Maintenance of genomic integrity is essential for the survival of an organism and its ability to pass genetic information to its progeny. However, DNA is constantly exposed to exogenous and endogenous sources of damage, which demands cells to possess DNA repair mechanisms. Of the many forms of DNA damage, double-strand breaks (DSBs) are particularly cytotoxic DNA lesions that cause genome instability and cell lethality, but also provide opportunities to manipulate the genome via repair. One of the major DSB repair pathways shared between single-celled yeast and humans is homologous recombination (HR). HR is initiated by the evolutionarily conserved Mre11-Rad50-Xrs2/Nbs1 (MRX in yeast, MRN in mammals) complex. The MRX complex has a multitude of functions such as damage sensing, adduct removal from DSB ends, and end tethering – a process to maintain the two ends of a DSB in close proximity. The role of the MRX complex has been uncovered by studying the repair of DSBs generated from meganucleases such as HO and I-SceI. However, it is unclear if this knowledge translates to the repair of DSBs from genome editing nucleases such as TALEN and CRISPR-Cas9 (Cas9), as these nucleases create DSBs with different end polarities. While the repair efficiencies and outcomes of TALEN and Cas9 are actively studied, less is known about the earlier stages of repair. The objective of this thesis is to examine the role of the MRX complex in repair processes at both ends of a DSB after cleavage with I-SceI, TALEN, and Cas9 in vivo using the model organism Saccharomyces cerevisiae. In Chapter 1, I describe the importance of DSB repair, a summary of HR and its sub-pathways, the functions of the MRX complex, and properties of I-SceI, TALEN, and Cas9. The materials and methods used in this thesis are detailed in Chapter 2. The work described in Chapter 3 focuses on end tethering and recruitment of downstream repair proteins in haploid cells. I find that DSB ends from the three nucleases all depend on the MRX complex for end tethering, and that initial end polarity does not affect tethering. DSBs created by Cas9 show greater dependence on the Mre11 nuclease of the MRX complex for Rad52 recruitment compared to DSBs from I-SceI and TALEN. Despite Mre11-dependent end processing and Rad52 recruitment at Cas9-induced DSBs, Cas9 stays bound to one DNA end after cleavage, irrespective of the MRX complex. These results suggest that Mre11 exonuclease activity required for adduct removal from DSB ends is not critical for Rad52 recruitment, and that Mre11 endonuclease activity may be driving processing of Cas9-bound DSBs. I also find that MRX tethers DSB ends even after Rad52 recruitment, and unexpectedly, untethered ends are processed asymmetrically in the absence of MRX for all three nucleases. In Chapter 4, I explore the interaction of DSB ends with their repair template, the intact homologous chromosome, in diploid cells. The primary goal is to monitor interhomolog contact in real time from homology search to completion of HR. Although technical limitations make it difficult to capture the entire HR program from DSB formation to repair, I show that untethered ends interact with the homolog separately in the absence of the MRX complex. Similar to haploids, diploid cells display defects in end tethering and end processing without the MRX complex. Repair outcomes of WT cells show an even distribution of G2 crossovers and non-crossovers, while pre-replication crossovers and break-induced replication are undetected. Overall, the results in this thesis provide insight into the functions of the MRX complex in repairing different DSB ends created by I-SceI, TALEN, and Cas9. In Chapter 5, I summarize all of these findings and discuss the motivation for future cell biology studies of HR.

