Reframing gene essentiality in terms of adaptive flexibility

Abstract Background Essentiality assays are important tools commonly utilized for the discovery of gene functions. Growth/no growth screens of single gene knockout strain collections are also often utilized to test the predictive power of genome-scale models. False positive predictions occur when computational analysis predicts a gene to be non-essential, however experimental screens deem the gene to be essential. One explanation for this inconsistency is that the model contains the wrong information, possibly an incorrectly annotated alternative pathway or isozyme reaction. Inconsistencies could also be attributed to experimental limitations, such as growth tests with arbitrary time cut-offs. The focus of this study was to resolve such inconsistencies to better understand isozyme activities and gene essentiality. Results In this study, we explored the definition of conditional essentiality from a phenotypic and genomic perspective. Gene-deletion strains associated with false positive predictions of gene essentiality on defined minimal medium for Escherichia coli were targeted for extended growth tests followed by population sequencing and transcriptome analysis. Of the twenty false positive strains available and confirmed from the Keio single gene knock-out collection, 11 strains were shown to grow with longer incubation periods making these actual true positives. These strains grew reproducibly with a diverse range of growth phenotypes. The lag phase observed for these strains ranged from less than one day to more than 7 days. It was found that 9 out of 11 of the false positive strains that grew acquired mutations in at least one replicate experiment and the types of mutations ranged from SNPs and small indels associated with regulatory or metabolic elements to large regions of genome duplication. Comparison of the detected adaptive mutations, modeling predictions of alternate pathways and isozymes, and transcriptome analysis of KO strains suggested agreement for the observed growth phenotype for 6 out of the 9 cases where mutations were observed. Conclusions Longer-term growth experiments followed by whole genome sequencing and transcriptome analysis can provide a better understanding of conditional gene essentiality and mechanisms of adaptation to such perturbations. Compensatory mutations are largely reproducible mechanisms and are in agreement with genome-scale modeling predictions to loss of function gene deletion events

Transposon and Exponential Mutagenesis Approaches for Determining Essential Genomic Functions

Transposon mutagenesis is an increasingly used technique in molecular microbiology, antimicrobial development, and bioproduction of compounds. However limited experimental evidence is available for the reproducibility and intrinsic biases of the technique. The reliance on annotated genomes for determination of essentiality is problematic as annotations can change rendering a gene list inaccurate. Genomic features form networks and functional redundancies can be overlooked using the technique, so methods producing double mutations in a similar fashion would provide better resolution of essential processes within a cell. In this thesis, the reproducibility of transposon library construction was assessed by generating mutant libraries. One library is demonstrated to be reproducible when using the BioTradis pipeline, as long as a sufficient density of mutants with insertions inside coding regions is achieved. Transposon insertion frequency was found to be dependent on factors other than gene essentiality, and these should be taken into account during insertion frequency analysis. Some of these insertion biases can be measured and accounted for and insertion counts normalised to reflect this. Also presented is an annotation-independent approach for determining essentiality using change point analysis that can accept normalised insertion data. This enables identification of essential regions that may or may not code for a known product. Finally, the development of Exponential Mutagenesis, the production of mutants containing multiple transposon insertions is demonstrated, producing preliminary data identifying known genetic interactions in metabolic pathways for folate biosynthesis; these are targeted by sulphonamides and trimethoprim, two therapeutic antimicrobials. A high throughput paired mutant approach will lead to a better understanding of the mechanisms and redundancy involved in bacterial survival and antimicrobial resistance. Investigating the full complement of double mutants should also allow us to understand gene interactions in both metabolic and biosynthetic pathways and highlight essential genomic functions rather than gene products

Machine Learning Approaches for the Prioritisation of Cardiovascular Disease Genes Following Genome- wide Association Study

