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Trade in CRISPR/Gene-Edited Wheat: A Partial Equilibrium AnalysisFosu, Prince January 2019 (has links)
Previous studies have analyzed how the adoption of genetically engineered or modified technologies have affected agricultural crops such as corn, soybeans, cotton, and barley without focusing on wheat. Also, given the negative impact of drought on wheat production, no studies have focused on the implications of drought tolerant (HB4) and CRISPR/gene-editing on wheat trade. To address these issues, this study employed the partial equilibrium analysis and analyzed the implications of drought tolerant (HB4) and CRISPR/gene-editing technology adoption on wheat trade under various scenarios. The study found that when Argentina, Australia, United States, Canada, and Russia adopt gene-editing wheat, all consuming countries experience a welfare gain except Japan, Korea, Belgium, Netherland, and Italy. More so, Argentina, Mexico, Nigeria, Brazil, Egypt, and Venezuela continue to consume CRISPR wheat in all scenarios. Also, all producing countries experience a gain in producer welfare.
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Leveraging DNA Damage Response Pathways to Enhance the Precision of CRISPR-Mediated Genome EditingNambiar, Tarun S. January 2020 (has links)
The ability to efficiently and precisely modify the genome of living cells forms the basis of genetic studies and offers great potential to research and therapy. With its unprecedented ease of use and efficiency, CRISPR-Cas9 has revolutionized genome editing at a stunning pace. Functioning like a pair of molecular scissors, the RNA-guided endonuclease Cas9 can cleave genomic DNA to generate double-stranded breaks (DSBs). DSBs trigger the DNA damage response (DDR), that sets into motion multiple cellular processes that attempt to repair these lesions. One such cellular pathway, named homology-directed repair (HDR), enables researchers to make desirable changes precisely to genomic DNA sequences. HDR facilitates nearly any genomic DNA change, from the replacement of a single nucleotide to the insertion of several thousands of nucleotides. Thus, the precision, as well as versatility at modifying genomic DNA, make HDR a particularly promising repair pathway for genome editing. However, competition with other error-prone DSB repair pathways reduces the efficiency of HDR and results in the generation of an excess of undesirable mutations. In this thesis, I address these two challenges associated with CRISPR-Cas9 genome editing: i) low efficiency of HDR and ii) large deletion mutations generated upon repair of Cas9-induced DSBs.
The first part of the thesis describes our study to identify genetic factors that stimulate HDR at Cas9 induced DSBs. Towards this goal, we individually express in human cells 204 open reading frames involved in the DDR and determine their impact on CRISPR-mediated HDR. From these studies, we identify RAD18 as a stimulator of CRISPR-mediated HDR. By defining the RAD18 domains required to promote HDR, we derive an enhanced RAD18 variant (e18) that stimulates HDR induced by CRISPR-Cas9 in multiple human cell types, including embryonic stem cells. Mechanistically, e18 suppresses the localization of the HDR-inhibiting factor 53BP1 to DSBs. Through this suppression of 53BP1, e18 promotes HDR at the expense of insertion and deletion mutations introduced by error-prone DSB repair pathways. Altogether, this study identifies e18 as an enhancer of CRISPR-mediated HDR and highlights the promise of engineering DDR factors to augment the efficiency of precision genome editing.
In the second part of the thesis I describe our study of the genetic mechanisms regulating large deletions that are generated upon repair of Cas9-induced DSBs. We perform a pooled CRISPR screen to interrogate the effect of knocking out 610 DDR genes on the frequency of CRISPR-mediated long deletions. The screen identifies genes that consistently affect the frequency of long deletions when knocked-out in different experimental conditions. Thus, our study lays the foundations for uncovering the mechanisms regulating CRISPR-mediated long deletions and has the potential to aid in the development of new strategies to limit their generation.
