sites, this study revealed multiple factors impacting loxPsym-mediated genomic rearrangements, including chromatin accessibility and genomic distance between sites. The impacts of large-scale genomic rearrangements on cell fitness under stress were investigated in progeny populations, and translocation and duplication events were detected that led to increased tolerance of nocodazole. SparLox83R was further enhanced with the SCRaMbLE system by crossing with a haploid strain containing synIII (JDY541) from the Sc2.0 project. Inducing SCRaMbLE in this heterozygous diploid strain resulted in complex genomic rearrangements, including numerous loss of heterozygosity (LOH) and aneuploidy events, as well as rapid adaptation of tolerance to acetic acid (HAc). The loxPsym sites in SparLox83R support the rapid development of new phenotypes by substantially boosting the SCRaMbLE system with large-scale genomic rearrangements.

Results
Genome-wide insertion of 83 loxPsym sites throughout the yeast genome
Cre/LoxP-based SCRaMbLE cannot yet be employed in the Sc2.0 project across all 16 yeast chromosomes because construction of a fully synthetic yeast genome is still underway. Previous studies employing SCRaMbLE in strains with multiple synthetic chromosomes reported the frequency of inter-chromosomal rearrangements as less than 10%31,32. To construct an engineered yeast strain suitable for effective introduction of genome-wide, large-scale genomic rearrangements, loxPsym-containing sequences were introduced by design into all the yeast chromosomes using a CRISPR-Cas9-based gene editing strategy (Supplementary Fig. 1a). All the insertion sites were designed at the intergenic regions which contained a PAM site, NGG, without disrupting any possible functional elements. Briefly, three loci were simultaneously targeted for integration of loxPsym sites in each cycle, eventually producing a strain with 83 sites (SparLox83) distributed across the 16 chromosomes, with at least two sites per chromosome (Fig. 1a). The presence of loxPsym at each locus was confirmed by whole-genome sequencing (WGS) (Supplementary Data 1, 2). The final strain exhibited four off-target insertions, namely II-3, II-8, IV-8, and V-2. Among these sites, three (II-3, II-8, and V-2) were located within the ORFs of non-essential genes. Since the strain did not display any discernible growth defects (Fig. 1b), we opted to retain the three loxPsym sites. Meanwhile, multiple loxPsym sites were identified at the same loci through self-duplication of the plasmid backbone sequence (Fig. 1a and Supplementary Fig. 1b). LoxPsym sites in SparLox83 were sparsely distributed, with significantly longer genomic distances between adjacent sites than in Sc2.0 synthetic chromosomes. The average genomic distance between adjacent loxPsym sites was ~145 kb in SparLox83, much larger than the 3 kb distance between sites in Sc2.0 chromosomes (Supplementary Fig. 1c). Furthermore, WGS and pulsed-field gel electrophoresis (PFGE) revealed a chromosome translocation between chrVIII and chrIX and an additional copy of chrIX in SparLox83 (Supplementary Fig. 2). In addition, we also identified 1197 SNPs/InDels in nuclear genome when we aligned the NGS reads to the yeast reference genome (GCF_000146045.2) (Supplementary Data 3). Excluding the sequences introduced by us, 305 SNPs/InDels were found to be associated with 158 overlapping open reading frames (ORFs). Notably, significant frameshift mutations were observed in YAL063C, YBR005W, YBR148W, and YEL042W.

The fitness of SparLox83 was evaluated by comparing growth in various conditions with its parental strain JDY524 (without loxPsym) and BY4741 in serial dilution tests. Growth of SparLox83 was indistinguishable from JDY524 or BY4741 on YPD and under stress conditions, including the presence of 8 μg/ml camptothecin (an inhibitor of topoisomerase I that causes genome instability) or 2.5 μg/ml nocodazole (a drug that affects mitosis by interference with microtubule polymerization) (Fig. 1b). Given the presence of multiple loxPsym sites in its genome, the genome stability of SparLox83 was checked using a previously described junction PCR-based assay33. None of the loxPsym sites were lost in 35 independent lineages after ~125 mitotic generations in YPD in the absence of Cre (Fig. 1c and Supplementary Fig. 3). These results confirmed the successful insertion of 83 loxPsym sites into the wild-type yeast genome without impairment of genome stability or fitness under various conditions.

Induction of Cre recombinase activity in SparLox83R leads to versatile rearrangements, particularly inter-chromosomal rearrangements
To facilitate the selection of clones with genomic rearrangements, the previously described ReSCuES system23 was integrated into SparLox83 to generate SparLox83R. ReSCuES was a reporter to efficiently identify SCRaMbLEd cells based on a loxP-mediated switch of “on” and “off” states of the two auxotrophic markers, URA3 and LEU2. Since there is at least one essential gene positioned between each pair of adjacent loxPsym sites, deletions will result in cell death, limiting the extent of all the possible rearrangements34. Therefore, an additional heterozygous diploid strain, JDY536, was generated by crossing SparLox83R with BY4742.

SparLox83R and JDY536 were induced to express Cre recombinase and selected using the ReSCuES system for five consecutive rounds before collection of SCRaMbLEants. No other selection was imposed. First, to confirm the presence of rearrangements, PCR was used to amplify some of the novel junctions that were not present in the original SparLox83R genome but which might be generated following loxPsym-derived rearrangements (Supplementary Fig. 4a, primers listed in Supplementary Data 4). Next, the pooled SCRaMbLEants were subjected to Nanopore MinION sequencing. To avoid PCR bias and obtain reads that were as long as possible, a ligation-sequencing strategy with no PCR amplification was used, and genome shredding steps were avoided. In total, 774,791 (8.06 Gb) and 2,523,754 (22.5 Gb) reads were generated from the SparLox83R and SparLox83R × BY4742 SCRaMbLEant populations, respectively.

A population-based strategy was adopted to detect and characterize rearrangement events in SCRaMbLEant populations (Fig. 2a). Flanking regions of loxPsym sites were extracted and assembled with all potential rearrangements in silico. Next, Nanopore reads containing loxPsym sequences were aligned to the potential rearrangements. Each filtered match between a read and a potential rearrangement was defined as a rearrangement event. This analysis revealed that 5721 and 936 rearrangement events took place across all loxPsym sites in the haploid and diploid strains, which accounted for 31.1% and 3.1% detected loxPsym-containing reads, respectively (Fig. 2b). Flanking region lengths were set to 500, 1000, 1500, and 2500 bp, with no significant differences seen in the analysis results (Supplementary Fig. 4b). The following results are those obtained using 1000 bp flanking regions. At least one rearrangement event was detected for every loxPsym site in both the haploid and diploid strains. Inter-chromosomal rearrangements predominated: 4852 (84.8%) and 595 (63.6%) of the rearrangement events were inter-chromosomal in haploid and diploid cells, respectively. Rearrangements were next assigned to their different chromosomes to test whether rearrangement events occurred evenly across the genome. In haploid cells, an increased number of rearrangement events was associated with a higher number of loxPsym sites on that chromosome (Fig. 2c, R2=0.9828, p<0.001).

These results obtained in the absence of selection beyond the requirement for at least one ReSCuES inversion event per cell showed that genome-wide insertion of loxPsym sites in SparLox83R produced versatile rearrangements, with a bias towards inter-chromosomal events, upon Cre induction. The preference for inter-chromosomal rearrangements suggests that SparLox83R presents an ideal model for studying the impacts of large-scale genomic rearrangements.

Quantitative analysis revealed an uneven distribution of rearrangements at different loxPsym sites
Previous studies used rearrangement event frequencies at particular loxPsym sites to quantify their rearrangement capacities31,32. Rearrangement event frequencies were, however, biased by the different sequencing depths at each loxPsym site. To overcome this, an Average Rearrangement Rate (ARR), a normalized rearrangement event frequency, was used to quantify the average rearrangement potential for each loxPsym site. ARRs were compared for intra- and inter-chromosomal rearrangements. Although intra-chromosomal ARRs were significantly higher than inter-chromosomal ARRs, the difference between the two categories was limited (Fig. 2d). Moreover, outlier ARRs represented particularly active sites. Seven outliers (II-7, II-6, VIII-2, VIII-1, XII-8, VIII-3, XII-7) were found for intra-chromosomal ARRs in diploid cells, two of which (II-7 and II-6) were also observed in haploid cells. Three sites (VIII-2, XII-7, XIII-7) were identified as inter-chromosomal outliers in diploid cells, but no outliers were observed for inter-chromosomal rearrangements in haploid cells.

Rearrangement Weight (RW) was derived to quantitatively describe the rearrangement event frequency between two specific sites and reduce the bias caused by sequencing depth. RW divides the number of rearranged events by the geometric mean of the sequencing depths of pairs of interacting sites. The RW matrix containing RWs of all possible loxPsym site pairs (2775 pairs) was generated. Of these, 2111 (76.1%) non-zero RWs were represented in haploid cells, suggesting that most of the potential rearrangement events occurred. Interaction maps of the RW matrix for intra- and inter-chromosomal rearrangements were generated (Supplementary Fig. 5). The RW between sites II-6 and II-7 (0.28) was significantly higher than other RW values, which were below 0.087 (Supplementary Fig. 5a). Inter-chromosomal RWs also exhibited an uneven distribution (Supplementary Fig. 5b), with events between site pairs located in the inner circle (RWs>0.02) occurring more frequently than with other site pairs (RWs<0.02). Together, these results showed that loxPsym sites in SparLox83R exhibited highly variable rearrangement event frequencies.

The genomic locations of loxPsym sites impact their rearrangement event frequencies
Previous studies using SCRaMbLE in strains harboring one or more synthetic chromosomes have confirmed that rearrangement frequency varied among loxPsym sites31,32. In synthetic chromosomes, where loxPsym sites are densely arranged, the rearrangement frequencies of loxPsym sites correlated with chromatin accessibility and spatial contact probability32. However, due to the preference for intra-chromosomal rearrangements in these synthetic chromosomes, factors impacting inter-chromosomal rearrangement frequencies remain less well understood. The genome-wide distribution of loxPsym sites in SparLox83R, with their frequent inter-chromosomal rearrangements and quantitatively defined RWs, indicate that SparLox83R is an ideal model to explore the features of rearrangement frequencies involved in sparsely arranged loxPsym sites.

Chromatin accessibility refers to the degree physical accessibility and availability of DNA within the chromatin structure for interaction with various regulatory factors, such as transcription factors and other proteins. It describes the ability of these factors to bind specific genomic regions and regulate gene expression35. Thus, it may be a factor to influence the interaction between the Cre recombinases and loxPsym sites. Consistent with previous findings32, the inter-chromosomal ARRs of sites positioned at open chromatin regions36 were significantly higher than sites outside open chromatin regions (Fig. 2e), while no significant correlation was observed for intra-chromosomal ARRs (Supplementary Fig. 6).

The highest RW was observed between sites II-6 and II-7, which possessed the shortest genomic distance among the 83 loxPsym sites. Next, the impact of genomic distance between two intra-chromosomal loxPsym sites on RW was examined. The RW between two loxPsym sites located on the same chromosome arm showed a negative correlation with genomic distance in haploid and diploid cells (Fig. 2f and Supplementary Fig. 7a). For two sites not on the same chromosome, spatial distance was determined using genome-wide chromosome conformation capture analysis (Hi-C), which facilitated the generation of a normalized contact map of the SparLox83R genome (Supplementary Fig. 7b). However, no correlation was found between the normalized contact counts and the RW of paired loxPsym sites (Supplementary Fig. 7c).

Together, these results suggest that chromatin accessibility and genomic distance partly impact the rearrangement frequency of loxPsym sites.

Large-scale genomic rearrangement and stress tolerance
Large-scale rearrangements drive phenotypic diversification and environmental adaptability37,38, but distinguishing the impact of large-scale rearrangements on fitness from other types of mutation in naturally evolved strains remains challenging. The SparLox83R strain can be used to introduce large-scale genomic rearrangements, allowing the investigation of the role of such rearrangements in cell fitness alterations under various conditions.

Rearrangements were induced in SparLox83R and strains were screened under a range of selective stresses including high alcohol (10% ethanol), high oxidation (3 mM H2O2), high osmotic stress (1 M NaCl), and non-fermentable sugar as a carbon source (3% glycerol). Experiments were repeated in the presence of reagents to perturb microtubules (10 μg/ml nocodazole and 40 μg/ml benomyl), cause DNA damage (30 μg/ml camptothecin), or inhibit the TOR pathway (10 ng/ml rapamycin) (Fig. 3a)23. Although no candidates conferring increased tolerance to ethanol, H2O2, NaCl, or glycerol were identified (Supplementary Fig. 8a), several clones were more resilient to nocodazole, benomyl, or rapamycin than the original strain (Fig. 3b and Supplementary Fig. 8b).

