CRISPR-based transistors for high throughput multiplexed monitoring of CRISPR-based editing efficiency for Sickle cells disease

基于 CRISPR 的晶体管，用于高通量多重监测镰状细胞病的基于 CRISPR 的编辑效率

• 批准号: 10346886
• 负责人: Kiana Aran
• 金额: $ 40.54万
• 依托单位: KECK GRADUATE INST OF APPLIED LIFE SCIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-07 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10346886
• 关键词: Affect; Alleles; Allogenic; Amino Acid Sequence; Binding; Biological Assay; Blood Transfusion; CRISPR/Cas technology; Cell Separation; Cells; Chromatin; Chromatin Structure; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Codon Nucleotides; Collaborations; Complex; Consumption; DNA; DNA analysis; Development; Disease; Employment; Engraftment; Ensure; Enzymes; Erythrocytes; Evaluation; Fetal Hemoglobin; Flow Cytometry; Gases; Genes; Genetic Models; Genomic DNA; Genomics; Glutamates; Goals; Guide RNA; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hemoglobinopathies; Heritability; Human; Human Genome; Immunologics; In Vitro; Infection; Infection prevention; Infertility; Journals; Libraries; Life Expectancy; Membrane Proteins; Mendelian disorder; Methods; Missense Mutation; Monitor; Morphologic artifacts; Mutation; Nature; Nucleotides; Optics; Organ; Orthologous Gene; Pathogenicity; Patients; Physiological; Point Mutation; Polymorphism Analysis; Population; Preparation; Prevalence; Process; Production; Publishing; Quality Control; Quality of life; RNA library; Resources; Safety; Sampling; Sickle Cell Anemia; Single Nucleotide Polymorphism; Source; System; Technology; Testing; Therapeutic; Time; Tissues; Transistors; Transplantation; Treatment Efficacy; Valine; Vendor; base; base editing; cost; cost effective; curative treatments; design; detection limit; early screening; flexibility; gene therapy; genome-wide; graft vs host disease; graphene; high risk; improved; in vivo; nanoelectronics; nanoscale; novel; nuclease; off-target site; programs; scale up; screening; sensor; success; tool; transcription factor

PROJECT SUMMARY/ABSTRACT SCD is a heritable disease, which affects a patient's red blood cells (RBCs). This monogenic disorder is caused by a single nucleotide polymorphism (SNP) within the HBB gene. Despite progress in the treatment of SCD regarding early screenings, prevention of infections, and blood transfusions, the life expectancy for SCD patients is still reduced by about 30 years. Currently, allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment available. Unfortunately, the process is invasive and associated with high risk of graft-versus-host-disease, infection, and infertility. CRISPR-based gene editing is a powerful therapeutic tool for potentially curing a wide variety of diseases. However, low editing efficiency can result in unedited HSPCs outcompeting edited ones, resulting in diminished therapeutic impact. Current methods for maximizing the percentage of edited cells rely on GFP or surface protein sequences to be contained within the homologous donor DNA, complex optical assays and cell sorting to establish cell populations with >85% editing efficiency. We propose to develop a versatile and easy-to-use platform to monitor and optimize the editing efficiency of CRISPR/Cas9 for SCD gene therapy applications. This in vitro platform utilizes multiplex CRISPR-transistors to quantify the amount of a specific sequence within an unamplified genomic DNA sample without the bias associated with the artifacts of library preparation like other sequencing-based methods. The electronic platform provides rapid readout with low sample input requirement. By combining the programmability of RNA-guided CRISPR-Cas technology with the scalability of nano-electronics, the proposed project provides a flexible, and simple to use ex-vivo monitoring solution for a comprehensive and effective gene therapy quality control. We will expand CRISPR-transistor design in Aim 1 to yield a sensor which employs a variety of gRNA designs and RNA-guided Cas nucleases to electronically detect and quantify single nucleotide changes using SCD as a genetic model. In Aim 2, we will scale up this technology design and fabricate a multiplex gFET capable of analyzing a single sample with up to 16 different RNA-guided Cas complexes simultaneously without amplification. In Aim 3, we will utilize this multi-plex CRISPR-transistor platform to rapidly assess the ex-vivo CRISPR/Cas9 HBB editing efficiency of HSPCs from patients with SCD. In addition, we will leverage the flexibility of CRISPR-transistor to establish an ON/OFF-target evaluation of the RNA-guided Cas nuclease in the presence of chromatin structures and compare against existing technologies for off- target screening, like CIRCLE-seq and genome wide. This project will demonstrate a facile, general platform for quantification of editing efficiency that has the potential to shorten the processing time, reducing sample and complexity necessary to ensure high quality of ex-vivo gene therapy.

项目概要/摘要 SCD 是一种遗传性疾病，会影响患者的红细胞 (RBC)。这种单基因疾病是 由 HBB 基因内的单核苷酸多态性 (SNP) 引起。尽管治疗取得进展 SCD 的早期筛查、感染预防和输血、预期寿命 SCD患者仍减少约30年。目前，异体造血干细胞 移植（HSCT）是唯一可用的治疗方法。不幸的是，这个过程是侵入性的，并且 与移植物抗宿主病、感染和不孕症的高风险相关。基于 CRISPR 的基因编辑 是一种强大的治疗工具，有可能治愈多种疾病。但编辑能力低下 效率可能导致未编辑的 HSPC 胜过编辑的 HSPC，从而导致治疗效果下降 影响。目前最大化编辑细胞百分比的方法依赖于 GFP 或表面蛋白 同源供体 DNA 中包含的序列、复杂的光学测定和细胞分选 建立编辑效率 >85% 的细胞群。我们建议开发一种多功能且易于使用的 用于监测和优化用于 SCD 基因治疗的 CRISPR/Cas9 编辑效率的平台 应用程序。该体外平台利用多重 CRISPR 晶体管来量化 未扩增的基因组 DNA 样本中的特定序列，没有与伪影相关的偏差 像其他基于测序的方法一样进行文库制备。电子平台提供快速读数 样品输入要求低。通过结合 RNA 引导的 CRISPR-Cas 的可编程性 技术具有纳米电子学的可扩展性，所提出的项目提供了一种灵活且简单的方法 使用离体监测解决方案进行全面有效的基因治疗质量控制。我们 将扩展 Aim 1 中的 CRISPR 晶体管设计，以产生采用多种 gRNA 设计的传感器 和 RNA 引导的 Cas 核酸酶，使用 SCD 以电子方式检测和量化单核苷酸变化 作为遗传模型。在目标 2 中，我们将扩大该技术设计并制造多路 gFET 能够同时分析具有多达 16 个不同 RNA 引导的 Cas 复合物的单个样品 无需放大。在目标 3 中，我们将利用这种多重 CRISPR 晶体管平台来快速评估 SCD 患者 HSPC 的离体 CRISPR/Cas9 HBB 编辑效率。此外，我们将 利用 CRISPR 晶体管的灵活性来建立 RNA 引导的 ON/OFF 目标评估 Cas 核酸酶在染色质结构存在下的情况，并与现有技术进行比较 目标筛选，如 CIRCLE-seq 和全基因组筛选。该项目将展示一个简单、通用的 量化编辑效率的平台，有可能缩短处理时间， 减少确保高质量离体基因治疗所需的样本和复杂性。