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

项目摘要/摘要 SCD是一种遗传性疾病，会影响患者的红细胞(RBC)。这种单基因紊乱是 由HBB基因内的单核苷酸多态(SNP)引起。尽管在治疗方面取得了进展 SCD在早期筛查、预防感染和输血方面的预期寿命 SCD患者的年龄仍然减少了大约30年。目前，异基因造血干细胞 移植(HSCT)是唯一可用的根治方法。不幸的是，这个过程是侵入性的 与移植物抗宿主疾病、感染和不孕不育的高风险相关。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。然而，低编辑量 效率可能导致未经编辑的HSPC胜过已编辑的HSPC，从而降低治疗效果 冲击力。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、复杂的光学分析和细胞分选 建立编辑效率为85%的细胞群。我们建议开发一种多功能的、易于使用的 监控和优化SCD基因治疗CRISPR/Cas9编辑效率的平台 申请。这个体外平台利用多路CRISPR晶体管来量化 未扩增的基因组DNA样本中的特定序列，没有与人工产物相关的偏见 文库制备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 该技术具有纳米电子的可扩展性，所提出的项目提供了一种灵活、简单的 利用体外监测溶液对基因治疗进行全面有效的质量控制。我们 将扩展目标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核酸酶使用SCD电子检测和量化单核苷酸变化 作为一种遗传模型。在目标2中，我们将放大这项技术设计并制造多路GFET 能够同时分析单个样品和多达16个不同的RNA引导的CaS络合物 不需要放大。在目标3中，我们将利用这个多路CRISPR晶体管平台来快速评估 SCD患者HSPC体外CRISPR/Cas9 HBB编辑效率的研究此外，我们还将 利用CRISPR晶体管的灵活性来建立RNA引导的开/关目标评估 存在染色质结构的CAS核酸酶，并与现有技术进行了比较。 靶向筛选，如循环序列和全基因组。这个项目将展示一种简单、通用的 用于量化可能缩短处理时间的编辑效率的平台， 减少样本和复杂性，以确保高质量的体外基因治疗。

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.