Clustered regularly interspaced short palindromic repeats (CRISPR) are derived from a bacterial and archaeal adaptive immune system. The core enzymes of CRISPR are RNA-guided endonucleases that sequence-specifically cleave foreign double-stranded DNA. Improving and controling the properties of the CRISPR system is a crucial step in advancing the therapeutic potential of CRISPR technology. Several classes of these enzymes exist and are being adapted for biotechnology, such as genome engineering. Cas12a (Cpf1) is a Type V CRISPR-associated (Cas) enzyme that naturally uses only one guide RNA, in contrast to Type II CRISPR-Cas9 enzymes. Thus, Cpf1 may represent a simpler, more practical tool for applications such as gene editing and therapeutics. This dissertation comprises four related studies in this area. To better understand the functional requirements for Cpf1-crRNA interaction and develop modified crRNAs suitable for synthetic biology and therapeutic applications, the first study performed nucleotide substitutions in the crRNA. It focused on the protein-interaction motif of the crRNA by incorporating base changes at the 2ʹ position that alter hydrogen-bonding capacity, sugar pucker, and flexibility. DNA substitutions in RNA can probe the importance of A-form structure, 2ʹ-hydroxyl contacts, and conformational constraints within RNA-guided enzymes. In addition, Chemical modifications include 2'-deoxy, 2'-fluoro, 2'- deoxy-arabinonucleic acid, and oxepane. Our study discovered that 2'-fluoro maintains the A-form structure and is compatible with AsCpf1 activity. Biochemical endonuclease activity, gene editing efficiency, Cpf1 binding affinity, and ribonucleoprotein stability were used to assess the tolerance and effects of modification. Characterizing structure-function requirements for Cpf1-crRNA interaction will facilitate better design and tuning of Cpf1 enzymes. The second study established a FRET-based assay in collaboration with a computational collaborator to identify small molecule inhibitors predicted by virtual docking and simulations. This study aims to lay the foundation for efficient, safe implementation of CRISPR-Cpf1. The third study used chemically modified Cas9-guide RNAs to offset known weaknesses of CRISPRi. It takes advantage of the high binding affinity and nuclease resistance of modified guides to potentially reduce the required components for CRISPRi.

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.

The development of a preimplantation embryo is a stepwise process consisting of morphological, biochemical, and genomic changes. Much remains unknown about the attainment of embryo competency to develop and establish pregnancy. To investigate this, we compared methods of selection at the oocyte or embryo level for improved blastocyst production. Brilliant cresyl blue staining was used to sort oocytes by their growth status (not fully grown vs. fully grown) and the timing of the first embryonic cell division to sort embryos. We found that an embryo's cleavage kinetics are more indicative of their competency than the growth status of the oocyte that gave rise to that embryo. We further investigated the cryopreservation survival of embryos with fast or slow cleavage kinetics and found no significant differences in their ability to hatch post-thawing. Next, we used the complete sequence of the cattle Y chromosome to identify oligonucleotides for efficient sexing of samples. These materials may be used to understand sexual dimorphism as a biological factor in future experiments. Finally, we designed a new method to induce targeted DNA sequence deletions and mRNA cleavage in zygotes using CRISPR-Cas. We targeted the gene OCT4, since the literature shows variable knockout outcomes. Our method improved deletion efficiency while accounting for preexisting or maternally inherited mRNA of the target gene. Our findings can be used to better understand early embryo development and biological drivers of quality, which can be leveraged to improve embryo production and transfer outcomes. / Master of Science / The development of an early embryo involves many biological and structural changes. Much remains unknown about the influences on embryo quality and ability to successfully develop. To investigate this, we compared methods for selecting the highest quality cattle eggs or embryos. We found that the observation of an embryo's development speed is better for selecting high quality embryos than egg quality. We further investigated the freezing survival of embryos with fast or slow growth. We found that the freezing survival of fast and slow growing embryos is not different. Next, we used the complete sequence of the cattle Y chromosome to identify PCR primers for determining sample sex. These resources can help us understand how an individual's sex can influence biological differences. Finally, we designed a new method for removing the total function of a gene in embryos. For this, we deleted segments of DNA and cut RNAs. Our findings can be used to better understand early embryo development and biological drivers of quality, which can be leveraged to improve embryo production and transfer outcomes.

No description available.

