Analysis of homolog-based CRISPR editing in somatic cells

体细胞中基于同源物的 CRISPR 编辑分析

• 批准号: 10343429
• 负责人: ETHAN BIER
• 金额: $ 31.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10343429
• 关键词: Adult; Affect; Agriculture; Alleles; Bacteria; Biology; CRISPR gene drive; Cell Cycle; Cell Line; Cells; Chromosome Mapping; Chromosome Pairing; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Culicidae; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; DNA Repair; DNA cassette; Disease model; Drosophila genus; Ectopic Expression; Elements; Endonuclease I; Event; Frequencies; G2 Phase; Gene Conversion; Genes; Genetic; Genome; Genomics; Grant; Guide RNA; Homologous Gene; Human; Human Cell Line; Induced Mutation; Insect Control; Insecta; Mammalian Cell; Mammals; Mediating; Medicine; Meiosis; Mitotic; Modeling; Mutation; Nonhomologous DNA End Joining; Oncogenes; Organism; Outcome; Pathway interactions; Phenotype; Pigmentation physiologic function; Population; Process; Reporter; Resistance; S phase; Sequence Homologs; Site; Somatic Cell; System; Technology; Testing; Variant; Visualization; Yeasts; base; base editing; design; developmental genetics; disease-causing mutation; expectation; experimental study; fly; gastrointestinal epithelium; gene correction; gene drive allele; gene drive system; gene therapy; genetic element; genetic variant; genome editing; genome integrity; genomic locus; improved; in vivo; insect disease vector; insight; mutant; next generation; notch protein; novel; novel strategies; nuclease; portability; precise genome editing; precursor cell; repair model; repair strategy; repaired; stem cells; tool; transgene expression

Recently developed CRISPR-based systems permit precise genome editing by inducing targeted DNA breaks at specific sites in the genome. Cellular DNA repair machinery can restore genome integrity by copying information from the intact homologous chromosome at the cleavage site via homology directed repair (HDR). While precise HDR-mediated DNA repair is the predominant pathway active during meiosis, the competing and potentially mutagenic non-homologous end-joining pathway (NHEJ) is typically thought to prevail in somatic cells. One reason for this bias is that the NHEJ pathway is active throughout somatic cell cycles, while HDR is primarily restricted to post-replicative S and G2 phases. Thus, achieving efficient HDR-based gene editing in somatic cells has proven challenging, which limits the in vivo use of this technology for human gene therapy. My group has contributed to developing the first CRISPR-based gene-drive (or active genetic) systems in flies, mosquitoes, mammals, and bacteria that bias germline inheritance of genetic elements programmed to cut the genome at their site of insertion. We also pioneered allelic-drive systems designed to promote biased inheritance of a favored allelic variant at a separate genetic locus. These germline drive systems also produce somatic phenotypes, which have generally been attributed to mutations induced by the NHEJ pathway. Recently, we developed genetic elements we refer to as “CopyCatchers” that permit visualization of HDR- mediated copying of gene cassettes. These studies have revealed an unexpectedly high frequency of somatic gene conversion (SGC) events in Drosophila (30-50%) wherein the chromosome homolog serves as a DNA repair template. Rates of SGC can be improved further by optimizing delivery of CRISPR components, or by reducing the expression of various genes encoding factors involved in DNA repair or chromosome pairing. Preliminary experiments indicate that interhomolog SGC can also take place in human cells and point to untapped strategies for repairing disease-causing mutations using intact sequences from the homologous chromosome. In this grant we propose to explore SGC repair mechanisms mediated by Cas9 and Nickase in somatic cells of Drosophila and then extend analysis of this interhomolog repair process to human cells. First, we will analyze the mechanisms underlying CRISPR dependent copying of gene cassettes or allelic variants to optimize their activities. Next, we will develop and optimize Drosophila models for homolog-based repair of disease-causing mutations in the Notch locus affecting mitotically active stem cells or in post-mitotic cells in the adult gut epithelium using a humanized Drosophila CFTR–/– disease model. Finally, we will assess whether insights gained in Drosophila are portable to human somatic cell lines, and whether interhomolog SGC can restore native gene activity in human cell-based models for cystic fibrosis. Enhancing homolog-based repair in mammalian cells could offer transformative possibilities for next-generation gene therapy strategies.

最近开发的基于 CRISPR 的系统可以通过诱导目标 DNA 进行精确的基因组编辑 基因组中特定位点的断裂。细胞DNA修复机制可以通过复制恢复基因组完整性 通过同源定向修复（HDR）从切割位点的完整同源染色体获取信息。 虽然精确的 HDR 介导的 DNA 修复是减数分裂期间活跃的主要途径，但竞争性和 潜在诱变的非同源末端连接途径（NHEJ）通常被认为在体细胞中普遍存在 细胞。造成这种偏差的原因之一是 NHEJ 通路在整个体细胞周期中都处于活跃状态，而 HDR 则在整个体细胞周期中处于活跃状态。 主要限于复制后 S 期和 G2 期。因此，实现高效的基于 HDR 的基因编辑 体细胞已被证明具有挑战性，这限制了该技术在人类基因治疗中的体内应用。 我的团队为开发第一个基于 CRISPR 的基因驱动（或活性遗传）系统做出了贡献 苍蝇、蚊子、哺乳动物和细菌，它们会偏向被编程的遗传元件的种系遗传 在插入位点切割基因组。我们还开创了等位基因驱动系统，旨在促进偏向 在单独的基因位点遗传偏好的等位基因变异。这些种系驱动系统还产生 体细胞表型，通常归因于 NHEJ 途径诱导的突变。 最近，我们开发了我们称之为“CopyCatchers”的遗传元素，它允许 HDR 可视化 介导的基因盒复制。这些研究揭示了出乎意料的高频率的体细胞 果蝇 (30-50%) 中的基因转换 (SGC) 事件，其中染色体同源物充当 DNA 修复模板。通过优化 CRISPR 组件的递送，或者通过 减少参与 DNA 修复或染色体配对的各种基因编码因子的表达。 初步实验表明，同源 SGC 也可以在人类细胞中发生，并指出 使用同源序列的完整序列修复致病突变的未开发策略 染色体。 在这笔资助中，我们建议探索体细胞中由 Cas9 和 Nickase 介导的 SGC 修复机制 果蝇细胞，然后将这种同源修复过程的分析扩展到人类细胞。首先，我们将 分析基因盒或等位基因变体的 CRISPR 依赖性复制的潜在机制 优化他们的活动。接下来，我们将开发和优化果蝇模型，用于基于同源物的修复 影响有丝分裂活性干细胞或有丝分裂后细胞的Notch位点的致病突变 使用人源化果蝇 CFTR–/– 疾病模型对成人肠道上皮进行研究。最后，我们将评估是否 在果蝇中获得的见解可以移植到人类体细胞系中，并且同源 SGC 是否可以 恢复人类囊性纤维化细胞模型中的天然基因活性。增强基于同源物的修复 哺乳动物细胞可以为下一代基因治疗策略提供变革的可能性。