Biology subjects

Olsson, H.

Publications and source records attributed to Olsson, H..

10 recordsLinked to original sources

bioRxiv2023

Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering of Komagataella phaffii

Komagataella phaffii (Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bio-production of valuable compounds is not yet fully exploited. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of non-conventional species including K. phaffii. Yet, improvements in gene-editing methods are desirable to further accelerate engineering of industrially and scientifically relevant K. phaffii strains. In this study, we have developed a versatile one vector-based CRISPR-Cas9 method and showed that it works efficiently at different genetic loci using linear DNA fragments with very short targeting sequences. Importantly, we show that by using our setup it is possible to catalyze single-stranded oligonucleotide-mediated mutagenesis and marker-free gene integrations. Notably, we performed site-specific point mutations and full gene deletions using single stranded 90-mers at very high efficiencies. Lastly, we present a strategy for transient inactivation of non-homologous end-joining (NHEJ) pathway, where KU70 gene is disrupted by a visual marker (uidA gene). The latter system enables precise CRISPR-Cas9 based editing (including multiplexing) and accelerates selection of the mutants that have simultaneously undergone a desired genetic modification(s) and restored NHEJ-proficient genotype. In conclusion, the tools presented in this study can be applied for easy and efficient engineering of K. phaffii strains and could potentially be coupled with high-throughput automated workflows.

synthetic biology↗

bioRxiv2023

Cortical changes during the learning of sequences of simultaneous finger presses

The cortical alterations underpinning the acquisition of motor skills remain debated. In this longitudinal study in younger adults, we acquired performance and neuroimaging (7T MRI) measures weekly over the course of 6 weeks to investigate neural changes associated with learning sequences of simultaneous finger presses executed with the non-dominant hand. Both the intervention group (n = 33) and the control group (n = 30) showed general performance improvements, but performance improved more and became more consistent for sequences that were intensively trained by the intervention group, relative to those that were not. Brain activity for trained sequences decreased compared with untrained sequences in the bilateral parietal and premotor cortices. No training-related changes in the primary sensorimotor areas were detected. The similarity of activation patterns between trained and untrained sequences decreased in secondary, but not primary, sensorimotor areas, while the similarity of the activation patterns between different trained sequences did not show reliable changes. Neither the variability of activation patterns across trials, nor the estimates of brain structure displayed practice-related changes that reached statistical significance. Overall, the main correlate of learning configural sequences was a reduction in brain activity in secondary motor areas.

neuroscience↗

bioRxiv2022

CRI-SPA, a mating based CRISPR-Cas9 assisted method for high-throughput genetic modification of yeast strain libraries

AbstractBiological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and the identification of phenotypic maximas would be of great benefit to synthetic biology. Thus, new research tools allowing simple and rapid mapping of sequence to function are required to forward these research fields. Here, we describe CRI-SPA, a method allowing the transfer of a chromosomal genetic feature from a donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion and Selective Ploidy Ablation and is executed within a week. We demonstrate the power of CRI-SPA by transferring four genes responsible for the production of betaxanthin, a yellow biosensor for the morphine precursor L-DOPA, into each strain of the yeast knock-out collection ({approx}4800 strains), providing a genome-wide overview of the genetic requirements for betaxanthin production. CRI-SPA is fast, highly reproducible, can be massively parallelized with automation and does not require selection for the transferred genetic feature.

systems biology↗

bioRxiv2020

Surveying the contribution of rare variants to the genetic architecture of human disease through exome sequencing of 177,882 UK Biobank participants

The UK Biobank (UKB) represents an unprecedented population-based study of 502,543 participants with detailed phenotypic data and linkage to medical records. While the release of genotyping array data for this cohort has bolstered genomic discovery for common variants, the contribution of rare variants to this broad phenotype collection remains relatively unknown. Here, we use exome sequencing data from 177,882 UKB participants to evaluate the association between rare protein-coding variants with 10,533 binary and 1,419 quantitative phenotypes. We performed both a variant-level phenome-wide association study (PheWAS) and a gene-level collapsing analysis-based PheWAS tailored to detecting the aggregate contribution of rare variants. The latter revealed 911 statistically significant gene-phenotype relationships, with a median odds ratio of 15.7 for binary traits. Among the binary trait associations identified using collapsing analysis, 83% were undetectable using single variant association tests, emphasizing the power of collapsing analysis to detect signal in the setting of high allelic heterogeneity. As a whole, these genotype-phenotype associations were significantly enriched for loss-of-function mediated traits and currently approved drug targets. Using these results, we summarise the contribution of rare variants to common diseases in the context of the UKB phenome and provide an example of how novel gene-phenotype associations can aid in therapeutic target prioritisation.