A nonsynonymous, loss of function variant (rs58542926, E167K) located in the gene encoding TM6SF2 was identified in multiple genetic association studies as significantly correlating with increased risk for non-alcoholic fatty liver disease (NAFLD) and decreased risk for hyperlipidemia. Given the pivotal role that lipoproteins play at the juncture of these two conditions, researchers hypothesize that the ER-membrane spanning TM6SF2 protein regulates the degree of lipidation of VLDL particles synthesized in the liver. However, not all published data supports this theory and contradictions regarding many aspects of the mechanistic function of TM6SF2 remain. The inconsistencies observed in the literature are in part due to drawbacks of the models used to study TM6SF2 activity; thus, there is an obvious need for an improved hepatocyte model to better understand how TM6SF2 impacts lipid metabolism.To address this need, we present an optimized protocol for the differentiation of inducible pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs), created in collaboration with the Leong Lab. We provide extensive validation of HLC maturity and hepatic functionality, including prolonged albumin secretion, evidence of membrane polarity, and cytochrome P450 induction. We also demonstrate that HLCs express proteins essential for lipoprotein metabolism, secrete authentic VLDL particles, and respond to metabolic perturbations, supporting their value for modeling hepatosteatosis and VLDL metabolism in vitro. To investigate the effect of TM6SF2 variant expression on hepatic lipid metabolism, we produced HLCs derived from 4 homozygous TM6SF2-carrier individuals (KK) and 4 age- and sex-matched unaffected siblings (EE). We describe the variability in differentiation efficiency that we observed in our sibling-matched HLC model and present the gene editing strategy we developed using CRISPR/Cas9 technology and transgene-induced expression to create isogenic iPSCs differing only in their TM6SF2 genotype [EE, KK, or knockout (KO)]. After extensive confirmation of successful gene editing, we explore the effect of TM6SF2 on lipid metabolism in the edited iPSCs. RNA-sequencing and qPCR validation reveal that the Sterol Regulatory Element Binding Protein 2 (SREBP2)-mediated transcriptional program regulating cholesterol synthesis is significantly increased in TM6SF2 KO iPSCs. However, lipidomics analysis and de novo lipogenesis functional assays show that free cholesterol (FC) levels are unchanged. In TM6SF2 KO iPSCs, we further show a reduction in the activities of Acyl-Coenzyme A: Cholesterol Acyltransferase 1 (ACAT1) and Phosphatidylserine Synthase 1 (PSS1), two enzymes that display optimal function when specifically localized to cholesterol enriched ER lipid raft-like domains. Our findings suggest that TM6SF2 may impact cholesterol localization within ER subdomains, which regulate expression levels of cholesterol synthesis genes and activities of ER lipid-raft associated enzymes. In summary, we present here methodological approaches for generating multiple cell culture models in which to investigate the function of TM6SF2, as well as novel evidence supporting a role for TM6SF2 in iPSC cholesterol metabolism.

