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靶向DNA进行精确的基因组编辑 基因组中特定位置的断裂。细胞DNA修复机制可以通过复制来恢复基因组的完整性 通过同源定向修复(HDR)从裂解部位完整的同源染色体中获得信息。 虽然精确的HDR介导的DNA修复是减数分裂中活跃的主要途径，但竞争和 潜在的诱变非同源末端连接途径(NHEJ)通常被认为在体细胞中占优势 细胞。这种偏见的一个原因是，NHEJ途径在整个体细胞周期中都是活跃的，而HDR是 主要限于复制后的S和G2期。因此，实现基于HDR的高效基因编辑 体细胞已被证明具有挑战性，这限制了这项技术在体内用于人类基因治疗。 我的团队致力于开发第一个基于CRISPR的基因驱动(或主动遗传)系统 苍蝇、蚊子、哺乳动物和细菌偏向生殖系遗传编程的遗传元素 在它们的插入部位切割基因组。我们还开创了等位基因驱动系统，旨在促进偏向 在单独的遗传座位上遗传一个受欢迎的等位基因变异。这些生殖系驱动系统还能产生 体细胞表型，通常被归因于由NHEJ途径诱导的突变。 最近，我们开发了被我们称为“拷贝捕捉器”的遗传元素，它可以实现HDR的可视化- 基因盒式磁带的中介复制。这些研究揭示了出乎意料的高频率的体细胞 果蝇的基因转换(SGC)事件(30%-50%)，其中染色体同源基因充当DNA 维修模板。可以通过优化CRISPR组件的交付或通过以下方式进一步提高SGC率 减少涉及DNA修复或染色体配对的各种编码因子的基因表达。 初步实验表明，同源SGC也可以在人类细胞中发生，并指向 使用同源基因的完整序列修复致病突变的未开发策略 染色体。 在这项资助中，我们建议探索由Cas9和Nickase介导的体细胞SGC修复机制 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CRISPR依赖的基因盒或等位基因变异复制的机制 优化他们的活动。接下来，我们将开发和优化果蝇模型，用于基于同源基因的修复 影响有丝分裂活性干细胞或有丝分裂后细胞的Notch基因座的致病突变 使用人源化果蝇CFTR-/-疾病模型的成年肠道上皮细胞。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，以及同源SGC之间是否可以 在囊性纤维化的人类细胞模型中恢复天然基因活性。增强基于同源的修复 哺乳动物细胞可以为下一代基因治疗策略提供变革性的可能性。