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

• 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
• 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

 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(申请人提供)：CRISPR/Cas9介导的定点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、TALENS、巨核酸酶和CRISPR/CAS9系统，使定点基因组工程成为可能。有针对性地进行基因修改的能力为工业界和学术界以及治疗和生物技术学科的科学家开辟了广泛的选择。在精确基因组工程过程中，通过核酸酶活性产生一种位点特异性的DSB。DSB通过容易出错的非同源末端连接(NHEJ)途径或同源定向修复(HDR)修复。在哺乳动物系统中，通过HDR途径进行基因重组是一个极其低效的过程，需要繁琐的实验室方法来识别准确的、所需的事件。竞争DNA修复途径NHEJ的活性进一步损害了这一点，NHEJ修复了大多数DNA DSB，并经常导致插入和缺失，从而导致突变事件。最近的研究表明，体内NHEJ活性的降低会导致HDR活性的增加，这一现象可以被用来提高HDR介导的CRISPR/Cas9精密基因组工程的效率。我们已经开发了一系列小分子化学抑制剂来抑制Ku的DNA结合活性，Ku是启动NHEJ途径所必需的蛋白质。初步的体外和细胞数据表明，在抑制剂的存在下，Ku DNA结合活性以一种有效和特定的方式被取消。我们将利用CRISPR/CAS9系统解决这些抑制剂降低NHEJ并随后增加HDR介导的基因组工程的能力。这些研究的完成将使我们能够推进商业化计划，将抑制剂推向对提高CRISPR/Cas9基因组工程的效率和特异性感兴趣的各方。