The pig has similar features to the human in aspects such as physiology, immunology, and organ size. Because of these similarities, genetically modified pigs have been generated for xenotransplantation. Also, when using the pig as a model for human diseases (e.g. cystic fibrosis transmembrane conductance regulator), the pig exhibited similar symptoms to those that human patients present. The main goal of this work was to examine the efficacy of direct injection of the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) in pigs and to overcome shortcomings that resulted after direct injection into the cytoplasm of developing zygotes. By using direct injection of CRISPR/Cas9 into developing zygotes, we successfully generated fetuses and piglets containing 9 different mutations. The total number of aborted fetuses was 20 and of live piglets was 55. Moreover, one issue that was encountered during the production of mutated pigs was that insertion or deletion (indel) mutations did not always introduce a premature stop codon because it did not interfere with the codon read. As a result of these triplet indel(s) mutations, a hypomorphic phenotype was presented; consequently, the mutated gene was partially functional. To prevent this hypomorphic phenotype, we introduced two sgRNAs to generate an intended deletion that would remove a DNA fragment on the genome by causing two double-strand breaks (DSB) during non-homologous end joining (NHEJ). The injection of two sgRNAs successfully generated the intended deletion on the targeted genes in embryos and live piglets. Results after using intended deletions, in IL2RG mutation pigs, did not show hypomorphic phenotypes even when a premature stop codon was not present. After using the intended deletion approach, function of the targeted genes was completely disrupted regardless of the presence or absence of a premature stop codon. Our next aim was to introduce (i.e. knock-in) a portion of exogenous (donor) DNA sequence into a specific locus by utilizing the homology direct repair (HDR) pathway. Because of the cytotoxicity of the linear form of the donor DNA, the concentration of the injected donor DNA was adjusted. After concentration optimization, four different donor DNA fragments targeting four different genes were injected into zygotes. Efficiency of knock-in was an average of 35%. Another donor DNA was used in this study which is IL2RG-IA donor DNA carried 3kb of exogenous cassette. It showed 15.6% of knock-in efficiency. IL2RG-IA Donor DNA injected embryos were transferred into surrogates, and a total of 7 pigs were born from one surrogate, but none of the 7 were positive for the knock-in. Future experiments need to be developed to optimize this approach. Overall, the direct injection of CRISPR/Cas9 is advantageous in cost, time, and efficiency for large animal production and for biomedical research. However, there are still unsolved challenges (off-targeting effects, low efficiency of knock-in, and monoallelic target mutation) that need to be elucidated for future application in humans and other species. / Doctor of Philosophy / The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system is commonly used to make genetically modified pigs. The CRISPR/Cas9 system can break the DNA on a desired gene region. During the DNA repair process, random DNA base pairs can be inserted or deleted on the broken regions, thus generating a mutation on the desired gene. Scientists have adopted new methods to disrupt genes in many species. One of these new methods is the direct injection of CRISPR/Cas9 into a fertilized oocyte. In our first project, we used direct injection of the CRISPR/Cas9 system into the fertilized one-cell embryo. A total of 55 live pigs and aborted 20 fetuses with specifically disrupted genes were produced for biomedical research model. During these studies, one critical drawback of the direct injection method was encountered. Partial function of the gene was possible. To prevent this problem, two DNA broken regions were generated by the CRISPR/Cas9 system to remove the middle part the DNA by two DNA breaking. This method successfully removed the middle portion of the DNA targeted region in the pig embryos. Embryos injected with the CRISPR/Cas9 system to cut the two specific DNA regions were transplanted into surrogate pigs, and a total of 15 piglets were produced. All 15 pigs confirmed that a specific part of the gene had been removed by two DNA breakage. Also, no function of the desired gene was found in the 15 pigs. The objective of the last experiment was to introduce a specific exogenous DNA sequences into specific region of DNA using the CRISPR/Cas9 system. For this study, four different exogenous DNA fragments were synthesized for four different genes. When injected, one exogenous DNA along with the CRISPR/Cas9 system, the average integration efficiency of the four exogenous DNA fragments was 35% in the embryo. Another exogenous DNA, which was longer than other four DNA fragments showed 15.6% integration efficiency. The embryos injected with the long exogenous DNA fragment, along with the CRISPR/Cas9 system, were transferred into surrogate pigs. The result was that a total of 7 piglets were born, but the exogenous DNA sequence was not found in none of the seven piglets. In conclusion, the CRISPR/Cas9 system showed effective removal of the entire gene function of specific genes in the pig. However, for future application in the human and other species, some problems (un-wanted region mutation and low efficiency of exogenous DNA integration) continue to emerge and need to be addressed in future experiments.

<p dir="ltr">Citrus greening has severely impacted orange trees in America, causing significant damage to farmers and industry. Gene editing has been proposed as a potential solution, but understanding consumer acceptance of the resulting gene edited products is essential to evaluate the marketability of this potential solution.</p><p dir="ltr">We conducted a hypothetical discrete choice experiment to analyze consumer preferences for gene edited orange juice. We further explored how consumer willingness-to-pay (WTP) for the gene edited attribute varies depending on the total number of attributes presented in the experiment and how information about the benefits of the technology influences WTP across the different attribute scenarios.</p><p dir="ltr">We find that, on average, consumers devalue the gene edited attribute, but their WTP for the attribute increases when information is provided. Importantly, we also observe that the number of attributes significantly impacts the WTP estimates, but the effect is moderated when information is provided.</p>

Targeted gene manipulation in a complex microbial community is an enabling technology for precise microbiome editing. This thesis introduces a new microbial therapeutic system dubbed Bacterial CRISPR-Transposase Reduction of Virulence In Situ (BACTRINS). BACTRINS is an in-situ microbiome engineering platform designed for efficient and precise genomic insertion of a desired payload and simultaneous knockout of target genes. When applied against a Shiga toxin-producing pathogen in the gut, this system delivers a CRISPR-associated transposase by bacterial conjugation for site-specific inactivation of the Shiga toxin gene and integration of a nanobody therapeutic payload to disrupt pathogen attachment. A single dose of this therapy resulted in high efficiency Shiga gene inactivation and improved survival in a murine infection model of Shiga-producing pathogen. This work establishes a new type of live bacterial therapeutic capable of reducing gut infections by transforming toxigenic pathogens into commensal protectors.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25339号 / 農博第2605号 / 新制||農||1106(附属図書館) / 学位論文||R6||N5511 / DFAM / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 大門 高明, 教授 松浦 健二, 教授 吉田 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM