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NK cell based anti-tumour therapies demonstrate high efficacy in targeting hematological malignancies, however, treatments for advanced solid tumours face challenges. The immunosuppressive environment produced by tumours prevents NK cells from maintaining cytotoxic activity and reducing tumour burden. Enhancing NK cell activation is essential to improve their function against solid tumours. Genetic manipulation of primary NK cells with viral and non-viral methods has seen a drastic improvement in recent years. Lentiviral vectors are being used to generate CAR-NK cells ex vivo, while refinement of electroporation protocols has allowed for the generation of stable gene knockouts in primary NK cells. To establish and validate the generation of a stable knockout in primary human NK cells we focused on targeting the NCAM-1 (CD56) surface adhesion molecule. The high surface expression of CD56 in NK cells makes it a suitable target to establish the knockout protocol. Furthermore, despite its levels of expression being correlated to different functional phenotypes, the role of CD56 in NK cell function is not understood. Here we have shown that current lentiviral transduction protocols are not viable methods to deliver the sgRNA/Cas9 system into primary NK cells. However, we found that nucleofection of the sgRNA/Cas9 complex into NK cells is an efficient method to generate gene knockouts. Using newly generated CD56KO NK cells we have shown that the expression of CD56 has no effect on NK cell cytotoxicity, cytokine production, proliferation, and in vivo tissue trafficking. In parallel, we have also identified an intracellular pathway that is active in the tumour microenvironment and could inhibit NK cell function. Recent studies on the intracellular signaling of the E3 ubiquitin-protein ligase Cbl-b have highlighted its role in inhibiting NK cell tumour lytic and anti-metastatic activity. Immunosuppressive factors produced by tumours activate the Cbl-b pathway, leading to the targeted degradation of signaling proteins required for NK cell activation. We have shown that Cbl-b is upregulated in ex vivo expanded NK cells cultured with GAS6 or ovarian cancer ascites. Therefore, the generation of human primary Cbl-bKO NK cells could be a beneficial asset to enhance NK cell cancer immunotherapy. / Thesis / Master of Science (MSc)

Clustered regularly interspaced palindromic repeats (CRISPR) and their associated (Cas) proteins co-opted as biotechnological tools have improved the simplicity and accessibility of gene editing for fields ranging from crop science to the treatment of human disease. These technologies, however, come with an inherent degree of risk associated with off-target events or direct misuse, accidental or intentional, leading to permanent genetic damage to ecosystems, livestock, or people. Naturally occurring anti-CRISPR proteins have been described, as well as synthetic small molecule inhibitors, but each of these approaches, while suitable for certain applications, leaves something to be desired in deliverability or efficacy in the face of many possible adverse CRISPR-related events. Inspired by strides in the field of oligonucleotide therapeutics, we developed the first reported anti-CRISPR nucleic acids for Streptococcus pyogenes (Sp)Cas9 to address the critical need for fail-safe inhibitors of Cas enzymes. These inhibitors, termed small nucleic acid-based inhibitors of Cas9 (SNuBs), comprise two modules which act in tandem to bind and disable the SpCas9 RNP. We have demonstrated that SNuBs inhibit Cas9 in vitro and in human cells. Successive rounds of optimization on our initial designs have yielded inhibitors capable of carrier-free uptake into human cells, high nuclease resistance, and robust inhibition at low stoichiometric concentrations relative to Cas9 and its RNA guide. In their current form, SNuBs quite possibly present the most tenable approach to inhibiting Cas9 in a variety of contexts including therapeutic applications in the near future.

Glucosyltransferases catalyze the transfer of glucose molecules from an active donor to acceptor molecules and are involved in many plant processes. SIP68, a tobacco glucosyltransferase protein, is a SABP2-interacting protein. It was identified in a yeast two-hybrid screen using SABP2 as bait and tobacco proteins as prey. SABP2, converts methyl salicylate to salicylic acid (SA) as a part of the signal transduction pathways in SA-mediated defense signaling. Subcellular localization is a crucial aspect of protein functional analysis to assess its biological function. The recombinant SIP68 tagged with eGFP was expressed transiently in Nicotiana benthamiana and observed under confocal microscopy. Fluorescent signals were observed in the epidermal cells. Subcellular fractionation of the tobacco leaves transiently expressing SIP68-+eGFP confirmed that SIP68 is localized in the cytosol. To study the role of SIP68 in plant stress signaling, transgenic lines with altered SIP68 expression were generated using RNAi and CRISPR Cas9 and analyzed.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22503号 / 農博第2407号 / 新制||農||1077(附属図書館) / 学位論文||R2||N5283(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 澤山 茂樹, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The field of synthetic biology seeks to use design principles of life to create new genes, organisms and populations to both better understand biology as well as generate species with useful properties. Budding yeast has been a workhorse for synthetic biology, as well as an important model organism in the broader fields of molecular biology and genetics. This thesis aimed to create genome engineering tools for the manipulation of genomes, with direct applications in yeast. I focused developing high-throughput and highly efficient methods for making genomic modifications in yeast to allow for the generation of large libraries of precisely modified yeast genomes. By manipulation of endogenous DNA recombinases and mismatch repair enzymes in yeast, we were able to develop an oligonucleotide only method for genome engineering to generate libraries as large as 10^5 individuals with a frequency of modification as high as 1%. Additionally, we validated the use of RNA-guided CRISPR/Cas9 endonucleases to make changes in yeast genomes, resulting in frequencies of genome modification >90% in transformed populations. We further optimized this method to generate larger libraries as high as 10^5 individuals and explored a proof of concept epistasis experiment involving thermotolerance. Lastly, the propagation of changes to successive generations is useful when engineering organisms on the population level. To this end we explored the use of RNA-guided gene drives to bias inheritance in S. cerevisiae. We show that inheritance of these selfish elements can be biased to over 99% and is reversible.

Riboflavin transport is mediated, in part, by riboflavin transporter proteins 2 and 3, encoded by SLC52A2 and SLC52A3, respectively. Biallelic mutations in SLC52A2 and SLC52A3 impair riboflavin transporter protein function and riboflavin transport, causing disruptions to mitochondrial metabolism which result in sensory and motor neurodegeneration and give rise to riboflavin transporter deficiency (RTD) in humans. RTD is a rare neurodegenerative disease characterised by respiratory compromise, muscle and limb weakness, and vision and hearing impairments. RTD patients are treated with high-dose riboflavin supplementation which is effective in over 70% of cases but can be ineffective due to rapid excretion of riboflavin when its plasma concentration exceeds 0.5 μM. To address the need for alternative or supplemental RTD treatment, this study generated morpholino-mediated knockdown and CRISPR/Cas9 models of RTD in zebrafish. An RTD-like phenotype is observed in these RTD models including hearing loss, decreased motor axon length, and impaired locomotor activity. The slc52a3 morphant phenotype was found to be specific via coinjection of slc52a3 morpholino/human SLC52A3 mRNA, which achieved effective rescue of the morphant phenotype, as well as slc52a3 morpholino/p53 morpholino coinjection, which maintains the slc52a3 morphant phenotype. In line with clinical findings, riboflavin supplementation resulted in some improvement of the morphant phenotype. Probenecid was selected as a candidate drug due to its inhibitory effect on OAT-3, which mediates riboflavin excretion. However, supplementing riboflavin treatment with probenecid provided no additional benefit to the slc52a3 knockdown model. Further development of CRISPR/Cas9-knockout lines of slc52a2 and slc52a3, as well continued therapeutic screening of riboflavin and probenecid and consideration of alternative therapeutics will provide more opportunities to uncover novel therapeutic strategies to improve RTD treatment.

CRISPR-Cas ist ein adaptives Immunsystem, dass Bakterien und Archaeen vor eindringenden Nukleinsäuren schützt. Es besteht aus einem sogenannten CRISPR-Array, der als genetisches Gedächtnis vorangegangene Infektionen speichert und einem cas Lokus, welcher für die Abwehr essentielle Proteine codiert. Das CRISPR-assoziierte Protein 9 (Cas9) des Typ II CRISPR-Cas Systems aus Streptococcus pyogenes ist heutzutage das Mittel der Wahl für Gentherapie und Genom-Modifikationen. Allerdings gibt es nach wie vor Probleme mit der Ungenauigkeit dieses Systems, welche für eben genannte Ansätze behoben werden müssen. Aus diesem Grund ist es besonders wichtig zu verstehen, in welcher Weise die Spezifität von Cas9 beeinflusst wird. In dieser Arbeit wurden die Voraussetzungen für eine spezifische Erkennung der Zielsequenz durch drei verschiedene Cas9 Proteine des Typs II-A und ein Cas12a Protein des Typs V-A CRISPR-Cas Systems untersucht. Wir zeigen, dass Arginin Seitenketten der sogenannten „bridge“ Helix in Cas9 von S. pyogenes eine wichtige Rolle in der Bindung und Spaltung der DNS spielen. Diese Seitenketten können in zwei Gruppen unterteilt werden, welche die Spezifität von Cas9 entweder vergrößern oder verkleinern. Die Aminosäuren R63 und R66 reduzieren die Spezifität von Cas9 indem sie den sogenannten R-loop in Anwesenheit einer Fehlpaarung stabilisieren. Wir zeigen außerdem, dass Q768 eine erhöhte Toleranz von Cas9 zu Fehlpaarungen an Position 15 der Zielsequenz vermittelt und dass das Entfernen dieser Aminosäure die Spezifität von Cas9 im Bereich der Zielsequenz, die am weitesten von der PAM entfernt ist, erhöht. Eine Kombination der Mutationen der oben genannten Arginin und Glutamin Seitenketten führt zur Erhöhung der Gesamtspezifität von Cas9. Die Ergebnisse dieser Arbeit tragen zum Verständnis bei, wie Cas9 Fehlpaarungen innerhalb der Zielsequenz detektiert und können dabei helfen weitere Strategien für eine verbesserte Spezifität von Cas9 zu entwickeln. / CRISPR-Cas (CRISPR-associated) systems are adaptive immune systems that have evolved in bacteria and archaea for protection against invading nucleic acids. They consist of a CRISPR array, where the genetic memory of the infection is stored and ultimately transcribed and processed into CRISPR RNAs (crRNAs), and of an operon of cas genes that encodes the Cas proteins. This thesis is focused on class 2 CRISPR-Cas systems that employ single RNA-guided nucleases in the interference phase. Dual-RNA guided CRISPR-associated protein 9 (Cas9) of the type II CRISPR-Cas system has become the tool of choice for genome editing applications in life sciences. However, off-target cleavage by Cas9 is one major issue that needs to be addressed for applications of the CRISPR-Cas9 technology for therapeutic purposes. Therefore, understanding the features that govern Cas9 specificity is of great importance. In this thesis, seed sequence requirements of three Cas9 proteins from the class 2 type II-A and one Cas12a protein from the class 2 type V-A CRISPR-Cas system have been investigated. We analyze the influence of mismatches and show that they affect target binding and/or cleavage by S. pyogenes Cas9. Additionally, we demonstrate that the arginine residues from the bridge helix of S. pyogenes Cas9 are important for target DNA binding and cleavage. Furthermore, these residues comprise two groups that either increase or decrease Cas9 sensitivity to mismatches i.e. specificity. R63 and R66 reduce Cas9 specificity by stabilizing the R-loop in the presence of mismatches. We also show that Q768 mediates Cas9 tolerance to a mismatch at target position 15 and removal of Q768 increases Cas9 specificity in the PAM-distal part of the target. Combination of arginine mutations and Q768A increased overall the sensitivity to mismatches. The results of this thesis elucidate how Cas9 senses PAM-adjacent mismatches and provide a basis to develop strategies for Cas9 variants with enhanced specificity.

Plants employ sophisticated molecular machineries to fine-tune their responses to growth, developmental, and stress cues. Plants cellular response influences gene expression through regulating processes like transcription and splicing. To increase the genome coding potential and further regulate the expression, pre-mRNA is alternatively spliced. Serine/Arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize splicing cis-elements and regulate both constitutive and alternative splicing. Recent studies reported only 22 SR proteins encoded in the genome of rice (Oryza sativa), which are classified into 6 subfamilies. Oryza s. SC subfamily 106 kDa (Os-Sc106) locus is homologous to the human SR protein SFSR11 (SRp54). Os-Sc106 contains SR proteins characteristics, and was not included among the rice SR proteins. The clustered regularly interspaced short palindromic repeats (CRISPR) and its associated protein 9 (Cas9) system, an RNA-guided endonuclease complex that introduces a double-strand break (DSB) into the DNA. Innovative scientific advances in genome engineering have made CRISPR/Cas9 an excellent system to conduct functional knockout studies of genes in most biological systems including plants. In this study, I targeted the rice Os-Sc106 locus at exon1, and 3 via CRISPR/Cas9 system. Genotyping analyses revealed the recovery of Os-Sc106 mutants including complete functional knockouts such as sf11h-2, sf11h-8, and sf11h-55. Phenotypic analyses show that Os-Sc106 mutants (sf11h-2, 8, 55, and 57) are oversensitive under abiotic stress in comparison to WT plants, suggesting that Os-Sc106 locus encodes a protein that is important for regulating plant stress responses.

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