One strain, JDY528, which gained tolerance to nocodazole, was selected for further analysis. Whole-genome sequencing was conducted using the Oxford Nanopore MinION platform. Genome assembly using Canu and correction using Nanopolish enabled comparison with the reference genome and identification of two loxPsym-mediated genome arrangements in the JDY528 strain. A translocation was observed between chromosomes IV and XIV (mediated by IV-5 and XIV-1), and a 484 kb duplication was found on chromosome IV (mediated by IV-7 and IV-9) (Fig. 3c). In addition, the additional copy of ChrIX in SparLox83R was lost in JDY528 (Supplementary Fig. 9a). The presence of these chromosomal rearrangements was confirmed by junction PCR (Supplementary Fig. 9b).

Major causal mutations in response to such selective forces are expected to change gene location/context and may involve the formation of new chromosomal junctions that affect higher-order genome regulation39. Therefore, we investigated the three-dimensional (3D) genome structure and transcription profile in these strains by Hi-C and RNA-seq analysis.

To determine the impact of rearrangements on spatial location, genome-wide Hi-C contact maps40 were compared between JDY528 and SparLox83R. Clustering of centromeres and telomeres (Supplementary Fig. 9c, red dots on the map) was seen in both strains, as previously reported41,42. Although one of the rearranged loxPsym sequences in JDY528 was near the centromere of chrIV, the contact maps showed that the translocation and duplication events in JDY528 did not disrupt localization and clustering of centromeres and telomeres (Supplementary Fig. 9d). The log2-ratio heatmap between JDY528 and SparLox83R was plotted43 to quantitatively compare the differences between the two strains. This analysis showed that contacts between chromosomes without genomic rearrangement in JDY528 and SparLox83R were similar (Fig. 3d). However, the duplication on chromosome IV led to elevated contacts with the duplicated region. The translocation between chromosome XIV and chromosome IV altered the contacts between these two chromosomes (Fig. 3d, black circle). Moreover, the contacts between chromosome IX and other chromosomes were weaker in JDY528 than in SparLox83R, which can be attributed to the loss of one copy in JDY528 (Fig. 3d, black arrow).

To investigate the genome-wide transcriptional impact of nocodazole treatment on cells cultured in YPD media, RNA sequencing was performed due to the tight relationship between chromosome organization and gene expression. A total of 197 gene transcripts with significantly altered expression levels (>2-fold increase or decrease, p value <0.0001) were identified in JDY528. Among the 82 upregulation genes, 73 genes were located within the duplication region (from YDR073W to YDR277C), while among the 115 downregulation genes, 75 genes were found on chromosome IX (Supplementary Fig. 10a). To determine whether these differentially expressed genes were associate with the large fragment rearrangement, we combined the RNA-seq data and Hi-C contact map, revealing that most of these genes clustered around either the rearrangement sites or chromosome IX (Fig. 3d). These findings strongly suggested that alterations in the 3D genome structure may contribute to changes in gene expression. Furthermore, we observed a decreased expression level of YDR001C which was located near the translocated junction (Supplementary Fig. 10a). However, its knock-out strain exhibited comparable tolerance to nocodazole when compared to wild-type strain (Supplementary Fig. 10b). Considering that changes in expression levels were not limited to regions surrounding translocation breakpoints, further analyses should explore a larger set of potentially involved genes. In addition, it was possible that nocodazole resistance could be influenced by both the large-scale duplication on ChrIV and the copy number variation of ChrIX.

To test whether the variation in chromosomal structure was sufficient to cause the observed nocodazole tolerance, a split-URA3 reporter strategy25 was used to reconstruct the chrIV/chrXIV translocation of JDY528 in JDY524 (the initial strain lacking loxPsym sites, Fig. 3e). The strain carrying only this translocation grew better than the split-URA3 inserted into JDY524 on medium with nocodazole (Fig. 3f), providing direct evidence that the chrIV/chrXIV translocation contributed to the observed nocodazole tolerance. These data revealed a connection among large-scale rearrangement, genome 3D structure, transcription, and phenotype, demonstrating that simply reconfiguring chromosome architecture was sufficient to provide fitness advantages in stressful growth conditions.

Chromosomes harboring sparsely arranged loxPsym sites combined with synthetic yeast chromosomes can generate complicated structural variations
SCRaMbLE is widely used in directed evolution to acquire strains with distinctive traits21–23,25,27,34. Here, to boost SCRaMbLE with genome-wide large-scale rearrangements, SparLox83R chromosomes were combined with a Sc2.0 synthetic chromosome in a single strain. As a proof of concept, SparLox83R was mated with JDY541, the strain carrying synIII33, to generate JDY544. After five rounds of SCRaMbLE using ReSCuES without selective pressures, nine SCRaMbLEant colonies were randomly isolated and sequenced. The presence of loxPsym sites and PCR Tags in synIII and chrIII allowed reads with loxPsym sites to be extracted and differentiated in SparLox83R and synIII.

First, SCRaMbLEd synIII was resolved in the sequenced strains. The arrangement of synIII in each strain was deconvoluted and visualized as described previously18 and each strain exhibited a unique structure (Fig. 4a). Rearrangement types in the nine strains were classified as deletions, inversions, duplications, duplicated inversions, inverted duplications, and complex rearrangements with respect to the parental synIII sequence. As an example, the pattern of chromosomes harboring sparsely arranged loxPsym sites rearrangements detected in JDY553 and JDY596 were illustrated as a dot-plot, wherein the segments of the rearranged genome were plotted against the corresponding segments of the parental genome in their original sequential order (Supplementary Fig. 11a). Meanwhile, the fate of each segment in each strain was determined (Supplementary Fig. 11b and Supplementary Table 1). By comparing sequence order and orientation in all strains except JDY550, several commonly conserved sequences (orange) and non-conserved sequences (gray) were identified (Supplementary Fig. 11c), most of which were consistent with previously reported regions30. The regions at the ends of synIII were prone to delete, but to different degrees in different strains (Supplementary Fig. 11c, light gray rectangles). Seven of the nine strains harbored a circular synIII, which was also observed in SCRaMbLEant cells previously44 (Fig. 4a, see asterisks). This circularization was confirmed by reads spanning the left and right ends of synIII. In addition, whole-chromosome duplications were identified in three strains (JDY550, JDY596, and JDY599).

For chromosomes other than chromosome III and synIII, reads containing loxPsym sites were aligned to the reference sequences of SparLox83R. Four types of structural variations were observed in seven of the nine strains, including one deletion event and six translocation events (Fig. 4b). In addition, a structural change was found in JDY550 mediated by IV-6 and loxPsym site 73, located between YCR057C and YCR059C on synIII. Read alignments revealed that IV-6-Up was combined with segment 74 on synIII, and IV-6-Down combined with segment 73 (Fig. 4c). A rearrangement between synIII and chrIII, in which the YCR061W gene was from synIII and its neighboring gene YCR065W was from chrIII (Fig. 4c), was also observed. However, no loxPsym sequence were detected at the breakpoint, which suggested that the rearrangement was generated by homologous recombination as synIII and chrIII shared significant sequence similarity. Three primer pairs were used to confirm these structure changes and the expected amplicons were directly sequenced to verify the specific translocation sites (Fig. 4c and Supplementary Fig. 12). These results demonstrated that loxPsym sites in SparLox83R were able to combine with synthetic chromosomes to generate a range of structural variants genome-wide, including rearrangements between SparLox83R-derived loxPsym sites and synIII-derived loxPsym sites and one example of an homologous recombination-mediated rearrangement (Table S1).

LOH and aneuploidy are frequently detected in SCRaMbLEd heterozygous diploids
As the rearrangement frequency was relatively lower with SparLox83R-derived loxPsym sites than with synIII-based sites, it was doubtful whether loss of heterozygosity (LOH) and aneuploidy, which were observed in previous SCRaMbLEd genomes20, were occurring in these strains. To examine this, the 2 kb upstream or downstream regions of each loxPsym site were designated as loxPsym site-Up and loxPsym site-Down and were analyzed as a unit. For example, X-1-Up and X-1-Down indicated the 2 kb upstream and downstream regions of X-1. Reads with or without loxPsym sites were mapped to the upstream/downstream regions and counted to deduce copy number variation of regions adjacent to loxPsym sites (Fig. 5a; where a blue square represents a copy of the upstream region or downstream region containing a loxPsym site and a white square represents a region lacking a loxPsym site). Using this strategy, copy numbers for all loxPsym upstream and downstream regions were assessed and some LOH events were observed in all nine strains (Fig. 5b and Supplementary Fig. 13). Several LOH events were likely caused by loxPsym site sequence deletion, such as the loxPsym sites on chromosome II (except for II-4 in JDY553 and JDY598), whereas other events were likely caused by loxPsym site sequence insertions into the counterpart of the other chromosome, such as IV-1 in all strains (except JDY552) (Fig. 5b). In addition, the structure of chrVIII in all nine strains differed from that of the parent strain JDY544. In JDY549, JDY550, and JDY596, only one copy of the wild-type chrVIII remained (Fig. 5b, c). In JDY551, JDY597, JDY598, and JDY599, two copies of the wild-type chrVIII were present instead of the heterozygous chrVIII in JDY544 (Fig. 5b, c). In JDY552, the loxPsym sequence of VIII-1 was inserted into the wild-type chrVIII and the remainder of SparLox83R-based chrVIII was lost (Fig. 5b, c). JDY553 contained three copies of the wild-type chrVIII, one of which contained an insertion of the VIII-1 loxPsym sequence (Fig. 5b, c). These variations were likely caused by structural differences in chrVIII between SparLox83R and the wild-type, in which a translocation of VIII-3 and IX-1 pre-existed prior to SCRaMbLEing (Supplementary Fig. 2).

Using the copy number variation information, the chromosomal structural variations detected by WGS were reconstructed and several non-reciprocal translocations were identified. In JDY549 and JDY596, an extra copy of IV-9-Down combined with X-5-Down and formed a new chromosome, with deletion of X-5-Up (Figs. 4b and 5d). In JDY551, JDY597, and JDY599, an extra copy of IV-1-Up combined with XIII-7-Up, forming a new chromosome and deleting XIII-7-Down (Figs. 4b and 5d). In JDY552, however, a duplication of VI-2-Up was observed with no change to IV-1-Up (Fig. 5b). To clarify the events at these sites, reads were aligned to reference chromosomes. A translocation occurred between loxPsym sites IV-1 and VI-2, and a duplication of an ~45-kb region upstream of VI-2 was inserted upstream of IV-1. In this case, it was uncertain whether the rearrangement was loxPsym-mediated (Fig. 5e).

Overall, the diverse rearrangements and resulting genomes indicated that sparsely arranged loxPsym sites in SparLox83R can be used to generate combinatorial diversity with synthetic chromosomes, including structural variation, LOH, and aneuploidy.

SparLox83R accelerates SCRaMbLE-mediated enhancement of acetic acid tolerance
The increased rearrangements observed in strains with synIII, as exemplified by the diversity of outcomes generated by SCRaMbLE of SparLox83R and synthetic chromosomes, suggested a potential strategy for strain improvement under stress conditions. To test this, the rate of variant evolution was compared between two heterozygous diploids, JDY546 (BY4741 × synIII) and JDY544 (SparLox83R × synIII), using tolerance to acetic acid (HAc) as a proof of principle. In total, 48 individual colonies from each strain were inoculated into a 96-well microplate and SCRaMbLE-Selection-based evolution cycles were performed. Mixed strains after each SCRaMbLE round were selected on YPD plates with a HAc gradient (Fig. 6a). The original strains (SparLox83R, JDY544, and JDY546) were tested for HAc tolerance to identify an appropriate level of stress. Severe growth defects were seen in the presence of 0.4% HAc (Supplementary Fig. 14a), indicating that this concentration was appropriate for selection of tolerant SCRaMbLEants. After the first round of SCRaMbLE (R1), most of the populations were able to grow on media with a maximum of 0.4% HAc (Supplementary Fig. 14b). After the second (R2) and third (R3) rounds, the HAc-tolerance of all populations increased significantly (Supplementary Fig. 14c, d). After the second round, most SCRaMbLEant populations were able to grow on 0.5% HAc media, with populations from JDY544 exhibiting better growth than those from JDY546 (Supplementary Fig. 14c). After the third round (R3), two different concentrations of cells were tested on YPD plates with a gradient of HAc ranging from 0.5% to 0.8% (Fig. 6b and Supplementary Fig. 14d). The SCRaMbLEant populations from 48 individual JDY544 colonies were able to survive on 0.7% HAc, whereas only some of the 48 individual JDY546 colonies could grow under the same conditions. These results suggested that the development of increased HAc tolerance was greatly accelerated in SCRaMbLEant populations derived from the JDY544 compared with those from JDY546, indicating that genomes with sparsely distributed loxPsym sites may be associated with rapid evolution of tolerance to HAc.

