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

项目总结/摘要 SCD是一种遗传性疾病，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。尽管治疗取得了进展 SCD的早期筛查，预防感染和输血， SCD患者仍减少约30年。目前，同种异体造血干细胞 造血干细胞移植（HSCT）是唯一可用的治愈性治疗。不幸的是，这个过程是侵入性的， 与移植物抗宿主病、感染和不育的高风险相关。CRISPR基因编辑 是一种强大的治疗工具，可以潜在地治愈各种疾病。低编辑 效率可能导致未编辑的HSPC胜过编辑的HSPC，导致治疗性降低。 冲击目前用于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， 建立具有>85%编辑效率的细胞群体。我们建议开发一种通用且易于使用的 监测和优化SCD基因治疗CRISPR/Cas9编辑效率的平台 应用.该体外平台利用多路CRISPR晶体管来定量CRISPR的量。 未扩增的基因组DNA样品中的特定序列，而没有与伪影相关的偏差 与其他基于测序的方法一样，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， 技术与纳米电子学的可扩展性，拟议的项目提供了一个灵活，简单， 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，以产生采用各种gRNA设计的传感器 和RNA引导的Cas核酸酶，以使用SCD电子检测和定量单核苷酸变化 作为一个基因模型。在目标2中，我们将扩大这种技术设计并制造多路gFET 能够同时分析具有多达16种不同RNA引导的Cas复合物的单个样品 没有放大。在目标3中，我们将利用这种多路CRISPR晶体管平台快速评估 来自SCD患者的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引导的靶向/脱靶评估， Cas核酸酶在染色质结构的存在下，并与现有技术进行比较， 靶向筛选，如CIRCLE-seq和全基因组。这个项目将展示一个简单的，一般的 量化编辑效率的平台，有可能缩短处理时间， 减少了确保高质量的离体基因治疗所必需的样品和复杂性。

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.