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: 10548152
• 负责人: Kiana Aran
• 金额: $ 40.92万
• 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/10548152
• 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; Electronics; 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; novel; nuclease; off-target site; 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.

项目摘要/摘要 SCD是一种可遗传的疾病，会影响患者的红细胞（RBC）。这种单基因障碍是 由HBB基因中的单个核苷酸多态性（SNP）引起。尽管治疗进展 SCD关于早期筛查，预防感染和输血，预期寿命 SCD患者仍减少约30年。目前，同种异体造血干细胞 移植（HSCT）是唯一可用的治疗方法。不幸的是，该过程是侵入性的， 与移植物 - 宿主疾病，感染和不育的高风险有关。基于CRISPR的基因编辑 是一种强大的治疗工具，可用于治疗多种疾病。但是，编辑较低 效率可能导致未经编辑的HSPC胜过编辑的效率，导致治疗性降低 影响。当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 序列包含在同源供体DNA中，复杂的光学测定和细胞排序 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 用于监视和优化CRISPR/CAS9的SCD基因疗法的编辑效率的平台 申请。这个体外平台利用多重CRISPR-trySistor来量化 没有与伪影相关的偏差的未放大基因组DNA样品中的特定序列 图书馆准备的类似于其他基于测序的方法。电子平台提供快速读数 较低的样本输入要求。通过结合RNA引导的CRISPR-CAS的可编程性 凭借纳米电子学的可扩展性的技术，该项目提供了灵活而易于 使用前体内监测解决方案进行全面有效的基因治疗质量控制。我们 将在AIM 1中扩展CRISPR-tranistor设计，以产生采用各种GRNA设计的传感器 RNA引导的CAS核酸酶可通过SCD来电子检测和量化单核苷酸变化 作为遗传模型。在AIM 2中，我们将扩展这种技术设计并制造多重GFET 能够同时分析具有多达16种不同RNA的CAS复合物的单个样品 没有放大。在AIM 3中，我们将利用这个多重CRISPR-Tranistor平台快速评估 来自SCD患者的HSPC的Ex-Vivo CRISPR/CAS9 HBB编辑效率。此外，我们将 利用CRISPR-tranistor的灵活性来建立对RNA引导的启动/离目标评估 在存在染色质结构的情况下CAS核酸酶，并与现有技术相比 目标筛选，例如圆形序列和基因组宽。该项目将展示一个巧妙的，一般的 量化编辑效率的平台，有可能缩短处理时间， 降低样品和复杂性，以确保高质量的前体基因治疗。

项目成果

Development of Single Molecule Techniques for Sensing and Manipulation of CRISPR and Polymerase Enzymes.

• DOI: 10.1002/smll.202300328
• 发表时间: 2023-05
• 期刊: Small
• 影响因子: 13.3
• 作者: Jose Chu;Andres Romero;Jeffrey Taulbee;Kiana Aran

CRISPR-Cas-Based Biomonitoring for Marine Environments: Toward CRISPR RNA Design Optimization Via Deep Learning.

• DOI: 10.1089/crispr.2023.0019
• 发表时间: 2023-07
• 期刊: The CRISPR journal
• 作者: B. Durán-Vinet;K. Araya-Castro;A. Zaiko;X. Pochon;S. Wood;Jo‐Ann L. Stanton;Gert‐Jan Jeunen;Michelle Scriver;Anya Kardailsky;Tzu-Chiao Chao;D. K. Ban;M. Moarefian;Kiana Aran;N. Gemmell