genomics↗

bioRxiv2020

A broad exome study of the genetic architecture of asthma reveals novel patient subgroups

IntroductionAsthma risk is a complex interplay between genetic susceptibility and environment. Despite many significantly-associated common variants, the contribution of rarer variants with potentially greater effect sizes has not been as extensively studied. We present an exome-based study adopting 24,576 cases and 120,530 controls to assess the contribution of rare protein-coding variants to the risk of early-onset or all-comer asthma. MethodsWe performed case-control analyses on three genetic units: variant-, gene- and pathway-level, using sequence data from the Scandinavian Asthma Genetic Study and UK Biobank participants with asthma. Cases were defined as all-comer asthma (n=24,576) and early-onset asthma (n=5,962). Controls were 120,530 UK Biobank participants without reported history of respiratory illness. ResultsVariant-level analyses identified statistically significant variants at moderate-to-common allele frequency, including protein-truncating variants in FLG and IL33. Asthma risk was significantly increased not only by individual, common FLG protein-truncating variants, but also among the collection of rare-to-private FLG protein-truncating variants (p=6.8x10-7). This signal was driven by early-onset asthma and did not correlate with circulating eosinophil levels. In contrast, a single splice variant in IL33 was significantly protective (p=8.0x10-10), while the collection of remaining IL33 protein-truncating variants showed no class effect (p=0.54). A pathway-based analysis identified that protein-truncating variants in loss-of-function intolerant genes were significantly enriched among individuals with asthma. ConclusionsAccess to the full allele frequency spectrum of protein-coding variants provides additional clarity about the potential mechanisms of action for FLG and IL33. Beyond these two significant drivers, we detected a significant enrichment of protein-truncating variants in loss-of-function intolerant genes.

genomics↗

bioRxiv2020

Identification of a novel missense variant in SPDL1 associated with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal disorder characterised by progressive, destructive lung scarring. Despite significant progress, the genetic determinants of this disease remain incompletely defined. Using next generation sequencing data from 752 individuals with sporadic IPF and 119,055 controls, we performed both variant- and gene-level analyses to identify novel IPF genetic risk factors. Our variant-level analysis revealed a novel rare missense variant in SPDL1 (NM_017785.5 p.Arg20Gln; p = 2.4 x 10-7, odds ratio = 2.87). This signal was independently replicated in the FinnGen cohort (combined p = 2.2 x 10-20), firmly associating this variant as a novel IPF risk allele. SPDL1 encodes Spindly, a protein involved in mitotic checkpoint signalling during cell division that has not been previously described in fibrosis. Our results highlight a novel mechanism underlying IPF, providing the potential for new therapeutic discoveries in a disease of great unmet need.

genetics↗

bioRxiv2020

PTENP1-AS contributes to BRAF inhibitor resistance and is associated with adverse clinical outcome in stage III melanoma

Approximately 50% of human cutaneous melanomas carry activating mutations in the serine/threonine protein kinase BRAF. BRAF inhibitors (BRAFi) selectively target the oncogenic BRAFV600E/K and are effective in approximately 80% of patients carrying the mutation. However, resistance to BRAFi is common and emerges within a median time of 6-7 months of treatment and is prolonged to 11 months when combined with MEK inhibitors. Better characterization of the underlying molecular processes is therefore needed to further improve treatments. Inactivation of the tumor suppressor gene PTEN has been suggested to occur during melanomagenesis and drug resistance development. We recently demonstrated that transcription of PTEN is negatively regulated by an antisense RNA from the PTEN pseudogene (PTENP1-AS) and here set out to investigate the impact of this molecular pathway on the resistance to BRAFi and clinical outcome. We used a panel of BRAFi resistant A375 sublines, and observed increased levels of PTENP1-AS associated with reduced expression of PTEN. Furthermore, this loss of PTEN expression was correlated to increased recruitment of Enhancer of zeste homolog 2 (EZH2) and formation of the transcriptional repression mark H3K27me3 at the PTEN promoter in the resistant cells. We demonstrated that targeting of PTENP1-AS was able to re-activate the expression of PTEN and sensitize resistant melanoma cells to BRAFi. Finally, we showed that PTENP1-AS is a promising prognostic marker for clinical outcome in melanoma patients as high expression of PTENP1-AS in regional lymph node metastases from stage III melanoma patients correlated with poor survival.

cancer biology↗

bioRxiv2020

Pleiotropy-guided transcriptome imputation from normal and tumor tissues identifies new candidate susceptibility genes for breast and ovarian cancer

