Two Refinements of the Template-Guided DNA Recombination Model of Ciliate Computing

To solve the mystery of the intricate gene unscrambling mechanism in ciliates, various theoretical models for this process have been proposed from the point of view of computation. Two main models are the reversible guided recombination system by Kari and Landweber and the template-guided recombination (TGR) system by Prescott, Ehrenfeucht and Rozenberg, based on two categories of DNA recombination: the pointer guided and the template directed recombination respectively. The latter model has been generalized by Daley and McQuillan. In this thesis, we propose a new approach to generate regular languages using the iterated TGR system with a finite initial language and a finite set of templates, that reduces the size of the template language and the alphabet compared to that of the Daley-McQuillan model. To achieve computational completeness using only finite components we also propose an extension of the contextual template-guided recombination system (CTGR system) by Daley and McQuillan, by adding an extra control called permitting contexts on the usage of templates. Then we prove that our proposed system, the CTGR system using permitting contexts, has the capability to characterize the family of recursively enumerable languages using a finite initial language and a finite set of templates. Lastly, we present a comparison and analysis of the computational power of the reversible guided recombination system and the TGR system. Keywords: ciliates, gene unscrambling, in vivo computing, DNA computing, cellular computing, reversible guided recombination, template-guided recombination

Ciliate Gene Unscrambling with Fewer Templates

One of the theoretical models proposed for the mechanism of gene unscrambling in some species of ciliates is the template-guided recombination (TGR) system by Prescott, Ehrenfeucht and Rozenberg which has been generalized by Daley and McQuillan from a formal language theory perspective. In this paper, we propose a refinement of this model that generates regular languages using the iterated TGR system with a finite initial language and a finite set of templates, using fewer templates and a smaller alphabet compared to that of the Daley-McQuillan model. To achieve Turing completeness using only finite components, i.e., a finite initial language and a finite set of templates, we also propose an extension of the contextual template-guided recombination system (CTGR system) by Daley and McQuillan, by adding an extra control called permitting contexts on the usage of templates.Comment: In Proceedings DCFS 2010, arXiv:1008.127

Using the CRISPR/Cas9 system to understand neuropeptide biology and regulation

Funding was provided by a Wellcome Trust ISSF starting grant (105625/Z/14/Z), Medical Research Scotland (PhD-719-2013), GW Pharmaceuticals (PhD-719-2013 - S.5242.001) and the BBSRC (BB/J012343/1).Peer reviewedPublisher PD

CRISPR/Cas9‐mediated genome editing: from basic research to translational medicine

The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome-editing tool has revolutionized basic science research. CRISPR/Cas9 system-based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient-specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease-focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients.Portuguese Foundation for Science and Technology: UID/BIM/04773/2013 1334 Spanish Ministry of Science, Innovation and Universities RTI2018-094629-B-I00 Portuguese Foundation for Science and Technology SFRH/BPD/100434/2014 European Union (EU) 748585 LPCC-NRS/Terry Fox grantsinfo:eu-repo/semantics/publishedVersio

Rewriting Human History and Empowering Indigenous Communities with Genome Editing Tools.

Appropriate empirical-based evidence and detailed theoretical considerations should be used for evolutionary explanations of phenotypic variation observed in the field of human population genetics (especially Indigenous populations). Investigators within the population genetics community frequently overlook the importance of these criteria when associating observed phenotypic variation with evolutionary explanations. A functional investigation of population-specific variation using cutting-edge genome editing tools has the potential to empower the population genetics community by holding "just-so" evolutionary explanations accountable. Here, we detail currently available precision genome editing tools and methods, with a particular emphasis on base editing, that can be applied to functionally investigate population-specific point mutations. We use the recent identification of thrifty mutations in the CREBRF gene as an example of the current dire need for an alliance between the fields of population genetics and genome editing

Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms

V_H replacement in rearranged immunoglobulin genes

Examples suggesting that all or part of the V<sub>H</sub> segment of a rearranged V<sub>H</sub>DJ<sub>H</sub> may be replaced by all or part of another V<sub>H</sub> have been appearing since the 1980s. Evidence has been presented of two rather different types of replacement. One of these has gained acceptance and has now been clearly demonstrated to occur. The other, proposed more recently, has not yet gained general acceptance because the same effect can be produced by polymerase chain reaction artefact. We review both types of replacement including a critical examination of evidence for the latter. The first type involves RAG proteins and recombination signal sequences (RSS) and occurs in immature B cells. The second was also thought to be brought about by RAG proteins and RSS. However, it has been reported in hypermutating cells which are not thought to express RAG proteins but in which activation-induced cytidine deaminase (AID) has recently been shown to initiate homologous recombination. Re-examination of the published sequences reveals AID target sites in V<sub>H</sub>-V<sub>H</sub> junction regions and examples that resemble gene conversion