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：
10864412
负责人：
Keith Thomas Gagnon
金额：
$ 24.19万
依托单位：
WAKE FOREST UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-05 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10864412
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 Structure Substrate Specificity System Technology Testing Therapeutic Therapeutic Research Time Translating chemical conjugate chemical synthesis design enzyme activity improved inhibitor microbiota model building model organism mutant new technology novel novel strategies novel therapeutics nuclease rational design 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.

项目摘要易于使用的CAS酶的精致，便捷且潜在的抑制剂仍然是主要的缺少基于CRISPR的生物医学和治疗研究的工具。 CRISPR-CAS的理由抑制剂包括需要更有效地生成模型细胞和生物，威胁基于CRISPR的疗法的危险脱离目标编辑，以及对更精确控制的愿望合成生物学和诊断应用中的CAS酶活性。已经开发了方法和改良的CAS酶来帮助解决这些问题和其他 CRISPR-CAS系统的缺点，尤其是化脓性链球菌（SPCAS9）的CAS9。这些包括具有较低靶向活性的CAS9突变体以及CAS9突变体和蛋白融合物，允许较小 Cas9的分子或基于光的激活。但是，这些修改通常具有伴随减少靶标活动，并不满足CAS9快速和特定关闭的需求。尽管改善了CAS酶，但需要特定，广泛适用且易于使用的需求抑制剂将仍然是不可用的必要工具。可以抑制cas9的小分子可能需要巨大的努力来发展和可能带来弊端，包括自己的脱靶效应和对生物体的微生物群的无意影响可能类似于抗生素副作用。最近已经发现了天然抗Crispr蛋白结合Cas9和其他CAS蛋白具有高亲和力。尽管有望作为抑制剂，但它们太大而无法具有类似毒品的特性及其最小化或优化不是一个明显的练习。它们必须通常编码用于单元格或组织。但是，它们为设计新的CRISPR-CAS抑制剂的设计提供了灵感。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，但可以与类似的亲和力结合，是化学合成，并很容易用常见方法引入细胞。在这个项目中，我们将通过优化其大小，化学，结合亲和力，抑制活性，开发NAB抑制剂技术，和细胞稳定性。我们将进一步开发有助于直接，无载体的方法和分子 NAB抑制剂的递送和定时释放。预计所得的NAB抑制剂将广泛适用而直接用于潜水生物医学研究。我们希望NAB抑制剂成为有价值的故障安全抑制剂，以克服基于CRISPR的治疗的关键安全障碍。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly.

DOI：
10.1016/j.jbc.2023.104700
发表时间：
2023-05
期刊：
JOURNAL OF BIOLOGICAL CHEMISTRY
影响因子：
4.8
作者：
Sudhakar, Sruthi;Barkau, Christopher L.;Chilamkurthy, Ramadevi;Barber, Halle M.;Pater, Adrian A.;Moran, Sean D.;Damha, Masad J.;Pradeepkumar, P. I.;Gagnon, Keith T.
通讯作者：
Gagnon, Keith T.

Small nucleic acids and the path to the clinic for anti-CRISPR.

DOI：
10.1016/j.bcp.2021.114492
发表时间：
2021-07
期刊：
Biochemical pharmacology
影响因子：
5.8
作者：
Barkau CL;O'Reilly D;Eddington SB;Damha MJ;Gagnon KT
通讯作者：
Gagnon KT

Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles.

DOI：
10.1038/s41467-021-26989-z
发表时间：
2021-11-15
期刊：
Nature communications
影响因子：
16.6
作者：
Ageely EA;Chilamkurthy R;Jana S;Abdullahu L;O'Reilly D;Jensik PJ;Damha MJ;Gagnon KT
通讯作者：
Gagnon KT