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Characterization of CRISPR-Cas12a Novel Small Molecule InhibitorsYinusa, Abadat 01 December 2022 (has links)
Cas12a (Cpf1) is a representative type V-A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) effector RNA-guided DNA endonuclease used widely for genome editing. Identification of Cas12a inhibitors is important for regulating gene editing, enhancing genome editing specificity, and safety for human therapeutics. This study used a fluorescence-based assay to screen diverse drug libraries at a core facility for potential small molecule candidates that can inhibit AsCas12a endonuclease activities. Further validation of the major hit compounds revealed that these small molecules inhibit Cas12a in vitro DNA cis and trans cleavage activities as well as gene editing in cells. IC50 values obtained from gene editing inhibition were even lower than primary screening reported IC50. We determined the impact of the small molecules on the thermal stability of Cas12a, possible binding sites, and binding affinity (Kd) using thermal denaturation experiments. Enzyme kinetics studies were used to investigate the effect of the inhibitors on ribonucleoprotein complex formation. The discovered molecules create a tool for achieving safer applications of CRISPR-Cas12a in biotechnology and therapeutics.
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CRISPR-Hybrid: A CRISPR-mediated intracellular selection platform for RNA aptamersSu-Tobon, Qiwen January 2024 (has links)
Thesis advisor: Jia Niu / In the last ten years, programmable CRISPR-Cas systems have been widely-used as genome editing tools for gene manipulation, epigenetic functionalization, and transcriptional regulation. Among them, fusing effector proteins directly to the Cas protein allows the resulting CRISPR machinery to direct these effector proteins to multiple sites of the same gene or multiple genes at once. Although they can be used to target multiple genetic loci simultaneously, these methods are often limited to applying one regulatory function (e.g., activation or repression) at a time. On the other hand, recruiting effector proteins via RNA aptamer-RNA-binding protein (RBP) recognition enabled multiplexed and multi-modular gene manipulations simultaneously. However, there are only a limited set of aptamer-RBP pairs that can function orthogonally and intracellularly, e.g., MS2 RNA aptamer with MS2 coat protein (MCP), and PP7 RNA aptamer with PP7 coat protein (PCP). The scarcity of orthogonal intracellular aptamer-RBP pairs imposes severe constraints on the CRISPR-mediated multifunctional manipulations of the genome and the epigenome. We established an intracellular selection platform for RNA aptamers, named CRISPR-Hybrid, and expanded the scope of aptamer-RBP toolkit for CRISPR transcription regulators. Using CRISPR-Hybrid, we successfully identified a highly active and specific aptamer for bacteriophage Qβ coat protein (QCP) in vivo, and characterized its binding affinity and specificity in vitro. We further validated the orthogonality of selected aptamer with QCP to other available intracellularly functional aptamer-RBP pairs including MS2-MCP and PP7-PCP in mammalian cells. Finally, we demonstrated the utility of this orthogonal pair in multiplexed and multi-modular regulations of endogenous genes. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
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Characterizing NgAgo and exploring its activities for biotechnological applicationsKok Zhi Lee (10725411) 29 April 2021 (has links)
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<p>Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene-
editing as they do not require sequence motifs adjacent to their targets for function. One promising
pAgo candidate from the halophilic archaeon Natronobacterium gregoryi (NgAgo) has been the
subject of intense debate regarding its potential in eukaryotic systems. NgAgo was initially
claimed to edit genes in mammalian cells, but the report was retracted due to replication failure.
Due to low solubility, subsequent studies refolded NgAgo and suggested that it cuts RNA but not
DNA; however, mutation of the conserved active site does not abolish cleavage activity, raising
the possibility of nuclease contamination. Another independent study demonstrated gene-editing
via NgAgo in bacteria. These inconsistent results underscore the knowledge gap and roadblock for
NgAgo-based gene-editing tool development.
