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  1. The flexibility and precision of CRISPR-Cas9 and related technologies have made these genome editing tools increasingly popular in agriculture, medicine, and basic science research for the past decade. Genome editing will continue to be relevant and utilized across diverse scientific fields in the future. Given this, students should be introduced to genome editing technologies and encouraged to consider their ethical implications early on in precollege biology curricula. Furthermore, instruction on this topic presents an opportunity to create partnerships between researchers and educators at the K-12 levels that can strengthen student engagement in science, technology, engineering, and mathematics. To this end, we present a 3-day student-centered learning program to introduce high school students to genome editing technologies through a hands-on base editing experiment in Escherichia coli, accompanied by a relevant background lecture and facilitated ethics discussion. This unique partnership aims to educate students and provides a framework for research institutions to implement genome editing outreach programs at local high schools. We have included all requisite materials, including lecture slides, worksheets, experimental protocols, and suggestions on active learning strategies for others to reproduce our program with their local communities. 
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    Free, publicly-accessible full text available June 1, 2024
  2. Base editors (BEs) are genome editing agents that install point mutations with high efficiency and specificity. Due to their reliance on uracil and inosine DNA damage intermediates (rather than double-strand DNA breaks, or DSBs), it has been hypothesized that BEs rely on more ubiquitous DNA repair pathways than DSB-reliant genome editing methods, which require processes that are only active during certain phases of the cell cycle. We report here the first systematic study of the cell cycle-dependence of base editing using cell synchronization experiments. We find that nickase-derived BEs (which introduce DNA backbone nicks opposite the uracil or inosine base) function independently of the cell cycle, while non-nicking BEs are highly dependent on S-phase (DNA synthesis phase). We found that synchronization in G1 (growth phase) during the process of cytosine base editing causes significant increases in C•G to A•T “byproduct” introduction rates, which can be leveraged to discover new strategies for precise C•G to A•T base editing. We observe that endogenous expression levels of DNA damage repair pathways are sufficient to process base editing intermediates into desired editing outcomes, and the process of base editing does not significantly perturb transcription levels. Overall, our study provides mechanistic data demonstrating the robustness of nickase-derived BEs for performing genome editing across the cell cycle. 
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  3. Abstract

    Base‐editing technologies enable the introduction of point mutations at targeted genomic sites in mammalian cells, with higher efficiency and precision than traditional genome‐editing methods that use DNA double‐strand breaks, such as zinc finger nucleases (ZFNs), transcription‐activator‐like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR‐associated protein 9 (CRISPR‐Cas9) system. This allows the generation of single‐nucleotide‐variant isogenic cell lines (i.e., cell lines whose genomic sequences differ from each other only at a single, edited nucleotide) in a more time‐ and resource‐effective manner. These single‐nucleotide‐variant clonal cell lines represent a powerful tool with which to assess the functional role of genetic variants in a native cellular context. Base editing can therefore facilitate genotype‐to‐phenotype studies in a controlled laboratory setting, with applications in both basic research and clinical applications. Here, we provide optimized protocols (including experimental design, methods, and analyses) to design base‐editing constructs, transfect adherent cells, quantify base‐editing efficiencies in bulk, and generate single‐nucleotide‐variant clonal cell lines. © 2020 Wiley Periodicals LLC.

    Basic Protocol 1: Design and production of plasmids for base‐editing experiments

    Basic Protocol 2: Transfection of adherent cells and harvesting of genomic DNA

    Basic Protocol 3: Genotyping of harvested cells using Sanger sequencing

    Alternate Protocol 1: Next‐generation sequencing to quantify base editing

    Basic Protocol 4: Single‐cell isolation of base‐edited cells using FACS

    Alternate Protocol 2: Single‐cell isolation of base‐edited cells using dilution plating

    Basic Protocol 5: Clonal expansion to generate isogenic cell lines and genotyping of clones

     
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