Familial, genome-wide association (GWAS), and sequencing studies and genetic correlation analyses have progressively unraveled the shared or pleiotropic germline genetics of breast and ovarian cancer. In this study, we aimed to leverage this shared germline genetics to improve the power of transcriptome-wide association studies (TWAS) to identify candidate breast cancer and ovarian cancer susceptibility genes. We built gene expression prediction models using the PrediXcan method in 681 breast and 295 ovarian tumors from The Cancer Genome Atlas and 211 breast and 99 ovarian normal tissue samples from the Genotype-Tissue Expression project and integrated these with GWAS meta-analysis data from the Breast Cancer Association Consortium (122,977 cases/105,974 controls) and the Ovarian Cancer Association Consortium (22,406 cases/40,941 controls). The integration was achieved through novel application of a pleiotropy-guided conditional/conjunction false discovery rate approach for the first time in the setting of a TWAS. This identified 14 new candidate breast cancer susceptibility genes spanning 11 genomic regions and 8 new candidate ovarian cancer susceptibility genes spanning 5 genomic regions at conjunction FDR < 0.05 that were > 1 Mb away from known breast and/or ovarian cancer susceptibility loci. We also identified 38 candidate breast cancer susceptibility genes and 17 candidate ovarian cancer susceptibility genes at conjunction FDR < 0.05 at known breast and/or ovarian susceptibility loci. Overlaying candidate causal risk variants identified by GWAS fine mapping onto expression prediction models for genes at known loci suggested that the association for 55% of these genes was driven by the underlying GWAS signal. SignificanceThe 22 new genes identified by our cross-cancer analysis represent promising candidates that further elucidate the role of the transcriptome in mediating germline breast and ovarian cancer risk.

genetics↗

bioRxiv2018

The association between weight at birth and breast cancer risk revisited using Mendelian randomisation

Observational studies suggest that higher birth weight (BW) is associated with increased risk of breast cancer in adult life. We conducted a two-sample Mendelian randomisation (MR) study to assess whether this association is causal. Sixty independent single nucleotide polymorphisms (SNPs) known to be associated at P < 5 x 10-8 with BW were used to construct (1) a 41-SNP instrumental variable (IV) for univariable MR after removing SNPs with pleiotropic associations with other breast cancer risk factors and (2) a 49-SNP IV for multivariable MR after filtering SNPs for data availability. BW predicted by the 41-SNP IV was not associated with overall breast cancer risk in inverse-variance weighted (IVW) univariable MR analysis of genetic association data from 122,977 breast cancer cases and 105,974 controls (odds ratio = 0.86 per 500 g higher BW; 95% confidence interval: 0.73--1.01). Sensitivity analyses using four alternative methods and three alternative IVs, including an IV with 59 of the 60 BW-associated SNPs, yielded similar results. Multivariable MR adjusting for the effects of the 49-SNP IV on birth length, adult height, adult body mass index, age at menarche, and age at menopause using IVW and MR-Egger methods provided estimates consistent with univariable analyses. Results were also similar when all analyses were repeated after restricting to estrogen receptor-positive or -negative breast cancer cases. Point estimates of the odds ratios from most analyses performed indicated an inverse relationship between genetically-predicted BW and breast cancer. Thus, there is little evidence from MR to suggest that the previously observed association between higher BW and increased risk of breast cancer in adult life is causal.

epidemiology↗

bioRxiv2018

Statin use and breast cancer survival: A Swedish nationwide study

BackgroundA sizeable body of evidence suggests that statins can cease breast cancer progression and prevent breast cancer recurrence. The latest studies have, however, not been supportive of such clinically beneficial effects. These discrepancies may be explained by insufficient power. This considerably sized study investigates the association between both pre- and post-diagnostic statin use and breast cancer outcome.\n\nMethodsA Swedish nation-wide retrospective cohort study of 20,559 Swedish women diagnosed with breast cancer (July 1st, 2005 through 2008). Dispensed statin medication was identified through the Swedish Prescription Registry. Breast cancer related death information was obtained from the national cause-of-death registry until December 31st, 2012. Cox regression models yielded hazard ratios (HR) and 95% confidence intervals (CI) regarding associations between statin use and breast cancer-specific and overall mortality.\n\nResultsDuring follow-up, a total of 4,678 patients died, of which 2,669 were considered breast cancer related deaths. Compared to non- or irregular use, regular pre-diagnostic statin use was associated with lower risk of breast cancer related deaths (HR=0.77; 95% CI 0.63-0.95, P=0.014). Similarly, post-diagnostic statin use compared to non-use was associated with lower risk of breast cancer related deaths (HR=0.83; 95% CI 0.75-0.93, P=0.001).\n\nConclusionThis study evidently supports the notion that statin use is protective regarding breast cancer related mortality in agreement with previous Scandinavian studies, although less so with studies in other populations. These disparities should be further investigated to pave the way for future clinical trials investigating the role of statins in breast cancer.

epidemiology↗