Nucleic Acid-Based Anti-CRISPR Inhibitors of Cas9

基于核酸的 Cas9 抗 CRISPR 抑制剂

基本信息

批准号：
10079496
负责人：
Keith Thomas Gagnon
金额：
$ 26.97万
依托单位：
SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-03 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10079496
关键词：
Address Affinity Animal Model Animals Antibiotics BCAR1 gene Binding Biochemical Biological Models Biomedical Research Breathing CRISPR therapeutics CRISPR/Cas technology Campylobacter jejuni Cell model Cells Chemicals Chemistry Chimeric Proteins Clustered Regularly Interspaced Short Palindromic Repeats DNA Dangerousness Development Diagnostic Engineering Enzymes Exercise Gene Expression Regulation Generations Genes Genetic Genome Genome engineering Goals Guide RNA Health Human In Vitro Length Light Measures Mediating Methods Modeling Modification Nucleic Acids Nucleotides Organism Pharmaceutical Preparations Property Proteins Safety Staphylococcus aureus Streptococcus pyogenes Structural Chemistry Substrate Specificity System Technology Testing Therapeutic Therapeutic Human Experimentation Time Translating base design enzyme activity improved inhibitor/antagonist microbiota mutant new technology novel novel strategies novel therapeutics nuclease side effect small molecule success synthetic biology tool

项目摘要

PROJECT SUMMARY Sophisticated, facile, and potent inhibitors of Cas enzymes that are simple to use are still a major missing tool for CRISPR-based biomedical and therapeutic research. The rationale for CRISPR-Cas inhibitors includes a need for more efficient generation of model cells and organisms, the threat of dangerous off-target editing in CRISPR-based therapeutics, and the desire for more precise control over Cas enzyme activity in synthetic biology and diagnostics applications. Methods and modified Cas enzymes have been developed to help address these issues and other shortcomings of CRISPR-Cas systems, particularly Cas9 from Streptococcus pyogenes (SpCas9). These include Cas9 mutants with lower off-target activity and Cas9 mutants and protein fusions that allow small molecule or light-based activation of Cas9. However, these modifications typically have a concomitant decrease in on-target activity and do not address the need for rapid and specific shut-down of Cas9. Despite improvements to Cas enzymes, the need for specific, broadly applicable, and easy-to-use inhibitors will remain a necessary tool that is not available. Small molecules that can inhibit Cas9 may take significant effort to develop and potentially come with downsides, including their own off-target effects and inadvertent impacts on the organism’s microbiota, possibly similar to antibiotic side-effects. Natural anti-CRISPR proteins have recently been discovered that bind Cas9 and other Cas proteins with high affinity. While promising as inhibitors, they are too large to possess drug-like properties and their minimization or optimization is not an obvious exercise. They must be genetically encoded for use in cells or organisms. However, they provide inspiration for the design of a new class of CRISPR-Cas inhibitors. Here we propose to develop a new technology, nucleic acid-based (NAB) inhibitors of Cas9 enzymes. These molecules are smaller than natural anti-CRISPR proteins but can bind with similar affinity, be chemically synthesized, and be readily introduced into cells with common methods. In this project, we will develop NAB inhibitor technology by optimizing their size, chemistry, binding affinity, inhibitory activity, and cellular stability. We will further develop methods and molecules that facilitate direct, carrier-free delivery and timed-release of NAB inhibitors. The resulting NAB inhibitors are expected to be broadly applicable and straightforward to use for diverse biomedical research. We expect NAB inhibitors to become valuable fail-safe inhibitors to overcome the critical safety hurdles in CRISPR-based therapeutics.

项目概要复杂、简便、有效且易于使用的 Cas 酶抑制剂仍然是主要的抑制剂基于 CRISPR 的生物医学和治疗研究缺少工具 CRISPR-Cas 的基本原理。抑制剂包括需要更有效地生成模型细胞和生物体，基于 CRISPR 的疗法中危险的脱靶编辑，以及对更精确控制的渴望 Cas 酶活性在合成生物学和诊断应用中的应用。已经开发出方法和修饰的 Cas 酶来帮助解决这些问题和其他问题 CRISPR-Cas 系统的缺点，特别是来自化脓性链球菌的 Cas9 (SpCas9)。包括具有较低脱靶活性的 Cas9 突变体以及允许小的 Cas9 突变体和蛋白融合然而，这些修饰通常伴随着 Cas9 的分子或光激活。靶向活性降低，并且不能解决 Cas9 快速和特定关闭的需要。尽管 Cas 酶有所改进，但仍需要特定的、广泛适用且易于使用的酶抑制剂仍将是一种无法获得的、可以抑制Cas9的小分子。付出巨大的努力来开发并可能带来负面影响，包括其自身的脱靶效应和对生物体微生物群的无意影响，可能类似于抗生素的副作用。最近发现天然抗 CRISPR 蛋白可以结合 Cas9 和其他 Cas 蛋白虽然具有高亲和力，但它们太大，不具备类似药物的特性及其作用。最小化或优化不是一个显而易见的练习，它们必须经过基因编码才能在细胞中使用。然而，它们为设计一类新型 CRISPR-Cas 抑制剂提供了灵感。在这里，我们建议开发一种新技术，基于核酸（NAB）的Cas9酶抑制剂。这些分子比天然抗 CRISPR 蛋白小，但可以以相似的亲和力结合，例如化学合成，并且可以通过常见方法轻松引入细胞中。通过优化 NAB 抑制剂的尺寸、化学性质、结合亲和力、抑制活性来开发 NAB 抑制剂技术，我们将开发进一步的方法和分子，以促进直接的、无载体的。 NAB 抑制剂的递送和定时释放预计将得到广泛应用。我们期望 NAB 抑制剂能够适用且直接用于各种生物医学研究。成为有价值的自动防故障抑制剂，以克服基于 CRISPR 的疗法中的关键安全障碍。