Genome-wide association studies (GWAS) have revealed thousands of genetic loci, establishing itself as a valuable method for unravelling the complex biology of many diseases. As GWAS has grown in size and improved in study design to detect effects, identifying real causal signals, disentangling from other highly correlated markers associated by linkage disequilibrium (LD) remains challenging. This has severely limited GWAS findings and brought the method’s value into question. Although thousands of disease susceptibility loci have been reported, causal variants and genes at these loci remain elusive. Post-GWAS analysis aims to dissect the heterogeneity of variant and gene signals. In recent years, machine learning (ML) models have been developed for post-GWAS prioritisation. ML models have ranged from using logistic regression to more complex ensemble models such as random forests and gradient boosting, as well as deep learning models (i.e., neural networks). When combined with functional validation, these methods have shown important translational insights, providing a strong evidence-based approach to direct post-GWAS research. However, ML approaches are in their infancy across biological applications, and as they continue to evolve an evaluation of their robustness for GWAS prioritisation is needed. Here, I investigate the landscape of ML across: selected models, input features, bias risk, and output model performance, with a focus on building a prioritisation framework that is applied to blood pressure GWAS results and tested on re-application to blood lipid traits

CRISPR/Cas9 genome editing of Recessive Dystrophic Epidermolysis Bullosa (RDEB) mutation hotspot

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe life-threatening skin adhesion disorder caused by loss-of-function mutations in the COL7A1-encoding type VII collagen (C7), a structural protein playing a crucial role in anchoring fibril (AF) formation at the dermal-epidermal junction (DEJ). Combinatorial cell and gene therapies based on the addition of a full-length copy of COL7A1 cDNA in RDEB keratinocytes, fibroblasts and skin equivalents have shown potential in preclinical and clinical settings although only modest and transient improvements have been reported. In parallel, induced pluripotent stem cells (iPSCs) are being investigated in preclinical studies for RDEB. iPSCs represent a valuable source of autologous patient material and can be differentiated into keratinocytes and fibroblasts for cellular therapy applications. Implementation of CRISPR/Cas9 and base editing-mediated gene correction in patient-derived iPSCs has allowed for the generation of autologous cellular models capable of overcoming barriers of conventional gene therapy. In this regards, the work described in this thesis aims to evaluate the feasibility of genome-editing approaches using CRISPR/Cas9 and Cytosine Base editing (BE) platforms to correct a mutation hotspot (c.425A>G, p.Lysl42Arg) within exon 3 of COL7A1 gene in patient-derived iPSCs. Gene repair by homology-directed recombination (HDR) following CRISPR/Cas9-induced double-strand breaks (DSBs) through viral and non-viral donor template deliveries resulted in a significant correction of the COL7A1 locus on genomic level. To avoid concerns surrounding the generation of DSBs, seamless BE-based G:C to A:T conversion resulted in a high restoration of the wild type COL7A1 sequence. Ultimately, capacity of gene- and base-corrected RDEB iPSCs to be differentiated in into keratinocytes (iKer) was evaluated in vitro and functional recovery of de novo C7 was assayed on protein level. Overall, this study explored the potential of CRISPR/Cas9 and BE site-specific correction of COL7A1 in RDEB-derived pluripotent stem cells. Furthermore, it demonstrated that gene-corrected iPSCs can be used as a source of epidermal progenitors thereby confirming their potential for future cell therapies for skin disorders

Advances in CRISPR/Cas gene therapy for inborn errors of immunity

Inborn errors of immunity (IEIs) are a group of inherited disorders caused by mutations in the protein-coding genes involved in innate and/or adaptive immunity. Hematopoietic stem cell transplantation (HSCT) is a mainstay definitive therapy for many severe IEIs. However, the lack of HLA-matched donors increases the risk of developing severe immunological complications. Gene therapy provides long-term clinical benefits and could be an attractive therapeutic strategy for IEIs. In this review, we describe the development and evolution of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) gene-editing systems, including double-strand break (DSB)-based gene editing and DSB-free base editing or prime editing systems. Here, we discuss the advances in and issues associated with CRISPR/Cas gene editing tools and their potential as therapeutic alternatives for IEIs. We also highlight the progress of preclinical studies for the treatment of human genetic diseases, including IEIs, using CRISR/Cas and ongoing clinical trials based on this versatile technology

Usher syndrome: A phenomenological study of adults across the lifespan living in England

