Discovery and applications of CRISPR-Cas inhibitor proteins

CRISPR-Cas抑制剂蛋白的发现及应用

• 批准号: 10006922
• 负责人: David Rabuka
• 金额: $ 25.21万
• 依托单位: ACRIGEN BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-02 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10006922
• 关键词: Address; Affinity; BCAR1 gene; Bacteria; Bacteriophages; Binding; Biochemical; Bioinformatics; Biological Assay; Biological Models; Biological Sciences; Blood Cells; Cells; Cleaved cell; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; Complex; DNA; DNA Binding; Dangerousness; Dependovirus; Development; Disease; Disease model; Elements; Engineering; Ensure; Evaluation; Event; Future; Gene Delivery; Genes; Genetic; Genetic Medicine; Genetic Variation; Goals; Government; Human; Human Cell Line; Human Genetics; Human Genome; In Vitro; Kinetics; Lead; Malignant Neoplasms; Measures; Medical; Mendelian disorder; Methods; Mission; Mutation; Oncogenic; Orthologous Gene; Outcome; Peptides; Persons; Phase; Process; Proteins; Proxy; Research; Risk; Safety; Staphylococcus aureus; System; Techniques; Technology; Testing; Text; Time; Toxic effect; Translating; Variant; Viral Vector; Western Blotting; Work; base; design; experimental study; genome editing; human disease; human genome sequencing; human model; in vivo; inhibitor/antagonist; innovation; new technology; novel; nuclease; precision genetics; preservation; prevent; screening; side effect; small molecule; small molecule inhibitor; tool

Abstract In the nearly 20 years since the groundbreaking sequencing of the human genome, the potential for precision genetic medicine has still not been realized. More recently, novel technologies such as CRISPR-Cas gene editing, which relies on DNA-cleaving nucleases discovered in bacteria, enable precise enzymatic editing of the human genome. Although this technique opens up opportunities to prevent or cure many diseases with unmet medical need, before this powerful technology can be translated into medical therapies it must be shown to be not only effective but safe. Off-target editing events with CRISPR-Cas systems occur due to prolonged nuclease activity resulting in unwanted side effects of the gene editing process, including the potential for oncogenic mutations or cellular toxicity. These safety issues can be addressed by designing and implementing more effective ways to control the gene editing process. Acrigen Biosciences is developing a solution to bring safe in vivo CRISPR-Cas gene editing to the market for the first time. This innovative approach encompasses robust anti-CRISPR small-protein off-switches for DNA-cleaving nucleases. This robust and universal approach to temporal control of the gene editing process will be key in translating the potential of CRISPR-Cas to the clinic, and aligns well with NCATS’ mission. The ultimate goal of this work is to identify proteins that are efficacious inhibitors of a Cas9 ortholog in human cells. The market for safe gene editing approaches will be very large: the technology can be applied to various monogenic diseases, cancers, and infectious diseases. The Phase I project has 3 specific aims. (1) Establish cell-based and in vitro quantitative experiments to assess inhibition in the function and potency of a Cas9 ortholog by (a) developing a candidate inhibitor screening platform and (b) using biochemical assays to characterize the mechanisms of anti-CRISPR proteins. Milestone: Titratable nuclease expression, as measured by Western blot and bacteriophage targeting in bacteria, looking at phage replication titers as a proxy for CRISPR activity. We will quantify the activity of candidate inhibitors with in vitro DNA-binding and DNA-cleavage experiments. (2) Identify candidate anti-CRISPRs by (a) detecting self-targeting in bacteria encoding homologs of a Cas9 ortholog and (b) identifying bacteriophages that are recalcitrant to the action of a Cas9 ortholog. Milestone: Select ~100 candidate proteins which are small in size, encoded by bacterial mobile elements, and associated with known anti-CRISPR marker genes. The discovery will be based on bioinformatics and phage-based approaches, such as phage replication quantification when targeted by CRISPR. Candidates to be tested in quantitative experimental system established in Aim 1. (3) Assess anti-CRISPR efficacy in two human cell lines (HEK293T and U2-OS-EGFP). Milestone: Identify 5 non-homologous proteins that can inhibit 90% of gene editing in model human cells, using a Cas9 ortholog to edit genes at three different loci.

摘要 在人类基因组突破性测序的近20年里， 基因医学尚未实现。最近，CRISPR-Cas基因等新技术 编辑依赖于在细菌中发现的DNA切割核酸酶，能够精确地酶促编辑 人类基因组尽管这项技术为预防或治愈许多疾病提供了机会， 未满足的医疗需求，在这种强大的技术可以转化为医疗疗法之前， 不仅有效而且安全。CRISPR-Cas系统的脱靶编辑事件发生的原因是 延长的核酸酶活性导致基因编辑过程的不希望的副作用，包括 致癌突变或细胞毒性的可能性。这些安全问题可以通过设计和 实施更有效的方法来控制基因编辑过程。Acrigen Biosciences正在开发一种 该解决方案首次将安全的体内CRISPR-Cas基因编辑推向市场。这一创新 该方法包括用于DNA切割核酸酶的稳健的抗CRISPR小蛋白关闭开关。这 对基因编辑过程进行时间控制的鲁棒和通用方法将是翻译 CRISPR-Cas在临床上的潜力，并与NCATS的使命保持良好的一致。这项工作的最终目标是 鉴定作为人细胞中Cas9直系同源物的有效抑制剂的蛋白质。安全基因市场 编辑方法将是非常大的：该技术可以应用于各种单基因疾病，癌症， 和传染病。第一阶段项目有三个具体目标。(1)建立基于细胞和体外 定量实验以评估Cas9直向同源物的功能和效力的抑制，通过（a）开发用于评估Cas9直向同源物的功能和效力的定量实验， 候选抑制剂筛选平台和（B）使用生物化学测定来表征 抗CRISPR蛋白。里程碑：可滴定的核酸酶表达，如通过蛋白质印迹和免疫印迹所测量的。 细菌中的噬菌体靶向，将噬菌体复制滴度作为CRISPR活性的代表。我们将 用体外DNA结合和DNA切割实验定量候选抑制剂的活性。（二） 通过（a）检测编码Cas9同源物的细菌中的自靶向， 直向同源物，和（B）鉴定对Cas9直向同源物的作用有抑制作用的噬菌体。里程碑： 选择约100个候选蛋白质，这些蛋白质尺寸小，由细菌移动的元件编码，并且相关 已知的抗CRISPR标记基因。这一发现将基于生物信息学和基于噬菌体的 方法，如通过CRISPR靶向时的噬菌体复制定量。候选人须在 目标1建立了定量实验体系。(3)在两种人类细胞系中评估抗CRISPR功效 （HEK 293 T和U2-OS-EGFP）。里程碑：确定5种可抑制90%基因的非同源蛋白质 在模型人类细胞中进行编辑，使用Cas9直系同源物在三个不同的基因座编辑基因。