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Small-cell lung cancer (SCLC) is speculated to harbor complex genomic intratumor heterogeneity (ITH) associated with high recurrence rate and suboptimal response to immunotherapy. Here, using multi-region whole exome/T cell receptor (TCR) sequencing as well as immunohistochemistry, we reveal a rather homogeneous mutational landscape but extremely cold and heterogeneous TCR repertoire in limited-stage SCLC tumors (LS-SCLCs). Compared to localized non-small cell lung cancers, LS-SCLCs have similar predicted neoantigen burden and genomic ITH, but significantly colder and more heterogeneous TCR repertoire associated with higher chromosomal copy number aberration (CNA) burden. Furthermore, copy number loss of IFN-γ pathway genes is frequently observed and positively correlates with CNA burden. Higher mutational burden, higher T cell infiltration and positive PD-L1 expression are associated with longer overall survival (OS), while higher CNA burden is associated with shorter OS in patients with LS-SCLC., Small-cell lung cancer (SCLC) is an aggressive disease with limited therapeutic options. Here the authors perform an immunogenomic analysis of limited-stage SCLC, revealing a homogeneous mutational landscape, but limited T-cell infiltration and a cold and heterogeneous T cell repertoire.

Fungal polyketides display remarkable structural diversity and bioactivity, and therefore the biosynthesis and engineering of this large class of molecules is therapeutically significant. Here, we successfully recode, construct and characterize the biosynthetic pathway of bikaverin, a tetracyclic polyketide with antibiotic, antifungal and anticancer properties, in S. cerevisiae. We use a green fluorescent protein (GFP) mapping strategy to identify the low expression of Bik1 (polyketide synthase) as a major bottleneck step in the pathway, and a promoter exchange strategy is used to increase expression of Bik1 and bikaverin titer. Then, we use an enzyme-fusion strategy to directly couple the monooxygenase (Bik2) and methyltransferase (Bik3) to efficiently channel intermediates between modifying enzymes, leading to an improved titer of bikaverin at 202.75 mg/L with flask fermentation (273-fold higher than the initial titer). This study demonstrates that the biosynthesis of complex fungal polyketides can be established and efficiently engineered in S. cerevisiae, highlighting the potential for natural product synthesis and large-scale fermentation in yeast., Bikaverin is a fungal-derived tetracyclic polyketide with antibiotic, antifungal and anticancer properties. Here, the authors employ various pathway engineering strategies to achieve high level production of bikaverin in baker’s yeast.

DNA data storage is a rapidly developing technology with great potential due to its high density, long-term durability, and low maintenance cost. The major technical challenges include various errors, such as strand breaks, rearrangements, and indels that frequently arise during DNA synthesis, amplification, sequencing, and preservation. In this study, a de novo strand assembly algorithm (DBGPS) is developed using de Bruijn graph and greedy path search to meet these challenges. DBGPS shows substantial advantages in handling DNA breaks, rearrangements, and indels. The robustness of DBGPS is demonstrated by accelerated aging, multiple independent data retrievals, deep error-prone PCR, and large-scale simulations. Remarkably, 6.8 MB of data is accurately recovered from a severely corrupted sample that has been treated at 70 °C for 70 days. With DBGPS, we are able to achieve a logical density of 1.30 bits/cycle and a physical density of 295 PB/g.

Vitiligo is an autoimmune disease in which melanocyte destruction causes skin depigmentation, with 49 loci known from previous GWAS. Aiming to define vitiligo subtypes, we discovered that age-of-onset is bimodal; one-third of cases have early onset (mean 10.3 years) and two-thirds later onset (mean 34.0 years). In the early-onset subgroup we found novel association with MHC class II region indel rs145954018, and independent association with the principal MHC class II locus from previous GWAS, represented by rs9271597; greatest association was with rs145954018del-rs9271597A haplotype (P = 2.40 × 10−86, OR = 8.10). Both rs145954018 and rs9271597 are located within lymphoid-specific enhancers, and the rs145954018del-rs9271597A haplotype is specifically associated with increased expression of HLA-DQB1 mRNA and HLA-DQ protein by monocytes and dendritic cells. Thus, for vitiligo, MHC regulatory variation confers extreme risk, more important than HLA coding variation. MHC regulatory variation may represent a significant component of genetic risk for other autoimmune diseases., GWAS have led to the identification of 49 genetic loci associated with vitiligo. Here, the authors observe a bimodal distribution of age-of-onset and find a novel genetic locus specifically associated with early-onset vitiligo, located in a regulatory element in the MHC class II region.

