Chemical enhancement of CRISPR/Cas9 mediated site-specific genome engineering

CRISPR/Cas9 介导的位点特异性基因组工程的化学增强

基本信息

批准号：
9140562
负责人：
Katherine Pawelczak
金额：
$ 29.3万
依托单位：
NERX BIOSCIENCES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9140562
关键词：
Academia Address Adjuvant Animals Biotechnology Bleomycin CRISPR/Cas technology Cell Culture Techniques Cells Chemicals Clustered Regularly Interspaced Short Palindromic Repeats Cultured Cells DNA DNA Binding DNA Double Strand Break DNA Repair Pathway DNA-Protein Interaction Data Discipline Disease model Double Strand Break Repair Engineering Event Genetic Genetic Materials Genetic Recombination Genome Genome engineering Health Human In Vitro Industry Ku Protein Laboratories Lead Marketing Measures Mediating Methods Modification Nonhomologous DNA End Joining Pathway interactions Process Proteins Research Research Personnel Scientist Sensitivity and Specificity Series Site Specificity Synthetic Genes System Technology Therapeutic abstracting base clinical application commercialization flexibility gene function genome editing in vivo inhibitor/antagonist insertion/deletion mutation interest novel nuclease public health relevance repaired small molecular inhibitor small molecule targeted agent technology development tool transcription activator-like effector nucleases zinc finger nuclease

项目摘要

DESCRIPTION (provided by applicant): Chemical enhancement of CRISPR/Cas9 mediated site-specific genome engineering. Abstract Programmable nucleases, including Zinc Finger Nucleases, TALENs, meganucleases and the CRISPR/Cas9 system allow for site-specific genome engineering. The ability to make targeted genetic modifications has opened up a wide variety of options for scientists in industry and academia and in both therapeutic and biotechnology disciplines. During precision genome engineering, a site specific DSB is generated through nuclease activity. The DSB is repaired by the error prone non-homologous end joining (NHEJ) pathway or by homology directed repair (HDR). Genetic recombination in mammalian systems through the HDR pathway is an extremely inefficient process and cumbersome laboratory methods are required to identify the accurate, desired events. This is further compromised by the activity of the competing DNA repair pathway, NHEJ, which repair the majority of DNA DSBs and can often lead to insertion and deletions resulting in mutagentic events. Recent studies have shown that decreasing NHEJ activity in vivo results in an increase in HDR activity, and this phenomena can be exploited to increase the efficiency of HDR mediated CRISPR/Cas9 precision genome engineering. We have developed a series of small molecule chemical inhibitors that inhibit the DNA binding activity of Ku, a protein necessary for initiation of the NHEJ pathway. Preliminary in vitro and cellular data has shown that Ku DNA binding activity is abolished in the presence of the inhibitors in a potent and specific fashion. I a single aim we will address the ability of these inhibitors to decrease NHEJ and subsequently increase HDR mediated genome engineering using the CRISPR/Cas9 system. Completion of these studies will allow us to move forward with a commercialization plan to market the inhibitors to parties interested in increasing the efficiency and specificity of CRISPR/Cas9 genome engineering.

描述（由申请人提供）：CRISPR/Cas9 介导的位点特异性基因组工程的化学增强摘要可编程核酸酶，包括锌指核酸酶、TALEN、大范围核酸酶和 CRISPR/Cas9 系统，具有进行位点特异性基因组工程的能力。靶向基因修饰为工业界和学术界以及治疗和生物技术学科的科学家提供了多种选择。 DSB 是通过核酸酶活性产生的。 DSB 通过容易出错的非同源末端连接 (NHEJ) 途径或通过 HDR 途径在哺乳动物系统中进行基因重组来修复，这是一个效率极低的过程，而且实验室也很繁琐。需要方法来识别准确的、所需的事件，而竞争性 DNA 修复途径 NHEJ 的活性进一步损害了这一点，NHEJ 修复大多数 DNA DSB，通常会导致插入和缺失。最近的研究表明，体内 NHEJ 活性的降低会导致 HDR 活性的增加，并且可以利用这种现象来提高 HDR 介导的 CRISPR/Cas9 精密基因组工程的效率。抑制 Ku 的 DNA 结合活性的分子化学抑制剂，Ku 是 NHEJ 通路启动所必需的蛋白质。初步的体外和细胞数据表明，在抑制剂存在下，Ku DNA 结合活性会被有效且特异性地消除。我们的一个目标是解决这些抑制剂减少 NHEJ 的能力，并随后使用 CRISPR/Cas9 系统增强 HDR 介导的基因组工程。这些研究的完成将使我们能够推进将抑制剂推向市场的商业化计划。对提高 CRISPR/Cas9 基因组工程的效率和特异性感兴趣的各方。