Usher syndrome is a rare inherited genetic condition which is one of the main causes of acquired deafblindness in the United Kingdom (UK). Although the condition is not life threatening, it is life altering and will have a significant impact on the lives of not only the person diagnosed with the condition, but also their families, friendship groups and new and existing relationships. The aim of the study was to develop an understanding of the experiences of diagnosis of and living with Usher syndrome, from the perspective of adults living in England. Specific objectives of the study were to explore the experience of being diagnosed with Usher syndrome; explore the transition from adolescence to adulthood for people who have Usher syndrome; to develop an understanding of the experience of living with Usher syndrome, including support, developmental opportunities and the role of the Deaf community; to disseminate original findings; inform future practice, service development, policy and education and make recommendations for further research relating to the experience of living with Usher syndrome. To address these aims and objectives, this qualitative, descriptive phenomenological study, conducted interviews with 20 males and females aged between 18-82 years from a variety of demographic locations. To contribute to the trustworthiness of the study, I developed a methodological innovation called ‘Multiple Sensory Communication and Interview Methods’ (MSCIM) which ensured that as far as possible communication and interview methods were participant led. Three overarching messages from findings were revealed: the importance of ensuring communication is timely, supportive and appropriate; Usher support at the right time: providing physical and virtual support networks and essentiality of Usher awareness: raising the profile. This study is unique because it is the first qualitative, descriptive phenomenological study to demonstrate new knowledge to better understand and support people living with Usher in England. Keywords Usher syndrome, sensory impairment, D/deafblindness, ‘Multiple Sensory Communication and Interview Methods’ (MSCIM), social work, qualitative research, descriptive phenomenolog

Predictability of Delinquency through Psychosocial and Environmental Variables across Three Generational Status Groups

Issues such as the rapid growth of the immigrant youth population and delinquency among adolescents generate public safety concerns among the U.S. population. However, delinquency intervention strategies for immigrant youth in the United States remain scant, which is problematic because these youth face acculturative challenges that increase their risk for maladaptive outcomes. This quantitative, cross-sectional study addressed a research gap regarding the differential influence of risk factors in predicting delinquency across 3 generational statuses. The theoretical framework guiding the study consisted of acculturation theory, the immigrant paradox, and differential association theory. Two research questions were evaluated using a stratified random sample of 255 U.S. adolescents from the Second International Self-Reported Delinquency Study Dataset. The bivariate correlation analyses show that delinquency was significantly related to self-control, neighborhood disorganization, and delinquent peers for the total adolescent sample, and family bonding and school climate at the generational status level. The multiple regression analyses show that delinquency was best predicted by self-control for first-generation immigrants, by neighborhood disorganization, school climate, and delinquent peers for second-generation immigrants, and by self-control, family bonding, and delinquent peers for native-born youth. The results demonstrate that immigrant and native-born youth have unique adaptive and developmental processes that impact their delinquency. By increasing knowledge of delinquency risk factors, the study findings may help advocates address public safety concerns, enhance the cultural responsiveness of interventions, and, ultimately, improve youths\u27 behavioral outcomes

The Machine–Organism Distinction

The idea that analysis of organisms can proceed by distinguishing organisms from machines is common to many areas of philosophy. This thesis argues that our search for a philosophy of organisms should not proceed by defining or relying on a Machine–Organism Distinction (MOD). We are often able to take biological theories that are thought to characterize organ- isms, such as theories of organismal autonomy and stability, and apply them to machines. I argue that we should not provide an analysis of organisms according to an MOD because there is no distinction available that holds up to scrutiny and evidence. There have been several major attempts to provide an MOD. I divide these in consecutive chapters according to the property of organisms offered as an MOD: teleology (Nicholson 2013), autonomy (Mossio and Moreno 2015), stochasticity (Skillings 2015; Godfrey-Smith 2016) and pro- cessual stability (Dupré and Nicholson 2018). I address these major attempts to provide an MOD by showing how each fails to provide an analysis of organisms that distinguishes them from machines. To do this, I examine a diversity of machines and organisms that serve as naturalistic counterexamples. Discoveries in molecular biology and ecology, as well as developments in robotics and biotechnology, show the failure of MODs in contemporary philosophy and biology. Moreover, not only does the MOD consistently fail, but philosophical arguments that rely upon MODs consistently misrepresent organisms themselves. I conclude with the idea that we should consider machines not as external to, or distinguished from, organisms, but as proper objects of biological science.Social Sciences and Humanities Research Council of Canada; Cambridge Commonwealth and European Trust