After a dorsal root crush injury, centrally-projecting sensory axons fail to regenerate across the dorsal root entry zone (DREZ) to extend into the spinal cord. We find that chemogenetic activation of adult dorsal root ganglion (DRG) neurons improves axon growth on an in vitro model of the inhibitory environment after injury. Moreover, repeated bouts of daily chemogenetic activation of adult DRG neurons for 12 weeks post-crush in vivo enhances axon regeneration across a chondroitinase-digested DREZ into spinal gray matter, where the regenerating axons form functional synapses and mediate behavioral recovery in a sensorimotor task. Neuronal activation-mediated axon extension is dependent upon changes in the status of tubulin post-translational modifications indicative of highly dynamic microtubules (as opposed to stable microtubules) within the distal axon, illuminating a novel mechanism underlying stimulation-mediated axon growth. We have identified an effective combinatory strategy to promote functionally-relevant axon regeneration of adult neurons into the CNS after injury., Central axons have limited regenerative ability following injury. Here, the authors show that chronic activation of DRG neurons results in highly dynamic microtubules at the distal axons and enhanced axonal regrowth and synaptogenesis in the spinal cord affecting functional recovery.

Self-propagating drive systems are capable of causing non-Mendelian inheritance. Here, we report a drive system in yeast referred to as a chromosome drive that eliminates the target chromosome via CRISPR-Cas9, enabling the transmission of the desired chromosome. Our results show that the entire Saccharomyces cerevisiae chromosome can be eliminated efficiently through only one double-strand break around the centromere via CRISPR-Cas9. As a proof-of-concept experiment of this CRISPR-Cas9 chromosome drive system, the synthetic yeast chromosome X is completely eliminated, and the counterpart wild-type chromosome X harboring a green fluorescent protein gene or the components of a synthetic violacein pathway are duplicated by sexual reproduction. We also demonstrate the use of chromosome drive to preferentially transmit complex genetic traits in yeast. Chromosome drive enables entire chromosome elimination and biased inheritance on a chromosomal scale, facilitating genomic engineering and chromosome-scale genetic mapping, and extending applications of self-propagating drives., Self-propagating drives allow for non-Mendelian inheritance. Here the authors use CRISPR to build a chromosome drive, showing elimination of entire chromosomes, endoreduplication of desired chromosomes and enabling preferential transmissions of complex genetic traits on a chromosomal scale in yeast.

Here, we identify iPLA2β as a critical regulator for p53-driven ferroptosis upon reactive oxygen species (ROS)-induced stress. The calcium-independent phospholipase iPLA2β is known to cleave acyl tails from the glycerol backbone of lipids and release oxidized fatty acids from phospholipids. We found that iPLA2β-mediated detoxification of peroxidized lipids is sufficient to suppress p53-driven ferroptosis upon ROS-induced stress, even in GPX4-null cells. Moreover, iPLA2β is overexpressed in human cancers; inhibition of endogenous iPLA2β sensitizes tumor cells to p53-driven ferroptosis and promotes p53-dependent tumor suppression in xenograft mouse models. These results demonstrate that iPLA2β acts as a major ferroptosis repressor in a GPX4-independent manner. Notably, unlike GPX4, loss of iPLA2β has no obvious effect on normal development or cell viability in normal tissues but iPLA2β plays an essential role in regulating ferroptosis upon ROS-induced stress. Thus, our study suggests that iPLA2β is a promising therapeutic target for activating ferroptosis-mediated tumor suppression without serious toxicity concerns., p53 is able to induce ferroptosis in response to reactive oxygen species (ROS)-induced stress and suppresses tumour growth. Here, the authors show that iPLA2β suppresses p53-medated ferroptosis by cleaving and detoxifying peroxidized lipids and that this is independent of canonical ferroptosis regulator GPX4.

