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.

This dissertation advances a new theory of the editor. Editors are pervasively influential for shaping our evaluations of all kinds of work, from literature, to news, to scholarship, and beyond. I test my theory on editorial influence in little magazines, providing close readings, recovering historical contexts, and identifying a wide variety of rhetorical affordances available to little magazines’ editors. I draw on rhetorical narrative theory, most especially the “implied author” concept, which I build on in order to theorize an “implied editor.” I furthermore demonstrate that understanding the authored and edited texts as distinct is important for teasing apart the full breadth of editorial work. This dissertation also proposes a new model of editorship. My model captures (1) the role of the editor, which is a simultaneously author- and audience-oriented role; (2) the importance of poetics; and (3) the dual-streamed creative role of editors. As such, my model captures dynamics not noted elsewhere and enables us to address such questions as: (1) How does editorship differ from authorship? (2) How can we think of the relationship among author, editor, and audience? and (3) How does the ethics of editorship differ from that of authorship? This dissertation further tests my model in conversation with a contemporary little magazine, issue 23 of A Public Space. In so doing, I explore how APS’s editor uses editorial textual resources, some of which overlap with authorial textual resources and some of which diverge from them. I also test my theory against the editorial role in Harlem Renaissance little magazines to elucidate the distinctive ways in which editors manifest their respective magazines’ purposes, both implicitly and explicitly. In so doing, these editors reveal why we must view the magazines as distinct and holistic entities with potentially competing priorities rather than merely collections of (then) up-and-coming writers and their discrete works. My model of editorship highlights Fire!!’s complex editorial significance. My theory of editorship and its application to these little magazines demonstrates the significance of editorship, particularly in relation to the crafting of uniquely edited texts.

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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.

<p> A survey of the main features and characteristics of text editors and formatters is given. An implementation of a text editing and formatting system is discussed. The Text Editor and Formatter (TEF) was designed to be easy to learn and use and to allow extensions of the present version with little modification to the existing system. TEF is a content (or context) oriented editing system with line organization and text formatting capabilities. </p> / Thesis / Master of Science (MSc)

H1 linker histones are structural components of chromatin, generally implicated in the formation of “higher order” chromatin states. With eleven non-allelic subtypes in mammals, the H1 family is highly diverse. While they are commonly associated with chromatin compaction and transcription repression, these histones also play crucial roles in mouse development and stem cell differentiation. Although the prevailing belief is that H1 subtypes have redundant functions, their distinct amino acid composition and differential expression throughout development suggest subtype-specific roles. Previous studies have explored the roles and interactions of linker histones, but limitations in model systems, cell types, and subtypes studied have hindered our comprehensive understanding of the implications and synergy of multiple H1 linker histones. To gain insight into the individual and combined roles of linker histones in human embryonic stem cells (hESCs), we conducted an extensive study in which we systematically removed each somatic linker histone and looked at all potential combinations. Using RNA-seq and in-depth bioinformatic analysis, we discovered that linker histones in hESCs exhibit partial non-redundancy. We classified them into three main groups associated with distinct biological processes, particularly related to development and stem cell differentiation. We observed that depleting H1.1 or H1.5 influenced the proportion of mesodermal progenitor cells, with further impact when combined with specific H1 subtypes, resulting in changes in ectodermal progenitor cells. Additionally, we demonstrated that linker histones synergistically regulate interconnected biological pathways, potentially affecting early stem cell differentiation. Based on our findings, we propose that H1 subtypes regulate specific transcriptional programs, which in conjunction, are fundamental in the coordination of essential cellular processes involved in early human embryonic development, both in the ground state of hESCs and during stem cell differentiation. We anticipate that the generation of the H1 KO library described in our study will provide a novel tool for studying the role of linker histones in later stages of human development and will facilitate the comprehension of specific roles of these chromatin proteins in other relevant cellular processes.

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