Analysis of homolog-based CRISPR editing in somatic cells

体细胞中基于同源物的 CRISPR 编辑分析

基本信息

批准号：
10676726
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
项目类别：
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/10676726
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 Development 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 S phase Sequence Homologs Site Somatic Cell System Technology Testing Visualization Yeasts base editing design disease-causing mutation expectation experimental study fly gastrointestinal epithelium gene correction 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 postmitotic precise genome editing precursor cell repair model repair strategy repaired resistance allele stem cells tool transgene expression transmission process

项目摘要

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的靶向DNA进行精确的基因组编辑在基因组中的特定部位中断。细胞DNA修复机械可以通过复制来恢复基因组完整性通过同源性修复（HDR），来自切割部位的完整同源染色体的信息。虽然精确的HDR介导的DNA修复是减数分裂，竞争和通常认为潜在的诱变非理论最终连接途径（NHEJ）在体细胞中占上风细胞。这种偏见的原因之一是NHEJ途径在整个体细胞周期中都活跃，而HDR为首先仅限于复制后的S和G2阶段。在此实现有效的基于HDR的基因编辑体细胞已被证明是挑战，这限制了该技术在人类基因治疗中的体内使用。我的小组为开发第一个基于CRISPR的基因驱动（或主动遗传）系统做出了贡献苍蝇，蚊子，哺乳动物和细菌偏向编程的遗传元素的种系遗传在其插入部位切割基因组。我们还开创了旨在促进有偏见的Allic-Drive系统在一个单独的遗传基因座上的偏爱等位基因变体的继承。这些种系驱动系统也会产生体型表型通常归因于NHEJ途径引起的突变。最近，我们开发了我们称为“模仿者”的遗传因素，可以可视化HDR- 基因盒的介导复制。这些研究表明，躯体的高频出乎意料果蝇（30-50％）中的基因转化率（SGC）事件，其中染色体同源物用作DNA 维修模板。可以通过优化CRISPR组件的交付或通过减少与DNA修复或染色体配对有关的各种基因的表达。初步实验表明，人体间SGC也可以在人类细胞中进行，并指向使用同源的完整序列修复致病突变的尚未开发的策略染色体。在这笔赠款中，我们建议探索由Cas9和Nickase介导的SGC修复机制果蝇的细胞，然后将这种本体间修复过程的分析扩展到人类细胞。首先，我们会的分析基因录音带或种族变体的CRISPR依赖性复制的基础机制优化他们的活动。接下来，我们将开发和优化果蝇模型，以基于同源性修复在影响有丝分裂活性干细胞或丝质后细胞的缺口基因座中引起疾病的突变使用人源化果蝇CFTR - / - 疾病模型的成年肠道上皮。最后，我们将评估是否在果蝇中获得的见解是人类体细胞系的便携恢复基于人类细胞的囊性纤维化模型中的天然基因活性。增强基于同源的维修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。