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的系统允许通过诱导靶向DNA进行精确的基因组编辑 在基因组中的特定位点断裂。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）从切割位点处的完整同源染色体获得信息。 虽然精确的HDR介导的DNA修复是减数分裂过程中活跃的主要途径，但竞争性的和非竞争性的DNA修复是主要途径。 潜在的致突变性非同源末端连接途径（NHEJ）通常被认为在体细胞中占优势， 细胞这种偏差的一个原因是NHEJ途径在整个体细胞周期中是活跃的，而HDR是活跃的。 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， 体细胞已被证明是具有挑战性的，这限制了该技术在人体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（或主动遗传）系统做出了贡献。 苍蝇、蚊子、哺乳动物和细菌，它们偏向于遗传元件的种系遗传， 在插入位点切断基因组。我们还开创了等位基因驱动系统， 在一个单独的基因座上遗传一个有利的等位基因变体。这些生殖系驱动系统还产生 体细胞表型，其通常归因于由NHEJ途径诱导的突变。 最近，我们开发了我们称为“CopyCatchers”的遗传元件，它允许HDR的可视化- 介导的基因盒复制。这些研究揭示了一个意想不到的高频率的体细胞 果蝇中的基因转换（SGC）事件（30-50%），其中染色体同源物充当DNA 修复模板通过优化CRISPR组分的递送，或 降低编码参与DNA修复或染色体配对的因子的各种基因的表达。 初步实验表明，同源物间的SGC也可以发生在人类细胞中，并指出 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，我们计划在体细胞中探索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。一是 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。下一步，我们将开发和优化果蝇模型，用于同源修复， 在Notch基因座中的致病突变影响有丝分裂活性干细胞或在有丝分裂后的细胞中， 使用人源化果蝇CFTR-/-疾病模型的成体肠上皮。最后，我们将评估 在果蝇中获得的见解可移植到人类体细胞系中，以及同源物SGC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。