Methods
Strains
The yeast strains used in this paper were derivatives of BY4741/BY4742. Standard methods for yeast culture and transformation were applied. The strains generated in this study are listed in Supplementary Data 5.

Consecutive insertion of loxPsym sites
SparLox83 was generated using CRISPR/Cas9-mediated multiplex DNA integration as previously described55. Briefly, three donor-gRNA cassettes were amplified and co-transformed into yeast cells alongside linearized plasmid (200 ng each) using the lithium acetate transformation method. Two plasmids were used, containing either LEU2 or URA3. Insertions were verified by PCR. Colonies containing repaired donor-gRNA-containing plasmids were eliminated by streaking onto YPGal plates (2% bacto-peptone, 1% yeast extract, 2% galactose, and 2% agar). Colonies were subsequently streaked onto SC-Ura, SC-Leu, and YPD plates to confirm loss of plasmid. Verified strains were used for the next round of loxPsym insertion.

Yeast colony PCR
Cells were resuspended in 20 μL of 20 mM NaOH and lysed in a thermocycler (94°C, 3 min; 4°C, 2 min, 5 cycles). Cell lysate (1 μL) was used as template for PCR using a site-specific forward primer upstream of each insertion site and a common reverse primer, MGO001 (Supplementary Data 4). PCR was performed using EasyTaq DNA polymerase (TransGen Biotech) and the conditions were as follows: 94°C for 5 min; 30 cycles of 94°C, 30 s; 55°C, 30 s; 72°C, 1 min; and final extension at 72°C for 7 min. Agarose gel analysis was used for visualization of PCR products.

Preparation of yeast genomic DNA
Yeast genomic DNA was prepared as previously reported56 with minor modifications. Briefly, collected yeast cells were washed once in sterile water and resuspended in 100 μL breaking buffer (50 mM Tris pH 8.0, 100 mM NaCl, 1% SDS, 2% Triton X-100, and 1 mM EDTA) before addition of an equal volume of glass beads (Sigma G8772) and 200 μL of Phenol-Chloroform-Isoamyl alcohol (PCI) (25:24:1). After vortexing for 10 min at room temperature, 100 μL sterile water was added and mixed by inverting the tube several times. After centrifugation at 12,000 × g for 10 min, the upper layer (150 μL) was transferred to a new microfuge tube. For DNA precipitation, 500 μL 100% ethanol was added and the sample was incubated at -20°C for 20 min. Genomic DNA was pelleted at 12,000 × g for 5 min at 4°C. The DNA pellet was dried at 45°C for 15 min in vacuum centrifugal concentrator (Eppendorf Concentrator Plus) and then resuspended in 50 μL sterile water.

Serial dilution assay
Yeast cells were cultured overnight in YPD medium with rotation at 30°C. Cultures were adjusted to the same cell concentrations after measuring the optical density at 600 nm (OD600) and then serially diluted 10-fold with water. Diluted cells were spotted onto selective media plates, incubated at 30°C for 48–72 h, and then imaged for analysis.

Assay for genomic stability
Yeast colonies were inoculated into 5 mL YPD medium and incubated at 30°C for 24 h with agitation. Cultures (5 μL) were transferred into new vessels containing 5 mL fresh YPD medium and cultured under the same conditions. These steps were repeated daily for ten days (~120 generations), after which cells were streaked onto YPD plates and incubated at 30°C for 1–2 days until colonies could be visualized. Single clones were isolated and subjected to PCR analysis to examine the presence of loxPsym sequences. In total, 35 independent clones were tested at 24 randomly chosen loxPsym sites.

Pulsed-field gel electrophoresis
Chromosomal DNA was prepared as previously described57. In brief, about 5 × 107 cells at the stationary phase were collected and mixed with a 0.6% low melting point agarose solution before being pipetted into a casting mold. The solidified inserts were then subjected to digestion using zymolyase and proteinase K enzymes. After washing, the samples were analyzed on a BioRad CHEF Mapper apparatus using a 0.9% agarose gel for 20 h at 14°C under an applied voltage of 6 V/cm and an angle of 120°, with switch times ranging from 10 to 60 s.

SCRaMbLE of yeast cultures
SparLox83R or the heterozygous diploids with ReSCuES sequence integrated were transformed with pGAL-Cre plasmid, which was constructed by integrating the NatMX cassette into the LYS2 coding sequence based on a previously described plasmid29. Single yeast colonies were inoculated into 5 mL YPD medium containing 0.1 mg/mL clonNAT and cultured overnight at 30°C. Cultures were diluted to OD600 = 0.3 in 5 mL fresh YEP medium with 2% w/v raffinose and 0.1% w/v glucose and cultivated at 30°C for 6 h. Galactose was added to a final concentration of 2% w/v and cultures incubated at 30°C for 2 h. Cultures were collected and plated onto solid YPD medium containing a selective agent.

For consecutive induction, five rounds of SCRaMbLE were induced as follows. SCRaMbLE was induced by the addition of galactose as described above. The single yeast colony was inoculated overnight and then transferred to fresh YEP medium with containing 2% w/v raffinose and 0.1% w/v glucose to cultivate for 6 h. Galactose was added to achieve a final concentration of 2% w/v, and the culture was incubated at 30°C for an additional 2 h. Subsequently, cells were collected, washed once with 1 mL sterile water, resuspended in either liquid SC-Leu or SC-Ura medium in alternative rounds, and cultivated to the stationary phase. After five rounds of SCRaMbLE, the cells were plated on a SC-Leu plate, from which nine colonies were selected for sequencing.

Split-URA3 mediated rearrangement
The URA3 gene was split in half by the intron of ACT1. A loxP sequence was inserted into the intron of ACT1. To integrate the two halves of the URA3 gene into the genome, a His marker was fused to one half of the URA3 gene and a Leu marker was fused to another half by PCR. Using transformation-associated recombination, the two halves of the URA3 gene with different markers were inserted into the location of XIV-1 and IV-5 of JDY524, respectively. A colony growing on the SC-His-Leu plate was picked and transformed with pGAL-Cre plasmid. The Cre expression was induced by the addition of galactose as described above. Put the inducted culture onto the SC-Ura plate and incubate at 30°C for 48 h. Colonies were verified by PCR.

Screening of HAc tolerance strains by continuous evolution
JDY544 and JDY546 were transformed with pGAL-Cre plasmid (clonNAT resistance) and several rounds of SCRaMbLE-assisted evolution were performed as follows. Each strain was evolved in 48 individual populations. Single colonies were cultured overnight in YPD containing 0.1 mg/mL clonNAT at 30°C and re-inoculated to obtain an OD600 = 0.3 in YEP medium with 2% w/v raffinose and 0.1% w/v glucose. After growing for 6 h at 30°C, galactose (2% w/v final concentration) was added and cultures incubated at 30°C for an additional 2 h. To inhibit Cre expression, glucose (2% w/v final concentration) was added and cultures incubated at 30°C until saturated. The SCRaMbLEd populations were diluted 1:10 and 1:100 into 100 μL fresh YPD. Diluted cells were plated on YPD agar plates and stress plates with series concentration of HAc. The strains incubated at 30°C for several days until visible colonies were formed for all the 96 populations on stress plates with low concentration of HAc. For the next round of evolution, the parent strains were picked from the stress plate with visible colonies for all the 96 populations. Repeat the incubation and induction processes.

Quantification for the growth of the evolved colonies
The growth of the strains after multiple rounds of directed evolution were quantitatively evaluated on YPD agar plates and stress plates with series concentration of HAc using CellProfiler v4.2.6 software. Given that the edges of the plates were relatively thick with no neighboring colonies, creating a nutrient-rich environment conducive to faster strain growth, colonies located at these plate edges were initially excluded. For the remaining colonies, a customized yeast patch identification workflow (https://cellprofiler.org/examples) were utilized to quantify the mean intensity of their forced spots. Specifically, we compressed the images into ~1200 × 800 png files and then cropped them based on colony positions in the images. The “Block size” parameter in the Correct Illumination Calculate step was set to 80, while for Identify Primary Objects step, we adjusted the “Typical diameter of objects” parameter to range between 8–80 units. In addition, in Filter Objects step, we modified both “Minimum Form Factor value” and “Minimum Area value” parameters to be 0.40 and 90, respectively. Finally, we conducted an unpaired two-tailed t-test to determine statistical significance regarding differences in growth.

Whole-genome Nanopore sequencing
DNA extraction and sequencing were performed as described previously26. Briefly, genomic DNA was extracted according to using a modified Qiagen Genomic-tip 100/g protocol with the Qiagen Genomic Buffer kit. DNA quality was assessed with agarose gel electrophoresis and with a NanoDrop™ 2000 Spectrophotometer and a Qubit 4 Fluorometer with dsDNA HS reagent. Oxford Nanopore Technology (ONT) sequencing library preparation was performed using SQK LSK108 or LSK109 kits with Native Barcoding kits EXP-NDB104 or EXP-NBD114, according to the manufacturer’s guidelines. Sequencing was performed on a MinION Mk1B device using FLO-MIN106D with R9.4.1 sequencing cells. Sequencing was performed for 48 h using MinKNOW (v3.6.5) software.

Junction detection of SCRaMbLEants
All possible combinations of the 83 loxPsym sites were assembled in silico using the yeast reference genome (GCF_000146045.2). Only 1 kb-long loxPsym neighboring sequences were retained. The assembled sequences were used as the reference for junction detection. LoxPsym-containing ONT sequence reads were identified using LAST software (v1250) and then aligned to the reference using Minimap2 (v2.20-r1061). Reads with MapQ>20 that covered at least one reference sequence were retained for subsequent analysis. Reads containing more than one loxPsym site were analyzed separately. The rearrangement rate (RR) of a particular loxPsym site, i, was defined as Nre i / (2Nnorm i + Nre i), where Nnorm i and Nre i were the number of normal and rearranged reads for loxPsym site i. The Rearrangement Rate (RR) was subsequently normalized to the total number of rearranged loxPsym sites to calculate Average Rearrangement Rate (ARR). The rearrangement weight (RW) of two particular loxPsym sites, i and j, was calculated by Eq. 1, which also considered bias of sequencing depth.

RW i,j = sqrt(Nre ij / (Nall i * Nall j)) (1)

Nre ij is the count of rearranged reads between loxPsym sites i and j, and Nall i = 2Nnorm i + Nre i. Networks of inter-chromosomal and intra-chromosomal rearrangements were constructed using Cytoscape software (v3.7.1).

Assembly-based structural variant calling
ONT reads (FASTQ format) were used with the Canu pipeline (v2.1.1) to assemble the SparLox83R and JDY528 genomes. Subsequently, Pilon software was employed to correct draft assemblies, utilizing the high-quality Illumina PE300 reads, for three consecutive rounds. The assembled genomes of JDY528 was aligned to SparLox83R using Minimap2 (v2.20-r1061) software with -x asm5 argument. The resultant PAF format files were used to display the structural variations.

Structural and copy number variant and loss of heterozygosity detection in hetero-diploid strains
ONT reads (FASTQ format) from hetero-diploid strains were filtered and trimmed using NanoFilt software. LoxPsym-containing ONT reads were obtained by LAST software (v1250) for structural variant (SV) detection. To avoid alignment bias due to the similarities of ChrIII and SynIII, LAST was used to separate loxPsym-containing reads according to whether they contained SynIII PCR Tags. Reads containing SynIII PCR Tags were mapped to SynIII and were used as the inputs to Euclidean path algorithms, which required only linear time to find a sequence consistent with the loxPsym junctions observed in the reads, to reconstruct SCRaMbLEd SynIII as previously described18. Reads without SynIII PCR Tags were aligned to the SparLox83R genome for SV calling using the NGMLR-sniffles (v1.0.12 for sniffles and v0.2.7 for ngmlr) pipeline. In addition, SynIII PCR Tag-containing reads were aligned to the SparLox83R genome to detect translocation between SynIII and SparLox83R. LoxPsym sites that were not identified in any reads were considered to be the result of Loss of Heterozygosity (LOH). We used a previously described iterative algorithm to estimate and refine the copy number variation (CNV) of loxPsym sites28. Briefly, the sequencing depth of the flanking 2000 bp of loxPsym sites was calculated for hetero-diploid strains and the depth of SCRaMbLEd strains were corrected according to the parental strain using the iterative algorithm.

Hi-C library preparation and data processing
Hi-C library preparation was performed as previously described58 at Beijing Novogene Bioinformatics Technology Co. Ltd. Samples, each with two biological repeatations, were flash-frozen and pulverized before formaldehyde cross-linking. Briefly, cell lysis and MboI digestion were performed after cross-linking, then DNA ends were labeled with biotin and joined. DNA was decrosslinked using Protease K and SDS. Free DNA was purified and extracted using Ampure XP beads. After quality control, ultrasonication was used to produce 200–500 bp DNA fragments. The biotin-labeled fragments were further enriched and sequenced by Illumina Novaseq. Hi-C data were mapped to the public S288C reference genome (GCF_000146045.2) using distiller-nf (v0.3.3). Read pairs that were not uniquely mapped (mapping score < 1) were discarded. Valid alignment files were transformed to .hic files for juicer using pre command. Hi-C map resolution was calculated as described previously59. Contact matrices used for heatmap visualization and further analysis were KR (Knight-Ruiz) normalized at 5 kb resolution. To compare the interaction frequencies between SparLox83R and other strains, the log2 ratio of a 5 kb binned contact map was computed and normalized by subtracting the median of its values.

For the construction of a 3D chromosome model, Hi-C data were mapped to the SparLox83R genome and binned at 5 kb resolution. Chromosome models were visualized using PyMOL. The 3D structure was learned using MCMC (v0.4).

RNA sequencing and analysis
SparLox83R and JDY528 strains, each with 3 biological replicates, were cultured overnight in YPD medium at 30°C. Subsequently, they were re-inoculated in 50 mL fresh YPD medium for further incubation until OD reached ~0.6. The cells were treated with DMSO or nocodazole at a concentration of 10 μg/mL for 2 h prior to harvesting. The RNA sequencing experiments were conducted at Beijing Novogene Bioinformatics Technology Co., Ltd. Libraries were prepared using the non-stranded Illumina NEBNext Ultra™ RNA Library Prep Kit. Raw data adapters were removed using Cutadapter (v3.4) software. Cleaned data was mapped using Hisat2 (v2.2.1), and PCR duplicates were identified and eliminated using picard (v2.25.7). Gene read counts were generated by htseq-count software (v0.13.5) with the intersection-nonempty option, while log2 (fold change) and padj values were calculated using DESEQ2 package (v1.30.1). TPM values for each gene were determined by Stringtie software (v2.1.7).

Statistics and reproducibility
Unless specially noted, two-tailed t tests were employed to compare different groups, while simple linear repression was used to assess the correlation between two groups in this paper. Statistical significance was considered at a p-value < 0.05 threshold. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Hi-C experiments were independently replicated twice, and RNA-seq analysis included three biological replicates.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors apply SCRaMBLE genome-wide in Saccharomyces cerevisiae. To make this experiment possible, they engineer a yeast strain containing 83 loxP sites distributed across all 16 nuclear chromosomes. They induce SCRaMBLE in this strain, as well as in an F1 produced by mating this strain to one containing synIII, a synthetic version of Chromosome III that also contains many loxP sites. These experiments show how SRaMBLE can be applied on a genome-wide scale to generate genomic changes within and between chromosomes, producing strains with diverse genome structures. This system can help improve understanding of genome structure and its relationship to traits of evolutionary and biotechnological interest.\nComments:\n1. In the Introduction’s sentence beginning ‘To overcome this problem’, the wording could be a little more precise. Both I-SceI and CRISPR/Cas9 systems involve induced DSB repair. Only the first is stated as involving induced DSB repair.\n2. In the following sentence, the word ‘pre-determined’ is used but arguably SCRaMBLE is still somewhat predetermined. In SCRaMBLE, recombination only happens where loxP sites are present.\n3. In the Results section entitled ‘Genome-wide insertion…’, the following modification would help: ‘Previous studies employing SCRaMBLE in strains with [multiple] synthetic chromosomes…’\n4. In the Results section entitled ‘Induction of Cre recombinase activity…’, succinctly explain the ReSCuES system (a couple sentences or so). Otherwise, the reader must look at the previous paper to understand why ReSCuES is mentioned.\n5. Figure 4a. I found the arrow notation insufficiently clear. Segments with very similar colors are indistinguishable. This impacts interpretation of SCRaMBLE strains, as it is hard to tell when a single segment has been duplicated multiple times as opposed to multiple neighboring segments having been duplicated. Perhaps in addition to the color gradient, using a mix of filled and unfilled arrows would help. There might be further options to increase the space of distinct arrows, such as crosshatching the fill.\n6. In the Methods section entitled ‘Split-URA3 mediated rearrangement’, ‘splitted’ should be ‘split’.\n7. In the Methods section entitled ‘Junction deletion…’, ‘((‘ should be ‘(‘.\n8. I might have missed them, but data and code availability statements could be missing.", "reviewer_2": "Li and coworkers report a new demonstration of the SCRaMbLE technology, where LoxPsym sites are integrated into the S. cerevisiae genome to yield controllable Cre-mediated chromosomal recombination events. Previous demonstrations of SCRaMbLE only involved LoxP sym sites integrated into one or a few (synthetic) chromosomes, which resulted in few inter-chromosomal rearrangements. In this new iteration of the technology, 83 LoxPSym sites were integrated in various positions distributed over all 16 S. cerevisiae chromosomes in the laboratory strain BY4741/s288c in an attempt to fuel more inter-chromosomal events.\nThe results show that the technology works and results in a broad range of intra- and inter- chromosomal recombination events between the different LoxPSym sites, with a marked increase in recombination frequency for sites that are located close to each other on the chromosome and sites located in open chromatin regions. The authors go on to demonstrate that recombination followed by selection for increased stress tolerance can in some (but not all) cases (i.e. different stress factors) quickly result in the selection of novel variants that show increased tolerance.\nThe authors have conducted a significant amount of high-quality experimental and computational work. Moreover, the text is well-written and the figures are generally clear.\nDespite perhaps not representing a major novel or innovative breakthrough compared to the previous papers that introduce and exploit SCRaMbLE, I feel that this study is interesting because it demonstrates directly just how important structural variation can be for quick evolutionary adaptation (an aspect that is perhaps not discussed in sufficient depth in the paper, see below) and opens new avenues to study this process in more detail in any strain, not just the Sc2.0 strains. Secondly, it provides an alternative route to employ SCRaMbLE to create novel, improved yeast variants.\nWhereas I am generally enthused about the work, I also believe that adding a more detailed analysis of the current results and a validation of some obtained results would improve the impact of the manuscript.\nMajor comments\nSome logical controls seem to be missing from the stress-adaptation experiments. As far as I can see, no wild-type cells (same strain background but not containing any LoxP sites) or uninduced SparLox83 (strain with the LoxP sites, but without induction of the Cre enzyme to induce recombination) were analyzed? This makes it difficult to estimate if and how the SCRaMbLE technology resulted in a quicker and/or better adaptation to stress, which in my mind is an essential piece of information to assess the usefulness of the technology for applications. Moreover, it would also be interesting to measure the potential fitness cost of the specific rearrangements that yield resistance to stress in non-stressful conditions (in other words: what is the tradeoff cost of the rearrangement) and compare this to resistant mutants obtained without SCRaMbLE. I also realize that this last suggestion might be outside the direct scope of this study, although I do think it would be a very interesting aspect that would allow us to better understand the potential of SCRaMbLE.\nWhy was the adaptive laboratory evolution experiment not carried out in the same way (using the same stress factors) for the haploid and diploid strains? It would be interesting to be able to compare the adaptation between these two starting strains…\nI do not agree with the following sentence: “These data indicate that LoxP-mediated large-scale genomic rearrangements changed the 3D genome structure, generating an advantageous phenotype”. I do not think that the data provide solid proof for the causative nature of the observed 3D-rearrangements. It is for example equally possible that changes in local chromatin structure (independent of changes in nuclear organization), readthrough of new neighboring genes, or copy number variation result in beneficial changes in expression of key genes…. In fact, I was surprised to see a Hi-C analysis, but no transcriptome analysis. I do not mean to suggest that it is essential for the authors to tease out what is causing the increased resistance, but I do think it is best to lower the claims and provide a better discussion of all factors that could play a role in the link between the rearrangements and the phenotypes.\nMinor:\n1. Introduction: it would be interesting to give a better overview of the different SCRaMbLE techniques, and also already mention why few inter-chromosomal rearrangements have been found and analyzed in these previous SCRaMbLE studies (namely because sites were often only present on one chromosome, and/or because recombination favored sites that are located close to each other, and those were always present in the dense arrays of LoxP sites in the Sc2.0 project…)\n2. Should this technique still be called SCRaMbLE “Synthetic Chromosome Rearrangement and Modification by LoxPsym-mediated Evolution”, since it does not involve synthetic chromosomes anymore?\n3. Results: It could be a good idea to re-evaluate the labeling of the strains. More informative names (instead of long, unintelligible codes) would avoid confusion and make the manuscript more understandable. Perhaps most importantly, consider re-naming JDY525, as it is the strain that contains 83 Lox sites, and SparLox83 being the code name for the same strain that now also harbors the ReSCuES system. A more systematic, unform naming would make it easier for the reader to follow. In addition, please indicate that JDY525 was constructed from strain BY4741 (this strain only pops up later in the text).\n4. Results: Briefly explain the ReSCuES system.\n5. In the results sections, the authors mention statistical terms in the main text for observations for which no statistical tests were performed, for example: ‘no correlation’ (Extended Data Fig.5b), ‘significant’ (Extended Data Fig.3b, Extended Data Figs. 8b&c).\n6. Summary: The claim that SparLox83xSynIII led to increased genomic rearrangements is not justified by the results; increased compared to which strain? SparLox83 and SynIII were not tested by themselves here.\n7. Summary: “analysis of these evolved strains…”; needs rephrasing, the authors refer to the wrong experiment (SparLox83xSynIII instead of SparLox83).\n8. Section 1: Construction of 83 genomic edits within one strain is very impressive and interesting. It would be very helpful to the field to gain insight in the number of unintended SNPs/InDels that were accumulated during strain construction.\n9. Section 1: It is confusing to me that strain JDY525 is used for these experiments, as the rest of the study is based on SparLox83. I suggest to add the results for this strain to Fig.1b.\n10. Section 1: Please add more information on the design of SparLox83: how were the insertion sites of LoxPsym sites chosen? Why were they not equally distributed amongst the chromosomes? Were they consistently inserted in 3’UTRs (as in the Sc 2.0 project) or also within ORFs/5’UTRs/…?\n11. Fig.1a: Only 74 LoxPsym sites are depicted, please add all sites.\n12. Fig. 1c: Could the authors provide the evidence for these claims as supplemental figure?\n13. Section 2: The authors stress that inter-chromosomal rearrangements pre-dominate over intra-chromosomal rearrangements. Although this is true for SparLox83, I wonder whether it is a fair general statement to make? In fact, intra-chromosomal rearrangements are more likely to occur (an observation which is also made in Fig. 2d and in section 6 (Table S6)), no?\n14. Section 3: It is unclear how ‘chromatin accessibility’ was defined/determined, this information should be added somewhere.\n15. Although I am convinced that the differences in recombination frequency that are observed between different LoxP sites are indeed mostly due to 1) their genomic distance and 2) the local chromatin structure, one cannot exclude other confounding factors, such as for example bias due to selection. Even in rich medium like YPD and optimal growth conditions, some rearrangements might cause a severe fitness defect or even cell death…. This could be pointed out more explicitly.\n16. Fig. 2e: I suggest adding the information for intra-chromosomal ARR.\n17. Section 4: The data for growth on ethanol, H2O2 and NaCl are missing from Extended Data Fig. 6a, please provide this information.\n18. Section 5: Could the authors analyze and describe the results in more detail? Information on how many recombination events were observed in total, how many per strain, how many inter-/intra-chromosomal and how many on synIII, how many of each type of recombination, ….. is not mentioned, but valuable info. I believe some of this data is given in Table S6, but I would strongly encourage the authors to add an extra panel to Fig. 4 to discuss this information and adapt their interpretation of the results.\n19. Fig. 4a: Define y-axis, as the figure shows the structural rearrangements in synIII, rearrangements between synIII and chromosomes from SparLox83 should also be shown in this panel.\n20. Fig. 5a: not very clear, please specify why some reads are grey/blue and add ChrA/B to all schematics.\n21. Fig. 5b: why is this data not shown for all chromosomes? Could the authors provide this information as supplemental material? And, add the data for ChrIII to the main figure?\n22. Section 7: not all populations show growth media with 0.4 % HAc (Extended Data Fig. 8b), please adapt the text.\n23. Discussion: It is very interesting that more recombination events were observed in haploid strains compared to the diploid. However, the first explanation of the authors (that recombination in haploids might lead to lethality) actually suggests the opposite. The second explanation should be rephrased (it is not the same experiment) and could in principle be investigated (the data is available to look for LOH and aneuploidy in these strains). Could you hypothesize more on potential explanations and mention whether this has been observed previously?\n24. Discussion: I am confused about the mentioning of a haploid strain with an essential gene plasmid. I believe this was not mentioned in the result section, could you please clarify this?\n25. Discussion: At the end of the section, the authors mention that an increased frequency of genomic rearrangements between SparLox83-derived chromosomes and synthetic chromosomes can be expected. I wonder why this is claimed, as the data in the manuscript suggests otherwise (e.g. the general decrease in genomic rearrangements for diploid strains (fig.1) and the low frequency of SparLox83 rearrangements in the presence of other synthetic chromosomes (Table S6))?\n26. Discussion: you mention that chromosomal rearrangements (or more commonly: structural variation) is a known, important driver of adaptive evolution. This is especially the case in yeast, where genomic rearrangements are often hypothesized to offer a “quick fix” to a sudden, harsh challenge (as they are often used in ALE). It would perhaps be appropriate to cite some of the pioneering work here, and discuss in more depth how SCRaMbLE could be used to study this process in more detail.\n27. Discussion: Please discuss if and how you could overcome the huge preference for recombination between proximal Lox sites in the complete Sc2/.0? If this is not resolved, the repertoire of rearrangements that will result from recombining the SCRaMbLE system in Sc2.0 will be quite limited?\n28. Methods, yeast colony PCR: indicate polymerase\n29. Methods, SCRaMbLE: provide information on the Cre-plasmid (Addgene ID/reference), specify if cells were kept in liquid cultures for consecutive rounds of SCRaMbLE or if they were plated in between.\n30. Data availability statement is missing and access of the WGS data is not provided.\n31. Table S2: specify the locus of ReSCuES integration in SparLox83.\nTypos:\n1. Summary: heterodiploid strains < strain; strains possessing synIII < strain\n2. Results, section 2: r=0.9828 >< fig.2c mentions R2\n3. Results, section 4: reference Luo et al., 2018 < reference 26\n4. Results, section 5: ‘often’ deleted: often should be removed if all 9 colonies were obtained from a single induced population. It should be mentioned clearly in the text how many biological repeats were used. If this was one biological repeat, also the discussion ‘similar biases’ should be adapted.\n5. Results, Fig.5: bule < blue\n6. Results, section 5: mention LOH=loss of heterozygosity\n7. Discussion, were existed < existed\n8. Methods, SCRaMbLE: SparL83<SparLox83\n9. Methods, Split-URA3: palte<plate, inducted<induced, other typo’s\n10. Methods, WGS: according<according\n11. Methods, junction detection: number 2 seems to be a mistake in formula Nrei/2Normi+Nrei\n12. Methods, Hi-C library: Spar83L<SparLox83", "reviewer_1_re-submission": "The authors apply SCRaMBLE genome-wide in Saccharomyces cerevisiae. To make this experiment possible, they engineer a yeast strain containing 83 loxP sites distributed across all 16 nuclear chromosomes. They induce SCRaMBLE in this strain, as well as in an F1 produced by mating this strain to one containing synIII, a synthetic version of Chromosome III that also contains many loxP sites. These experiments show how SRaMBLE can be applied on a genome-wide scale to generate genomic changes within and between chromosomes, producing strains with diverse genome structures. This system can help improve understanding of genome structure and its relationship to traits of evolutionary and biotechnological interest."}, "author_response": "Response: We appreciated the reviewer for recognizing the values and significance of our work and providing the helpful comments. The point-to-point responses are as follows.\nComments:\n1. In the Introduction’s sentence beginning ‘To overcome this problem’, the wording could be a little more precise. Both I-SceI and CRISPR/Cas9 systems involve induced DSB repair. Only the first is stated as involving induced DSB repair.\nResponse: Thank you for pointing this out. We have revised the text as follows: “To quantify the impact of genomic rearrangement independently, several methods have been developed to construct targeted long-range rearrangements in model organisms, including, Cre/loxP recombination, I-SceI-induced and CRISPR/Cas9-induced DSB repair.”\n2. In the following sentence, the word ‘pre-determined’ is used but arguably SCRaMBLE is still somewhat predetermined. In SCRaMBLE, recombination only happens where loxP sites are present.\nResponse: We thank for the reviewer’s careful reading. We agree with the reviewer’s comment and have deleted the word ‘pre-determined’.\n3. In the Results section entitled ‘Genome-wide insertion…’, the following modification would help: ‘Previous studies employing SCRaMBLE in strains with [multiple] synthetic chromosomes…’\nResponse: We appreciate the reviewer for the comments and have revised the sentence according to the reviewer’s suggestion.\n4. In the Results section entitled ‘Induction of Cre recombinase activity…’, succinctly explain the ReSCuES system (a couple sentences or so). Otherwise, the reader must look at the previous paper to understand why ReSCuES is mentioned.\nResponse: We appreciate the reviewer for pointing this out. We have explained the ReSCuES system in the revised manuscript as follows: “ReSCuES was a reporter to efficiently identify SCRaMbLEd cells based on a loxP-mediated switch of “on” and “off” states of the two auxotrophic markers, URA3 and LEU2.”\n5. Figure 4a. I found the arrow notation insufficiently clear. Segments with very similar colors are indistinguishable. This impacts interpretation of SCRaMbLE strains, as it is hard to tell when a single segment has been duplicated multiple times as opposed to multiple neighboring segments having been duplicated. Perhaps in addition to the color gradient, using a mix of filled and unfilled arrows would help. There might be further options to increase the space of distinct arrows, such as crosshatching the fill.\nResponse: We are sorry for the confusing writing in the manuscript. The arrow notation with gradient rainbow colors was followed the “SCRaMbLEgrams” which was a tool to depict the recombination events observed in SCRaMbLEd strains in Sc.2.0 project (Genome Research, 2016, PMID: 26566658). In response to the reviewer’s suggestion, we have regenerated the graph (Figure RL1a). However, accurately discerning the precise location of each segment within the rearranged chromosome remains challenging. Nevertheless, it was evident from both the previous and modified graphs that each strain exhibited a unique synIII structure. Considering no significant improvements were achieved and to maintain consistency with other studies in the Sc.2.0 project, this graph has not been revised accordingly.\nIn addition, detailed information can be found in Extended Data Fig. 11a&b. Taking JDY553 and JDY596 as examples, the segments of the rearranged genome were plotted against the corresponding segments of the parental genome in their original sequential order (Figure RL1b). As visualization, rearrangement types were classified as deletions, inversions, duplications, duplicated inversions, inverted duplications, and complex rearrangements with respect to the parental synIII sequence. Then, the fate of each segment in each strain could be determined (Figure RL1c). By combining this information together, a comprehensive depiction of synIII's specific rearrangement could be achieved.\nFigure RL1. a. The arrangement of synIII in each strain modified according to the reviewer’s suggestion.\nb. Dot-plots illustrating synIII rearrangements in JDY553 and JDY596. Each dot represents a fragment between two loxPsym sites. Black dots represent fragments arranged in synIII sequence order and blue dots represent fragments inverted with respect to the synIII sequence. c. The fate of each segment in each strain was classified as deletion (gray), inversion (blue), duplication (yellow), duplicated inversion (dark green), inverted duplication (light green), multiple duplication (red) or identical to the parent strain (white).\n6. In the Methods section entitled ‘Split-URA3 mediated rearrangement’, ‘splitted’ should be ‘split’.\nResponse: As suggested, we have changed the “splitted” to “split”.\n7. In the Methods section entitled ‘Junction deletion…’, ‘((‘ should be ‘(‘.\nResponse: We sincerely thank the reviewer for careful reading and this has been corrected.\n8. I might have missed them, but data and code availability statements could be missing.\nResponse: Thanks for pointing this out and we have supplemented date availability statement in the revised manuscript.\nResponse: We appreciated the reviewer for recognizing the values and significance of our work and providing the constructive and insightful comments. The point-to-point responses are as follows.\nMajor comments\nSome logical controls seem to be missing from the stress-adaptation experiments. As far as I can see, no wild-type cells (same strain background but not containing any LoxP sites) or uninduced SparLox83 (strain with the LoxP sites, but without induction of the Cre enzyme to induce recombination) were analyzed? This makes it difficult to estimate if and how the SCRaMbLE technology resulted in a quicker and/or better adaptation to stress, which in my mind is an essential piece of information to assess the usefulness of the technology for applications. Moreover, it would also be interesting to measure the potential fitness cost of the specific rearrangements that yield resistance to stress in non-stressful conditions (in other words: what is the tradeoff cost of the rearrangement) and compare this to resistant mutants obtained without SCRaMbLE. I also realize that this last suggestion might be outside the direct scope of this study, although I do think it would be a very interesting aspect that would allow us to better understand the potential of SCRaMbLE.\nResponse: Thank you for the reviewer’s comments. Regarding the absence of control cells, there are two possible explanations. Firstly, it should be noted that the SCRaMbLE system is an inherent feature of Sc 2.0 and its effectiveness in facilitating adaptive evolution has been demonstrated in previous studies. Comparative colonies with increased ethanol tolerance were observed when β-estradiol was added to induce Cre recombinase expression, while no colony growth was observed on stress plates without β-estradiol induction (Figure RL2a from Nature Communications, 2018, PMID:29789540). Similar results have also been reported in other synthetic chromosome-based SCRaMbLEing studies. Therefore, we believe that the induction of Cre is necessary to generate strains with enhanced HAc tolerance.\nSecondly, our aim in conducting stress-adaptation experiments was to demonstrate that genomes containing sparsely distributed loxPsym sites could serve as a potential strategy for enhancing strain performance under stressful conditions. To achieve this goal, we employed two different genotypic strains: SparLox83R×synIII and BY4741×synIII. Both strains contained a synthetic chromosome and one copy of 16 chromosomes with or without loxPsym sites, respectively. The diploid strain BY4741×synIII was used as a control because previous studies have shown that heterozygous diploid strains carrying a synthetic chromosome can rapidly generate new phenotypes through environmental selection (Figure RL2b from Nature Communications, 2018, PMID:29789590). After three rounds of evolution, populations derived from SparLox83R×synIII exhibited superior growth on media containing 0.6% HAc compared to those derived from BY4741×synIII. Considering the genotypic differences between these two strains, we concluded that the accelerated rate of evolution may be associated with rearrangements mediated by sparsely distributed loxPsym sites.\nIn addition, thank you for the later suggestion, we would be pursued late.\nFigure RL2. a. SynXII strain with or without inducing SCRaMbLE by estradiol was plated onto SC-Ura plates containing 8% of ethanol. The cited figure was derived from the article PMID:29789540. b. Serial dilution assay comparing the growth of SCRaMbLEd strains to their non-SCRaMbLEd parent (yMS521) on high caffeine YPD plates. The cited figure was derived from the article PMID:29789590.\nWhy was the adaptive laboratory evolution experiment not carried out in the same way (using the same stress factors) for the haploid and diploid strains? It would be interesting to be able to compare the adaptation between these two starting strains…\nResponse: Thank you for the reviewer’s comments. We have indeed conducted a similar experiment using the haploid and diploid strains. Initially, we tested the original strains, SparLox83R and JDY536, for HAc tolerance (Figure RL3a). Subsequently, 12 individual colonies from each strain were inoculated into a 96-well microplate, and SCRaMbLE-Selection based evolution cycles were performed. Following the first round of SCRaMbLE (Round 1), three different cell concentrations ranging from OD 1.0 to 0.01 were tested on YPD plates with a gradient of HAc (Figure RL3b). 600 Compared to the parental strains, both haploid and diploid populations exhibited increased tolerance to HAc. However, the diploid populations demonstrated superior growth compared to those derived from SparLox83R. After the second round, most SCRaMbLEant populations of the diploid strains were able to grow on 0.6% HAc media at an OD =1.0, while populations from SparLox83R still 600 displayed growth defect on 0.5% HAc media at an OD =0.1 and 0.01 (Figure RL3c). Further 600 evolution was not pursued as the diploid populations exhibited better growth than those derived from SparLox83R.\nAlthough a differential evolution rate was observed between the haploid and diploid strains, we aimed to investigate the potential of enhancing strain performance under stressful conditions by combining genomes containing sparsely distributed loxPsym sites and synthetic chromosomes, which theoretically could generate greater genomic heterogeneity. Therefore, these results were not included in the revised manuscript.\nFigure RL3. Rapid development of HAc tolerance using SCRaMbLE in the haploid and diploid strains.\na. Serial dilution assay on YPD medium with/without HAc. Cells at log phase were diluted to OD 600 =1.0, and then 10-fold dilution. Two strains, SparLox83R and JDY536, showed growth defect on YPD with 0.4% and 0.5% HAc. b&c. Growth of 24 SCRaMbLEant populations after first and second round SCRaMbLE. After SCRaMbLE, the saturated culture was diluted to OD =1.0, 0.1 or 0.01 and spotted 600 onto YPD with/without HAc.\nI do not agree with the following sentence: “These data indicate that LoxP-mediated large-scale genomic rearrangements changed the 3D genome structure, generating an advantageous phenotype”. I do not think that the data provide solid proof for the causative nature of the observed 3D-rearrangements. It is for example equally possible that changes in local chromatin structure (independent of changes in nuclear organization), readthrough of new neighboring genes, or copy number variation result in beneficial changes in expression of key genes…. In fact, I was surprised to see a Hi-C analysis, but no transcriptome analysis. I do not mean to suggest that it is essential for the authors to tease out what is causing the increased resistance, but I do think it is best to lower the claims and provide a better discussion of all factors that could play a role in the link between the rearrangements and the phenotypes.\nResponse: Thank you for the reviewer to point this out. According to the reviewer’s suggestion, we have revised the sentence to “These data revealed a connection among large-scale rearrangement, genome 3D structure, transcription, and phenotype, demonstrating that simply reconfiguring chromosome architecture was sufficient to provide fitness advantages in stressful growth conditions.”\nIn addition, we performed the transcriptome analysis comparing JDY528 and SparLox83R. As shown in Figure RL4a, the duplicated region (from YDR073W to YDR277C) exhibited significant up-regulation of the majority of genes. Conversely, a decrease in copy number from two to one on chrIX resulted in decreased expression levels for most genes. The differentially expressed genes were further mapped to the 3D model of JDY526 genome and showed strong colocalization with 3D structure alterations. Only one gene, YDR001C, located nearby the junctions, showed differential expression. However, its knock-out strain showed comparable tolerance to nocodazole when compared to wild-type strain (Figure RL4b). Considering that changes in expression levels were not limited to neighboring regions of the translocation breakpoint, it may explore a larger set of genes in subsequent analyses. Furthermore, it was possible that nocodazole resistance could be influenced by both the large-scale duplication on ChrIV and the copy number variation of ChrIX. The information has been added in the revised Extended Data Fig. 10.\nFigure RL4. Transcriptome analysis between JDY528 and SparLox83R. a. Volcano plot of global expression changes of JDY526 compared to SparLox83R. b. Serial dilution assay comparing the knock-out strain and the wild-type strain.\nMinor:\n1. Introduction: it would be interesting to give a better overview of the different SCRaMbLE techniques, and also already mention why few inter-chromosomal rearrangements have been found and analyzed in these previous SCRaMbLE studies (namely because sites were often only present on one chromosome, and/or because recombination favored sites that are located close to each other, and those were always present in the dense arrays of LoxP sites in the Sc2.0 project…)\nResponse: We appreciate the reviewer for the constructive comments. Following the reviewer’s suggestions, we have supplemented an overview of the different SCRaMbLE techniques in the introduction section as follows:\n“Moreover, intra-chromosomal rearrangements dominate, with inter-chromosomal rearrangements rarely detected in individual SCRaMbLEd strains despite the utilization of a strain with four synthetic chromosomes31. By sequencing SCRaMbLEd pools derived from inducing a strain harboring 5.5 synthetic chromosomes, comparable frequencies of intra- and inter-chromosomal events were detected at 47.24% and 52.67%, respectively32. However, only 9.87% of the total reads accounted for inter-chromosomal recombination, indicating that the occurrence of inter-chromosomal recombination was significantly lower than that of intra-chromosomal recombination in cells containing multiple synthetic chromosomes. Considering that the spatial proximity between loxPsym sites plays a crucial role in chromosome interactions, it can be inferred that translocation events would be more frequent when loxPsym sites are distributed throughout the entire genome.”\nPlease see line 86-96 in page 5.\n2. Should this technique still be called SCRaMbLE “Synthetic Chromosome Rearrangement and Modification by LoxPsym-mediated Evolution”, since it does not involve synthetic chromosomes anymore?\nResponse: Thank you for the reviewer to point this out. We agree with the reviewer that SCRaMbLE is a synthetic chromosome engineering technique which was first introduced as part of the Sc2.0-project. It is based on Cre/loxPsym recombination system by integration of loxPsym sequences into synthetic chromosomes. As the Cre/loxPsym recombination system is a universal and functional way to generate structural variation, the general concept of SCRaMbLE can be exploited to other yeast strains equipped with recombination sites (Nature Communications, 2018, PMID:29789533). In this study, we employed a CRISPR/Cas9-based strategy to efficiently integrate loxPsym sequences into multiple genomic loci, thereby inducing structural variations across the entire genome through the Cre/loxPsym recombination system. Thus, we still called this technique as SCRaMbLE.\n3. Results: It could be a good idea to re-evaluate the labeling of the strains. More informative names (instead of long, unintelligible codes) would avoid confusion and make the manuscript more understandable. Perhaps most importantly, consider re-naming JDY525, as it is the strain that contains 83 Lox sites, and SparLox83 being the code name for the same strain that now also harbors the ReSCuES system. A more systematic, unform naming would make it easier for the reader to follow. In addition, please indicate that JDY525 was constructed from strain BY4741 (this strain only pops up later in the text).\nResponse: Thanks for the reviewer’s great suggestion. JDY525 is the strain with 83 Sparsely distributed LoxPsym sites and SparLox83 is generated by integrating ReSCuES system into JDY525. To make the manuscript more understandable, we have modified JDY525 to SparLox83 and SparLox83 in the previous version to SparLox83R in the revised manuscript.\n4. Results: Briefly explain the ReSCuES system.\nResponse: We appreciate the reviewer for the comments and we apologize for the confusing writing in the manuscript. We have explained the ReSCuES system in the revised manuscript as follows: “ReSCuES was a reporter to efficiently identify SCRaMbLEd cells based on a loxP-mediated switch of “on” and “off” states of the two auxotrophic markers, URA3 and LEU2.”\n5. In the results sections, the authors mention statistical terms in the main text for observations for which no statistical tests were performed, for example: ‘no correlation’ (Extended Data Fig.5b), ‘significant’ (Extended Data Fig.3b, Extended Data Figs. 8b&c).\nResponse: Thanks for the reviewer to point this out. According to the reviewer’s suggestions, the correlation between the RW values and the distances of two loxPsym sites on different chromosomes were determined (Figure RL5a), as showed in revised Extended Data Fig. 7c.\nAs for Extended Data Fig.3b, the absence of data replication precluded the accurate application of a one-way ANOVA test. Thus, Pearson correlation coefficients (R package: psych, v2.3.9) were computed within the ARRs of the four different lengths in haploid and diploid cells, yielding P values below 0.01 (Figure RL5b). Please see revised Extended Data Fig. 4b.\nAs for Extended Data Figs. 8b&c, we quantitatively assessed the growth of the strains before and after multiple rounds of directed evolution on YPD and YPD + HAc plates using CellProfiler v4.2.6. As the edges of the plates were relatively thick and had no neighboring colonies, providing a nutrient-rich environment and faster growth for the strains, we first removed the colonies located at the edges of the plates. For the remaining colonies, we utilized a customized yeast patch identification workflow (https://cellprofiler.org/examples) to quantify the mean intensity of their forced spots. Specifically, we first compressed the images into approximately 1200x800 png files. Then, we cropped them based on the positions of the colonies in the images. The \"Block size\" parameter in the CorrectIlluminationCalculate step was adjusted to 80, the \"Typical diameter of objects\" parameter in the IdentifyPrimaryObjects step was adjusted to 8-80, and the \"Minimum FormFactor value\" and \"Minimum Area value\" in the FilterObjects step were adjusted to 0.40 and 90, respectively. Finally, we performed an unpaired two-tailed t-test to assess the significance of the growth differences (Figure RL5c). Please see revised Extended Data Fig. 14b&c&d.\nFigure RL5. Examples for statistical tests. a. Correlation between RW in haploid cells and 1/genomic distance between loxPsym sites on the different chromosome. b. ARR calculated using different flank region lengths in haploid cells. Pearson correlation coefficients within the four lengths in haploid cells were analyzed. c. The growth of the strains on YPD of Round 3 were quantitatively evaluated using CellProfiler v4.2.6.\n6. Summary: The claim that SparLox83xSynIII led to increased genomic rearrangements is not justified by the results; increased compared to which strain? SparLox83 and SynIII were not tested by themselves here.\nResponse: We appreciate the reviewer for the comments and we apologize for the confusing writing in the manuscript. To demonstrate that the loxPsym sites in SparLox83R could combine with synthetic chromosomes to generate genomic rearrangements, we detected the structural variations in nine SCRaMbLEd SparLox83RxSynIII colonies. Rearrangements were not only observed within SparLox83R-derived loxPsym sites and synIII-derived loxPsym sites, but also between them. Thus, we concluded that SparLox83RxSynIII led to increased genomic rearrangements compared with BY4741 × synIII, which was a strain with only synIII.\nTo avoid vagueness, we have revised the text as follows: “Moreover, SCRaMbLE of the hetero-diploid strains derived from crossing SparLox83R with strains possessing synthetic chromosome III (synIII) from the Sc2.0 project led to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to a hetero-diploid strain with only synIII.”\n7. Summary: “analysis of these evolved strains…”; needs rephrasing, the authors refer to the wrong experiment (SparLox83xSynIII instead of SparLox83).\nResponse: We appreciate the reviewer for the comments and sorry for the mistake. We have corrected the description as: “Analysis of the SCRaMbLEd strain with increased tolerance to nocodazole demonstrated that genomic rearrangements can perturb the transcriptome and 3D genome structure and can consequently impact phenotypes.”\n8. Section 1: Construction of 83 genomic edits within one strain is very impressive and interesting. It would be very helpful to the field to gain insight in the number of unintended SNPs/InDels that were accumulated during strain construction.\nResponse: We appreciate the reviewer for the valuable suggestion, and we have indeed performed the SNPs/InDels analysis in SparLox83 using the short illumina reads derived from whole-genome shotgun sequencing. Totally, we identified 1197 SNPs/InDels in nuclear genome when we aligned the NGS reads to the yeast reference genome (GCF_000146045.2). These SNPs/InDels include 83 loxPsym insertion sites, a ReSCueS model, as well as the laboratory modifications when constructed the parental strain of SpaLox83. Furthermore, we detected 158 overlapping open reading frames (ORFs) associated with 305 SNPs/InDels. Notably, YAL063C (FLO9), YBR005W (RCR1), YBR148W (YSW1), and YEL042W (GDA1) exhibited significant frameshift mutations. The detailed information of SNPs/InDels is added in Table S3.\n9. Section 1: It is confusing to me that strain JDY525 is used for these experiments, as the rest of the study is based on SparLox83. I suggest to add the results for this strain to Fig.1b.\nResponse: We appreciate the reviewer for the valuable suggestion. Initially, we employed a CRISPR-Cas9 based gene editing strategy to integrate loxPsym sites, resulting in the generation of JDY525 strain with 83 inserted sites. Given the addition of these 83 loxPsym sites, it was important to evaluate the fitness and genome stability of the final strain. Subsequently, JDY525 was utilized to evaluate the phenotype on various types of media and perform PCRTag analysis of 35 independent lineages after ~120 mitotic generations. After confirming that JDY525 remained unaffected in terms of genome stability and growth fitness, we integrated ReSCuES as a reporter into JDY525, generating SparLox83 strain to identify SCRaMbLEd cells. In the following SCRaMbLE experiments, we used SparLox83. We agreed with the reviewer that the phenotype of SparLox83 should also be contained in Fig. 1b. The serial dilution tests were performed as showed in the revised Fig. 1b.\nFigure RL6. Revised Fig. 1b. SparLox83 and SparLox83R phenotyping on various media.\n10. Section 1: Please add more information on the design of SparLox83: how were the insertion sites of LoxPsym sites chosen? Why were they not equally distributed amongst the chromosomes? Were they consistently inserted in 3’UTRs (as in the Sc 2.0 project) or also within ORFs/5’UTRs/…?\nResponse: We appreciate the reviewer’s comments and we are sorry for the vague description. The principle of the loxPsym sites we chose is that these sites are located at the intergenic regions which contained a PAM site, NGG, and the insertions should not disrupt any possible functional elements. However, four off-target insertions were detected in the final strain, which were II-3, II-8, IV-8 and V-2. Three of the four sites (II-3, II-8, and V-2) were located at the ORFs of non-essential genes. As the strain did not show distinguishable growth defect, the three loxPsym sites were retained. The information on the design of SparLox83 was added in the revised text. Please see line 125-136 in page 7.\nWe have revised the text as follows: “All the insertion sites were designed at the intergenic regions which contained a PAM site, NGG, without disrupting any possible functional elements. Briefly, three loci were simultaneously targeted for integration of loxPsym sites in each cycle, eventually producing a strain with 83 sites (SparLox83) distributed across the 16 chromosomes, with at least two sites per chromosome (Fig. 1a). The presence of loxPsym at each locus was confirmed by whole genome sequencing (WGS; Table S1 and S2). The final strain exhibited four off-target insertions, namely II-3, II-8, IV-8, and V-2. Among these sites, three (II-3, II-8, and V-2) were located within the ORFs of non-essential genes. Since the strain did not display any discernible growth defects (Fig. 1b), we opted to retain the three loxPsym sites. Meanwhile, multiple loxPsym sites were identified at the same loci through self-duplication of the plasmid backbone sequence (Fig. 1a and Extended Data Fig. 1b).”\n11. Fig.1a: Only 74 LoxPsym sites are depicted, please add all sites.\nResponse: We appreciate the reviewer’s carefully reading and we are sorry for the vague description. According to the results of whole-genome sequencing, a sequence containing the sgRNA plasmid backbone sequence with two loxPsym sites was inserted into the targeted sites, including IV-12/IV-13, IX-2/IX-3, X-1/X-2, X-3/X-4 and XV-2/XV-3. Moreover, three duplications of the sgRNA plasmid backbone sequence with four loxPsym sites were detected at XIII-1/XIII-2/XIII-3/XIII-4. Revisions have been made to Fig. 1a and specific descriptions have been incorporated into Extended Data Fig. 1b of the revised manuscript in order to provide a comprehensive presentation of these sites.\nFigure RL7. Revised Fig. 1a and Extended Data Fig. 1b. Distribution of 83 loxPsym sites in the SparLox83 strain. a. A comprehensive overview of the 83 loxPsym sites across 16 chromosomes. b. The detailed information for the multiple sites at the same loci through self-duplication of the plasmid backbone sequence.\n12. Fig. 1c: Could the authors provide the evidence for these claims as supplemental figure?\nResponse: According to the reviewer for the suggestion, we have supplemented the PCR verification results to Extended Data Fig. 3, which were amplified by using the genomic DNA of 35 independent colonies after ~120 mitotic generations as templates (Figure RL8).\nFigure RL8. Revised Extended Data Fig. 3. PCR verification for 24 loxPsym sites in 35 independent colonies after ~120 mitotic generations. The absence of amplicons in the initial amplification was indicated by the red rectangles, while subsequent repetitions revealed the presence of correct amplicons, displayed at the bottom right corner.\n13. Section 2: The authors stress that inter-chromosomal rearrangements pre-dominate over intra-chromosomal rearrangements. Although this is true for SparLox83, I wonder whether it is a fair general statement to make? In fact, intra-chromosomal rearrangements are more likely to occur (an observation which is also made in Fig. 2d and in section 6 (Table S6)), no?\nResponse: We appreciate the reviewer's question. Our sequencing analysis of the mixed culture indeed revealed a higher frequency of inter-chromosomal events compared to intra-chromosomal ones. However, we observed that there are more inter-chromosomal candidates than intra-chromosomal candidates for each loxPsym site, potentially introducing bias. To accurately quantify the preference for rearrangements, it is necessary to normalize this bias. Thus, we introduced the Average Rearrangement Rate (ARR) to quantify the average rearrangement potential for each loxPsym site as showed in Fig. 2d. In the table S6, intra-chromosomal rearrangements were dominated because the events occurring between the loxPsym sites on synIII were included. The distances of synIII-derived sites were much closer than those of SparLox83R-derived sites, resulting a preference for interaction.\n14. Section 3: It is unclear how ‘chromatin accessibility’ was defined/determined, this information should be added somewhere.\nResponse: Thanks for the reviewer’s suggestion. The definition of chromatin accessibility has been added to the revised manuscript as follows: “Chromatin accessibility refers to the degree physical accessibility and availability of DNA within the chromatin structure for interaction with various regulatory factors, such as transcription factors and other proteins. It describes the ability of these factors to bind specific genomic regions of the genome and regulate gene expression (Nucleus., 2022, PMID: 36404679).” Please see line 248-253 in page 12.\n15. Although I am convinced that the differences in recombination frequency that are observed between different LoxP sites are indeed mostly due to 1) their genomic distance and 2) the local chromatin structure, one cannot exclude other confounding factors, such as for example bias due to selection. Even in rich medium like YPD and optimal growth conditions, some rearrangements might cause a severe fitness defect or even cell death…. This could be pointed out more explicitly.\nResponse: Thank you for the reviewer’s constructive suggestion. We agree with the reviewer that it should be taken into consideration that the cells with different rearrangements varies in growth rate and bias may be introduced by phenotypic selection. However, it is difficult to distinguish whether the rearrangement preference is due to the fact that some pairs of loxPsym sites are prone to rearrange than other pairs or growth advantages brought about by rearrangements in some cells because both of them could result in increased cell amounts in the SCRaMbLEd pool for sequence. On the contrary, rearrangements which cause severe fitness defect may be covered in the cell pool as limited cells could spread in the population.\nWe have supplemented the discussion in the revision as: “Additionally, the distribution of cells with different rearrangements in the final population for sequence can be influenced by growth advantages or disadvantages resulting from structural variations. This factor also contributes to rearrangement preferences when utilizing the SCRaMbLEd pool for sequencing, although it is not easily distinguishable on its own.” Please see line 509-513 in page 22.\n16. Fig. 2e: I suggest adding the information for intra-chromosomal ARR.\nResponse: Following the reviewer’s suggestion, the information for intra-chromosomal ARR has been added to Extended Data Fig. 6. As no significant correlation was observed for intra-chromosomal ARRs, this information was not provided in the previous manuscript.\nFigure RL9. Revised Extended Data Fig. 6. Comparison of intra-chromosomal ARRs between loxPsym sites within (+) and outside (-) open chromatin regions in haploid cell.\n17. Section 4: The data for growth on ethanol, H2O2 and NaCl are missing from Extended Data Fig. 6a, please provide this information.\nResponse: According to the reviewer’s suggestion, the results have been added to Extended Data Fig. 8a of the revised manuscript.\nFigure RL10. Revised Extended Data Fig. 8a. SCRaMbLEd cells exhibited similar tolerance to the parent strain SparLox83R under stress conditions. Cells at log phase were 10-fold serially diluted onto YPD agar plates containing 10% ethanol, 3 mM H O , 1 M NaCl, or 3% glycerol and incubated for 2 2 2 days at 30°C.\n18. Section 5: Could the authors analyze and describe the results in more detail? Information on how many recombination events were observed in total, how many per strain, how many inter-/intra-chromosomal and how many on synIII, how many of each type of recombination, ….. is not mentioned, but valuable info. I believe some of this data is given in Table S6, but I would strongly encourage the authors to add an extra panel to Fig. 4 to discuss this information and adapt their interpretation of the results.\nResponse: According to the reviewer’s suggestion, the status of each segment in the 9 strains were exhibited in the panel and the figure has been added to Extended Data Fig. 11b. In the figure, recombination events were classified as deletions (gray), inversions (blue), duplications (yellow), duplicated inversion (dark blue), inverted duplication (light blue) and complex rearrangements (red) with respect to the parental synIII sequence according to previous report (Genome Research, 2016, PMID: 26566658). Based on this figure, we quantified and presented the counts of each type of recombination event on synIII in Table S5. Since it was challenging to determine whether adjacent segments underwent simultaneous rearrangement, we considered each segment as an individual unit for counting. Relevant information regarding the rearrangements of synIII has been included in the supplementary figures, as it was not deemed to be the focal point of this paper.\nFigure RL11. Revised extended Data Fig. 11a and 11b. Rearrangements of synIII were observed in SCRaMbLE cells. a. Dot-plots illustrating synIII rearrangements in JDY553 and JDY596. Each dot represents a fragment between two loxPsym sites. Black dots represent fragments arranged in synIII sequence order and blue dots represent fragments inverted with respect to the synIII sequence. b. The fate of each segment in each strain was classified as deletion (gray), inversion (blue), duplication (yellow), duplicated inversion (dark green), inverted duplication (light green), multiple duplication (red) or identical to the parent strain (white).\n19. Fig. 4a: Define y-axis, as the figure shows the structural rearrangements in synIII, rearrangements between synIII and chromosomes from SparLox83 should also be shown in this panel.\nResponse: We are sorry for the vague description. The figure 4a represented the segment order and orientation of rearranged synIII for the nine strains which were generated by SCRaMbLEgrams, a tool to depict the recombination events observed in SCRaMbLEd strains in Sc.2.0 project (Genome Research, 2016, PMID: 26566658). The y-axis indicates the name of a SCRaMbLEd strain and the x-axis was the arrangement of synIII which was visualized as a sequence of arrows. The arrows represent segments which are flanked by loxPsym sites on synIII. The color of each arrow indicates the segment number in the parental chromosome, and the direction of the arrow represents the orientation.\nThere were two reasons that the rearrangement between synIII and chromosomes from SparLox83R was not shown in the figure. Firstly, the segment sizes flanked by two adjacent loxPsym sites of SparLox83R were significantly larger than those of synIII, resulting in distinct arrow representations for chrIII compared to synIII. The rearrangement between synIII and chrIII was an inter-chromosome event while the rearrangements displayed in Fig. 4a all belonged to intra-chromosomal events. Furthermore, a homologous recombination-mediated rearrangement occurred between synIII and chrIII, which was not mediated by loxPsym sites and could not be visualized as a sequence of arrows. Additionally, the comprehensive depiction of the rearrangements occurring between synIII and chrIII has been provided in Fig. 4c, encompassing all the detected rearrangements between synIII and chromosomes from SparLox83.\n20. Fig. 5a: not very clear, please specify why some reads are grey/blue and add ChrA/B to all schematics.\nResponse: We apologize for the lack of clarity in our graphical representation. The color of ChrA has been modified from blue to gray, aligning it with the hue of the associated reads (1&2 in the Figure RL12). The four reads depicted in the figure correspond to the four types identified by sequencing: the reads 1 represents a sequence containing a loxPsym site from ChrA; the reads 2 represents a sequence without loxPsym sites from ChrA; the reads 3 represents a translocation mediated by a loxPsym site between ChrA and ChrB, as half of it could be mapped to ChrA and the remaining portion could be mapped to ChrB; the reads 4 represents a sequence without loxPsym sites from ChrB (the part within the dotted box). The copy number of each fragment of ChrA and ChrB can be determined by calculating based on the sequencing depth, which are represented as 4 reads in the figure. The modified figure has been updated in the revised manuscript.\nFigure RL12. Revised Fig. 5a. The modified workflow of copy number variation analysis.