자료유형  

 학위논문

Control Number  

0017163113

International Standard Book Number  

9798384466451

Dewey Decimal Classification Number  

574

Main Entry-Personal Name  

Yan, Jun.

Publication, Distribution, etc. (Imprint  

[S.l.] : Princeton University., 2024

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2024

Physical Description  

138 p.

General Note  

Source: Dissertations Abstracts International, Volume: 86-04, Section: B.

General Note  

Includes supplementary digital materials.

General Note  

Advisor: Adamson, Britt.

Dissertation Note  

Thesis (Ph.D.)--Princeton University, 2024.

Summary, Etc.  

요약Engineering of CRISPR-Cas systems has produced a suite of genome editing tools including programmable nucleases, base editors and prime editors. During my Ph.D., I studied how cellular processes regulate these tools and affect editing outcomes. Chapter 2 focuses on CRISPR nucleases capable of generating DNA double-strand breaks (DSBs) at target sequences. The DSBs are repaired by a complex multi-pathway repair network, leading to heterogenous outcomes. To systematically profile how DNA repair regulates these outcomes, we developed a CRISPR screening platform called Repair-seq; my part in this work was to characterize outcomes uncaptured by Repair-seq including large deletions. I discovered that Cas9-induced DSBs on lentiviral vectors can lead to large deletions through single-strand annealing. Chapter 3 focuses on prime editors which enable precise modifications of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs. To identify cellular determinants of prime editing, I performed Repair-seq screens in K562 cells and identified DNA mismatch repair (MMR) as a major suppressor of small prime edits in those cells. Together with our collaborators, we then developed and validated a dominant negative MLH1 (MLH1dn) whose transient expression enhances prime editing by inhibiting MMR. Chapter 4 features genome-scale CRISPRi screens to identify additional prime editing determinants. A single factor emerged as the strongest mediator: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches, edit types, endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. I found that La functionally interacts with and stabilizes the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, these studies provide key insights into how cellular environment regulates genome editing and suggest useful strategies for improvement.

Subject Added Entry-Topical Term  

Molecular biology.

Subject Added Entry-Topical Term  

Systematic biology.

Subject Added Entry-Topical Term  

Genetics.

Index Term-Uncontrolled  

Genome editing

Index Term-Uncontrolled  

pegRNA

Index Term-Uncontrolled  

Prime editing

Index Term-Uncontrolled  

Double-strand breaks

Added Entry-Corporate Name  

Princeton University Molecular Biology

Host Item Entry  

Dissertations Abstracts International. 86-04B.

Electronic Location and Access  

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Control Number  

joongbu:657264