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<p>In this work, I revisit this enzyme and characterize its function in vitro and in a bacterial
system. The halophilic features of NgAgo have been neglected in the literature, leading to
inconclusive results. Like other halophilic proteins, NgAgo has modified amino acid composition,
leading to failure of domain identification/function prediction via sequence alignment. Indeed,
using more sensitive structural alignments, I identified a new single-stranded DNA binding domain,
repA, in NgAgo and other halophilic pAgos. Due to its halophilic nature, NgAgo expresses poorly
in low-salt environments, with the majority of protein being insoluble and inactive even after
refolding. However, soluble NgAgo indeed cuts DNA. NgAgo DNA-cleaving activity can only be
abolished via mutation in the canonical PIWI domain and repA deletion, revealing a new catalytic
behavior in pAgos. Moreover, NgAgo requires both repA and PIWI domains to create double-
stranded DNA breaks, leading to cell death or enhancing homologous recombination, or gene-
editing, at a modest level in bacteria. Rational protein engineering of NgAgo was also pursued to
increase solubility. Although three out of seven mutants showed significant increases in solubility,
they lost the ability to cleave DNA in E.coli. Structural modeling revealed some subtle but
important differences in the protein structures, explaining why the mutants lose their function.
Besides, a selection system for improving endonuclease activity was optimized for future pAgo
optimization. Collectively, this work revealed that NgAgo possesses unique catalytic behavior in
the pAgo family and has some gene-editing application potential. More importantly, this work expands knowledge of the pAgo family, providing a foundation for future pAgo-based gene-
editing tool development. </p>
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Nanosystems for Gene Editing and Targeted TherapyLao, Yeh-Hsing January 2019 (has links)
Nanomedicine has emerged in the past decades, and a variety of designs for drug/gene delivery have been reported since the concept of nanomedicine was first demonstrated. However, with the exception of a few notable successes, the clinical translation of nanomedicine has been slow. Specificity and delivery efficiency are the major obstacles; only a few nanomedicine systems can effectively reach and release the therapeutic payload at the target site, thereby limiting the therapeutic efficacy. To tackle these issues, this work aims to design new strategies to improve nanomedicine systems at the gene-, protein- and tissue- levels.
We applied CRISPR/Cas9 technology for gene targeting. Delivering CRISPR/Cas9 elements, including Cas9 endonuclease and a corresponding guide RNA, allows for specific gene mutagenesis. A conventional gene delivery carrier often has a highly positive charge density for higher transgene expression, but this may result in unfavorable effects on the Cas9 plasmid transfection. As a large plasmid, strong interaction between the Cas9 plasmid and the polycation with high charge density may hinder the plasmid’s intracellular release. Moreover, high Cas9 expression usually leads to undesirable off-target effects. We addressed these two major obstacles by designing a low-charged density micelle, composed of quaternary ammonium‐terminated poly(propylene oxide) and amphiphilic Pluronic F127. We tested this design on a human papillomavirus (HPV)-induced cervical cancer model to target the HPV oncogene, E7. Our micellar carrier enabled effective Cas9 transfection with a transient Cas9 expression, which offered enhanced Cas9 on-target specificity. This nonviral Cas9‐mediated E7 mutagenesis resulted in significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo.
Although CRISPR/Cas9 technology is a powerful toolkit for gene manipulation, gene editing might not be practical for therapeutics in the cancers that develop from endogenous mutations, which may vary among patients and disease stages. Protein-targeting, therefore, may be a more efficient approach. Aptamer and its selection technology, namely SELEX, offer direct evolution to obtain a nucleic acid ligand that specifically recognizes the protein target. Yet, aptamer screening remains unsatisfactory, and the success rate of SELEX is limited. We designed two approaches to improve the aptamer screening. We first employed a microarray platform to deconvolute the aptamer sequence and identified the aptamer functional motif. The resulted protein-targeting motif with an optimal length and showed enhanced structural and functional characteristics compared with its parental sequence. In addition to sequence optimization, conjunction of two distinct aptamers that recognize different epitopes of the protein target is another approach to improve the aptamer’s affinity. In looking for a rapid way to screen this bivalent aptamer pair, we designed a quantum dot (QD)/ Förster resonance energy transfer (FRET) sensor. Using a thrombin aptamer as a model system, we conjugated an anti-thrombin aptamer with QD and stained the other one with the intercalation dye, YOYO-3. If the two aptamers recognized different epitopes of thrombin, the conformational change of the two aptamers would take place when interacting with thrombin, and this would induce YOYO-3 dye’s translocation. YOYO-3 would be transferred from the aptamer to QD surface, resulting in a strong FRET signal. In contrast, if they recognized the same epitope, binding competition between two aptamers would inhibit dye translocation, thereby giving a minimal FRET signal. By measuring the FRET signal, we can verify if the two aptamers may form a bivalent pair.
Lastly, we integrated mesenchymal stem cell (MSC) with a nanomedicine system to achieve active tissue-targeting. MSC is known to migrate toward certain types of cancer cells by chasing the chemotaxis release from the cancer cells, but the therapeutic payload that MSC can carry is limited. Forming an MSC spheroid allowed the loading of the nanomedicine system with another type of anti-cancer drug. We therefore designed a hybrid MSC/nanomedicine spheroid, which functioned as an active tumor-targeting platform, enabling effective delivery for both cytotoxic protein and chemotherapeutic drugs. In a heterotopic glioblastoma model, the hybrid spheroid significantly improved the retention of the nanomedicine system at the tumor site, leading to enhanced tumor inhibition in vivo.
Collectively, this work demonstrated the effective approaches for gene, protein and tissue targeting by addressing the issues of low specificity and limited delivery efficiency that many current nanomedicine systems face. Particularly, the results may add to the armamentarium of cancer therapeutics, which remains largely challenging and intractable.
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Application of CRISPR/Cas9 to edit genes affecting seed morphology traits in wheatPan, Qianli January 1900 (has links)
Master of Science / Genetics Interdepartmental Program / Eduard D. Akhunov / The CRISPR/Cas9-based genome editing system holds a great promise to accelerate wheat improvement by helping us to understand the molecular basis of agronomic traits and providing means to modify genes controlling these traits. CRISPR/Cas9 is based on a synthetic guide-RNA (gRNA) that can guide Cas9 nuclease to specific targets in the genome and create double strand breaks (DSB). The DSB are repaired through the error-prone non-homologous end joining process causing insertions or deletions that may result in loss-of-function mutations. Here, we have developed an effective wheat genome editing pipeline. We used next-generation sequencing (NGS) data to estimate genome editing efficiency of many gRNAs using the wheat protoplast assay and choose the most efficient gRNAs for plant transformation. We successfully applied this pipeline to five wheat orthologs of the rice yield component genes that have been identified previously. We obtained edited plants for all these genes and validated the effect of the mutations in TaGW7 on wheat traits, which showed trends similar to those in rice. These results suggest that transferring discoveries made in rice to improve wheat is a feasible and effective strategy to accelerate breeding.
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A genetic and epigenetic editing approach to characterise the nature and function of bivalent histone modificationsBrazel, Ailbhe Jane January 2018 (has links)
In eukaryotes, DNA is wrapped around a group of proteins termed histones that are required to precisely control gene expression during development. The amino acids of both the globular domains and unstructured tails of these histones can be modified by chemical moieties, such as methylation, acetylation and ubiquitination. The ‘histone code’ hypothesis proposes that specific combinations of these and other histone modifications contain transcriptional information, which guides the cell machinery to activate or repress gene expression in individual cell types. Chromatin immunoprecipitation (ChIP) experiments using undifferentiated stem cell populations have identified the genomic co-localisation of histone modifications reported to have opposing effects on transcription, which is known as bivalency. The human α-globin promoter, a well-established model for the study of transcriptional regulation, is bivalent in embryonic stem (ES) cells and this bivalency is resolved once the ES cells terminally differentiate (i.e. only activating or repressing marks remain). In a humanised mouse model, the deletion of a bone fide enhancer within the human α-globin locus results in heterogeneous expression patterns in primary erythroid cells. Notably, this correlates with an unresolved bivalent state at this promoter in terminally differentiated cells. Using this mouse model it is not feasible to ascertain whether the transcriptional heterogeneity observed in the cells lacking an α-globin enhancer is reflective of epigenetic heterogeneity (i.e. a mixed population of cells) rather than co-localisation of bivalent histone modifications within the same cells. Furthermore, the functional contribution of bivalency to development has yet to be described. To address these difficulties, I aimed to generate a fluorescent reporter system for human α-globin to facilitate the separation of transcriptionally heterogeneous erythroid cells. This model will provide material for ChIP studies on transcriptionally active and inactive populations to determine whether the epigenetic bivalency is reflective of a mixed cell population or true bivalency. In addition, I aimed to produce epigenetic editing tools to target bivalent promoters, which in combination with in vitro differentiation assays would provide an interesting framework to test the function of bivalency during development. In this study, I extensively tested gene-editing strategies for generating a fluorescent reporter knock-in in humanised mouse ES cells. I validated the suitability of humanised mouse ES cell lines for gene targeting studies and optimised a robust in vitro differentiation protocol for studying erythropoiesis. I utilised both recombineering and CRISPR/Cas9 gene editing tools in tandem with PiggyBac transposon technology, to knock-in the reporter gene. I made significant steps in gene targeting and successfully inserted the reporter downstream of the α-globin gene. I also generated a cloning system to express site-specific DNA-binding domains (TALEs) fused to epigenetic regulators with the aim to resolve bivalent histone modifications in vitro. From preliminary tests using these fusion proteins targeting Nrp1, a bivalent promoter in mES cells, I observed mild but significant changes in gene expression although histone modifications were unchanged. The various tools generated and tested in this study provide a solid foundation for future development of genetic and epigenetic editing at the human α-globin and other bivalent loci.
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Characterizing human regulatory genetic variation using CRISPR/Cas9 genome editingBrandt, Margot January 2020 (has links)
Rare gene-disrupting variants and common regulatory variants play key roles in rare and common disease, respectively. These variants are of great interest for investigation into genetic contributions to disease, but experimental methods to validate their impact on gene expression levels are lacking. In this study, we utilized CRISPR/Cas9 genome editing to validate regulatory variants including cis-eQTLs, rare stop-gained variants in healthy and disease cases and one immune-response trans-eQTL master regulator.
For investigation into common and rare regulatory variants within transcribed regions, we developed a scalable CRISPR-based polyclonal assay for experimental assessment. First, we applied this assay to nine rare stop-gained variants found in the general population, in GTEx. After editing, the stop-gained variants show a significant allele-specific depletion in transcript abundance, as expected. Next, we utilized the assay to validate 33 common eQTLs found in GTEx. After editing, the eQTL variants show higher variance in effect size than control variants, indicating a regulatory effect. Finally, we applied the polyclonal editing approach to clinical and new stop-gained variants in two disease-associated genes. The results follow the expected trend, with NMD being triggered by variants upstream of the NMD threshold but not by those beyond. This method demonstrates scalable experimental confirmation of putative causal regulatory variants, and improved interpretation of regulatory variation in humans.
Next, we sought to experimentally validate an immune-response eQTL for IRF1 in cis and many genes in trans under LPS stimulation. We used CRISPRi to repress the enhancer locus and found that the enhancer is active in our immune cell system. Next, we used CRISPR-Cas9 genome editing and isolation of monoclonal cell lines to target this variant locus. After LPS stimulation, we performed RNA-sequencing on wild type and edited clones, showing that the effect size of the genes which are associated with the trans-eQTL are correlated with differential expression between the edited and wild type cell lines for the same genes. Additionally, we find that the differential expression between edited clones is correlated with CRISPRi repression of the IRF1 promoter and enhancer. In this way, we were able to identify a common genetic variant which modifies the transcriptomic immune response to LPS and validate the trans-eQTL signal.
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Mre11-Rad50-Xrs2 Complex in Coordinated Repair of DNA Double-Strand Break Ends from I-SceI, TALEN, and CRISPR-Cas9Lee, So Jung January 2022 (has links)
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.
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