\n21. Fig. 5b: why is this data not shown for all chromosomes? Could the authors provide this information as supplemental material? And, add the data for ChrIII to the main figure?\nResponse: We apologize for the confusing writing. Indeed, the copy numbers of all loxPsym upstream and downstream regions have been assessed. A blue square represents a copy of the upstream or downstream region containing a loxPsym site, while a white square represents a region lacking such a site. Consequently, all regions of chromosomes could be represented by either a white square, or a blue square, or a combination. However, only a subset of regions on several chromosomes showed difference compared to the parent strain as presented in Fig. 5b. According to the reviewer’s suggestion, the information of the rest chromosomes was provided as a supplemental figure, Extended Data Fig. 13.\nAs for chrIII, the presence of PCRTags in synIII enables the differentiation of reads from chrIII and synIII, thereby enabling to determinate their respective copy numbers. Additionally, the complex structural variations on synIII make it impossible to depict the upstream or downstream region of the loxPsym sites as presented in Fig. 5a. Moreover, the copy number variation of synIII has been illustrated in Fig. 4a and Extended Data Fig. 12. Therefore, only the data for ChrIII was provided in Extended Data Fig. 13.\nFigure RL13. Revised Extended Data Fig. 13. Copy numbers of 2 kb upstream/downstream regions flanking the loxPsym sequence were determined for chromosomes without rearrangements in the nine diploid strains. The panels exhibited identical in all the nine strains but with varied synIII.\n22. Section 7: not all populations show growth media with 0.4 % HAc (Extended Data Fig. 8b), please adapt the text.\nResponse: Thanks for pointing this out and we have modified the text as follow: “After the first round of SCRaMbLE (R1), most of the populations were able to grow on media with a maximum of 0.4% HAc (Extended Data Fig. 14b).”\n23. Discussion: It is very interesting that more recombination events were observed in haploid strains compared to the diploid. However, the first explanation of the authors (that recombination in haploids might lead to lethality) actually suggests the opposite. The second explanation should be rephrased (it is not the same experiment) and could in principle be investigated (the data is available to look for LOH and aneuploidy in these strains). Could you hypothesize more on potential explanations and mention whether this has been observed previously?\nResponse: We are sorry for our pool writing for this section. In theory, more possibilities of recombination events could be detected in diploid cells because essential genes positioned between each pair of adjacent loxPsym sites would lead to the loss of viable cells and deletions are only occurred in diploid cells. Unexpectedly, we detected more rearrangement events detected in the haploid pool rather than in the diploid pool. Thus, we firstly speculated that the loss of loxPsym sites after SCRaMbLEing was the major reason. Therefore, we provided two possibilities to cause the loss of loxPsym sites. One was the deletion events which could only be occurred in diploid cells and the other was the LOH and aneuploidy events. According to the reviewer’s comment, we have rephrased the second explanation. Additionally, we provided another potential explanation and discussed the differences between our findings and those based on synthetic chromosomes.\nThe revised text was as follows: “Characterization of genetic rearrangements in SparLox83R and SparLox83R × BY4742 revealed that the proportion of rearrangement events was 10-fold higher in haploid than in diploid cells. There are several possible reasons behind this phenomenon. First, the loss of loxPsym sites could only be occurred in diploid strains. In haploid strains, loxPsym-mediated deletion of fragments might lead to lethality because of essential gene loss47,48 while deletions can occur in the diploid without lethal effects because of the additional presence of the wild-type genome34. Additionally, the possibility of the occurence of LOH and aneuploidy events have been demonstrated by sequencing nine SCRaMbLEant colonies (Fig. 5). This might also be happened in the diploid strain of SparLox83R × BY4742 and further decrease the total number of loxPsym sites49. Moreover, five rounds of induction-selection were performed before sequencing, and this may have further lowered the total number of loxPsym sites. Second, the additional copy of chromosome without loxPsym site may hinder the rearrangement capacity of the inter-chromosomal interaction. Although diploid colonies based on synthetic chromosomes of Sc2.0 exhibited a higher recombinational frequency compared to haploid colonies, no inter-chromosomal events were detected among the hundreds of sequenced diploid colonies31. We also detected a related lower inter-chromosomal interaction proportion in diploid strains (63.6%) than in haploid strains (84.8%) while the expected proportion is 7% for intra-chromosomal events (210/2775) and 93% for inter-chromosomal events (2565/2775) if there is no preference for intra- or inter-chromosomal recombination. Additionally, haploid strains may have high tolerance to gross inter-chromosomal events as its plasticity. However, in a diploid strain, recombination events between different chromosomes may give rise to two distinct genotypes of the homologous chromosome pairs, possibly causing genomic instability and severe growth detect50. More experimental validations are needed.” Please see line 514-538 in page 22.\n24. Discussion: I am confused about the mentioning of a haploid strain with an essential gene plasmid. I believe this was not mentioned in the result section, could you please clarify this?\nResponse: We are sorry for our careless that the literature was not cited in the manuscript. The strain harboring an essential gene plasmid was reported by Wang et. al. (PMID: 32268063). They put all essential genes of yeast chromosome III to a centromeric plasmid and transformed to the strain with synIII. Deletion frequencies of synIII chromosomal segments were revealed by investigation of SCRaMbLEd strains. Several segments with no deletion were detected in all strains, most of which were consistent with our results. Three segments were deleted in our strains which harboring a copy of wild type chrIII, but not in their strains.\nTo avoid misunderstanding, we have added the citation and modified the sentence as follows:\n“Segments 65, 66, and 67 were deleted in our strains but were not retained in the haploid strain previous reported30, which was possessed an essential gene plasmid, indicating that synthetic lethal interactions can be bypassed when a copy of wild type chrIII is present.”\n25. Discussion: At the end of the section, the authors mention that an increased frequency of genomic rearrangements between SparLox83-derived chromosomes and synthetic chromosomes can be expected. I wonder why this is claimed, as the data in the manuscript suggests otherwise (e.g. the general decrease in genomic rearrangements for diploid strains (fig.1) and the low frequency of SparLox83 rearrangements in the presence of other synthetic chromosomes (Table S6))?\nResponse: We apologize for the confusion and thank you for the comment. The SCRaMbLE system is firstly applied for synthetic chromosomes. Several previous studies have revealed that there is a preference for intra-chromosome recombination than the inter-chromosome recombination and the potential of SCRaMbLE to study genomic rearrangements is currently hindered. Here, a strain with 83 loxPsym sites distributed across the 16 chromosomes was constructed. A variety of large-scale genomic rearrangements, especially inter-chromosomal events, were detected in the resultant SCRaMbLEd yeast population. In addition, we demonstrated that the large-scale genomic rearrangements could generate advantageous phenotypes and the loxPsym sites can interact with those from synthetic chromosome. Although decreased genomic rearrangements were identified in diploid strains (Fig. 2), more rapid environmental adaptation was observed in diploid strains than in haploid strains (Figure RL3) and LOH and aneuploidy were detected in single-colony sequencing which were missed in population sequencing. Similarly, strains harboring SparLox83-derived chromosomes and synthetic chromosomes showed an accelerated gain of HAc tolerance (Fig. 6).\nOur results indicated that diploid strains with SparLox83-derived loxPsym sites and synthetic chromosomes can generate more structural variations than haploid strains and diploid strains with only synthetic chromosomes when exposure to the stress because genotypic diversity is an important driver of adaptation to new environments. In addition, a translocation was observed between chromesome III and synIII (Fig. 4c). If the strain with 16 synthetic chromosomes is available, the possibility that interactions between SparLox83-derived chromosomes and synthetic chromosomes would be increased as two copies of any chromosomes harbor loxPsym sites. Based on these, we speculate that the frequency of translocation events using the final Sc2.0 yeast strains harboring all synthetic chromosomes would be increased.\n26. Discussion: you mention that chromosomal rearrangements (or more commonly: structural variation) is a known, important driver of adaptive evolution. This is especially the case in yeast, where genomic rearrangements are often hypothesized to offer a “quick fix” to a sudden, harsh challenge (as they are often used in ALE). It would perhaps be appropriate to cite some of the pioneering work here, and discuss in more depth how SCRaMbLE could be used to study this process in more detail.\nResponse: Thank you for the suggestion. According to the reviewer’s advice, we have further discussed this in the discussion section of the revised version as follows:\n“Previous studies have demonstrated the potential of SCRaMbLE for directed evolution24,26,27. The SCRaMbLE experiment typically involves inducing Cre recombinase in yeast cells containing one or more synthetic chromosomes to generate a population with genomic heterogeneity. This population is then subjected to selective growth conditions for the screening of viable cells52. Compared with classical adaptive laboratory evolution via causal mutations, SCRaMbLE facilitates rapid and extensive genetic rearrangements, including deletion, inversion and duplication. Additionally, aneuploidy is also considered as a quick response to stress53. Our findings suggested that SCRaMbLE based on the loxPsym sites across the 16 chromosomes in SparLox83R led to the potential occurrence of aneuploidy, such as whole chromosome deletion and duplication (Fig. 5c), which were rarely observed in cells with synthetic chromosomes. Thus, these two types of SCRaMbLE could complement each other and be orthogonal to other directed evolution methods as they provide different types of genetic changes54.” Please see line 580-593 in page 24.\n27. Discussion: Please discuss if and how you could overcome the huge preference for recombination between proximal Lox sites in the complete Sc2.0? If this is not resolved, the repertoire of rearrangements that will result from recombining the SCRaMbLE system in Sc2.0 will be quite limited?\nResponse: Thank you for the suggestion. The SCRaMbLE system in Sc2.0 is designed to generate population-level diversity in yeast strains through the utilization of the Cre/loxPsym system. The synthetic chromosomes facilitate large-scale genomic rearrangements between any two loxPsym sites within or across them. Recent studies investigating SCRaMbLE without selection pressure have highlighted the significant influence of Cre enzyme abundance, genome ploidy, and chromatin structures on recombination frequencies and resulting outcomes. These findings suggested there is a preference for interaction with specific loxPsym sites. However, when employing SCRaMbLE as part of a directed evolution strategy, it typically requires exposing yeast cells to challenging environments such as selective pressures or phenotypic screening to identify cells with beneficial genomic rearrangements that enhance adaptation to environmental stress over multiple generations. Furthermore, while rearrangement events are more likely to occur at short distances, long-range recombination events have also been observed. This can be attributed partially to multiple recombination events undergone by SCRaMbLEd genomes (bioRxiv, ID: 507906). Additionally, SCRaMbLE has the capability to reshape both content and order of chromosomes. Thus, each round of SCRaMbLE introduces different proximal loxPsym site configurations which may help to overcome inherent recombination preferences.\nAccording to the reviewer’s suggestion, the related discussion have been added: “Although limited interactions were observed between SparLox83-derived chromosomes and synthetic chromosomes, strains harboring two genotypes of chromosomes exhibited an accelerated acquisition of HAc tolerance (Fig. 6). These findings suggested that exposure to challenging environments was crucial for identifying cells with genomic rearrangements when utilizing the SCRaMbLE system in directed evolution experiments. The composition and arrangement of chromosomes can be reshaped, resulting in different interaction preferences within loxPsym sites during each round of SCRaMbLE. By increasing the number of SCRaMbLE rounds, variants with enhanced fitness can be enriched.” Please see line 570-578 in page 24.\n28. Methods, yeast colony PCR: indicate polymerase\nResponse: Thanks for pointing this out and the polymerase used for yeast colony PCR have been indicated.\n29. Methods, SCRaMbLE: provide information on the Cre-plasmid (Addgene ID/reference), specify if cells were kept in liquid cultures for consecutive rounds of SCRaMbLE or if they were plated in between.\nResponse: The plasmid was constructed by integrating the NatMX cassette into the LYS2 coding sequence based on the plasmid reported by Luo et. al. (Genome Biology, 2021, PMID: 33397424). For the consecutive rounds, cells were kept in liquid cultures during SCRaMbLE. We have modified the related information in the revised manuscript. Please see line 893-895 in page 45.\n“SparLox83R or the heterozygous diploids with ReSCuES sequence integrated were transformed with pGAL-Cre plasmid, which was constructed by integrating the NatMX cassette into the LYS2 coding sequence based on a previously described plasmid21.”\n30. Data availability statement is missing and access of the WGS data is not provided.\nResponse: Thanks for pointing this out and we have supplemented date availability statement in the revised manuscript. Please see line 1041-1048 in page 50.\n31. Table S2: specify the locus of ReSCuES integration in SparLox83.\nResponse: We appreciate the reviewer for pointing this out and we have added the information to table S2.\nTypos:\n1. Summary: heterodiploid strains < strain; strains possessing synIII < strain\nResponse: This has been corrected.\n2. Results, section 2: r=0.9828 >< fig.2c mentions R2\nResponse: This has been corrected.\n3. Results, section 4: reference Luo et al., 2018 < reference 26\nResponse: This has been corrected.\n4. Results, section 5: ‘often’ deleted: often should be removed if all 9 colonies were obtained from a single induced population. It should be mentioned clearly in the text how many biological repeats were used. If this was one biological repeat, also the discussion ‘similar biases’ should be adapted.\nResponse: According to the reviewer’s suggestion, ‘often deleted’ has been revised to “prone to delete”. “Similar biases” has been revised to “high similarity”.\n5. Results, Fig.5: bule < blue\nResponse: This has been corrected.\n6. Results, section 5: mention LOH=loss of heterozygosity\nResponse: This has been corrected.\n7. Discussion, were existed < existed\nResponse: This has been corrected.\n8. Methods, SCRaMbLE: SparL83<SparLox83\nResponse: This has been corrected.\n9. Methods, Split-URA3: palte<plate, inducted<induced, other typo’s\nResponse: This has been corrected.\n10. Methods, WGS: according<according\nResponse: This has been corrected.\n11. Methods, junction detection: number 2 seems to be a mistake in formula Nrei/2Normi+Nrei\nResponse: We thank the reviewer for the careful reading. We have confirmed that the “number 2” was right.\n12. Methods, Hi-C library: Spar83L<SparLox83\nResponse: This has been corrected.\nREVIEWERS' COMMENTS\nReviewer #2 (Remarks to the Author):\nDear authors,\nIn general, I am very happy to see how you addressed the comments of myself as well as the other reviewer. I believe that these adaptations have made an already impressive manuscript even stronger and, above all, more accurate. As such, I support publication.\nThat said, I am less satisfied by the response to my very first comment, namely that no \"no-recombination / no-SCRAMBLE\" controls have been used in this manuscript. I understand that a previous SCRAMBLE paper has reported these controls, but this is not a good reason to not include the proper controls in this new study, which uses a different SCRAMBLE setup. I would ask the authors to at least mention explicitly that their data do not prove that the SCRAMBLEing leads to quicker adaptation. It is of course OK to also mention that the results reported in PMID:29789540 do suggest that SCRAMBLE is indeed essential.\nkind regards,\nKevin Verstrepen\nResponse: Thank the reviewer for their recognition of our work. According to the reviewer’s suggestion, we have supplied the statement that our data did not prove quicker adaptation by SCRaMbLE in the revised discussion section.\nThe revised text is as follows: “However, further investigation is necessary to determine whether SCRaMbLE could provide a more rapid response to stress than classical adaptive laboratory evolution.” Please see page 25, line 591-593."}]

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