Structural variations (SVs) exert important functional impacts on biological phenotypic diversity. Here we show a ring synthetic yeast chromosome V (ring_synV) can be used to continuously generate complex genomic variations and improve the production of prodeoxyviolacein (PDV) by applying Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE) in haploid yeast cells. The SCRaMbLE of ring_synV generates aneuploid yeast strains with increased PDV productivity, and we identify aneuploid chromosome I, III, VI, XII, XIII, and ring_synV. The neochromosome of SCRaMbLEd ring_synV generated more unbalanced forms of variations, including duplication, insertions, and balanced forms of translocations and inversions than its linear form. Furthermore, of the 29 novel SVs detected, 11 prompted the PDV biosynthesis; and the deletion of uncharacterized gene YER182W is related to the improvement of the PDV. Overall, the SCRaMbLEing ring_synV embraces the evolution of the genome by modifying the chromosome number, structure, and organization, identifying targets for phenotypic comprehension.

Compatibility between host cells and heterologous pathways is a challenge for constructing organisms with high productivity or gain of function. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity. Here we construct a genetic AND gate to enable precise control of the SCRaMbLE method to generate synthetic haploid and diploid yeast with desired phenotypes. The yield of carotenoids is increased to 1.5-fold by SCRaMbLEing haploid strains and we determine that the deletion of YEL013W is responsible for the increase. Based on the SCRaMbLEing in diploid strains, we develop a strategy called Multiplex SCRaMbLE Iterative Cycling (MuSIC) to increase the production of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE. This strategy is potentially a powerful tool for increasing the production of bio-based chemicals and for mining deep knowledge.

SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) is a genome restructuring technique that can be used in synthetic genomes such as that of Sc2.0, the synthetic yeast genome, which contains hundreds to thousands of strategically positioned loxPsym sites. SCRaMbLE has been used to induce rearrangements in yeast strains harboring one or more synthetic chromosomes, as well as plasmid DNA in vitro and in vivo. Here we describe a collection of heterozygous diploid strains produced by mating haploid semisynthetic Sc2.0 strains to haploid native parental strains. We subsequently demonstrate that such heterozygous diploid strains are more robust to the effects of SCRaMbLE than haploid semisynthetic strains, rapidly improve rationally selected phenotypes in SCRaMbLEd heterozygous diploids, and establish that multiple sets of independent genomic rearrangements are able to lead to similar phenotype enhancements. Finally, we show that heterozygous diploid SCRaMbLE can also be carried out in interspecies hybrid strains., SCRaMbLE has been used to rearrange synthetic chromosomes that have been introduced into host yeast. Here the authors produce semi-synthetic heterozygous diploid strains for rapid selection of phenotypes and map the rearrangements underlying selected phenotypes such as thermoresistance and caffeine resistance.

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The “in vitro SCRaMbLE system” uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a β-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve., SCRaMbLE allows for the rapid and large scale rearrangement of genetic data in yeast carrying synthetic chromosomes. Here the authors demonstrate an in vitro use of the method to generate DNA libraries for optimization of biochemical reactions.

Compatibility between host cells and heterologous pathways is a challenge for constructing organisms with high productivity or gain of function. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity. Here we construct a genetic AND gate to enable precise control of the SCRaMbLE method to generate synthetic haploid and diploid yeast with desired phenotypes. The yield of carotenoids is increased to 1.5-fold by SCRaMbLEing haploid strains and we determine that the deletion of YEL013W is responsible for the increase. Based on the SCRaMbLEing in diploid strains, we develop a strategy called Multiplex SCRaMbLE Iterative Cycling (MuSIC) to increase the production of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE. This strategy is potentially a powerful tool for increasing the production of bio-based chemicals and for mining deep knowledge., The SCRaMbLE system integrated into Sc2.0’s synthetic yeast chromosome project allows rapid strain evolution. Here the authors use a genetic logic gate to control induction of recombination in a haploid and diploid yeast carrying synthetic chromosomes.