CAREER:Defining and improving Class 2 CRISPR-Cas endonuclease sequence specificity

大多数高等生物都有复杂的免疫系统来帮助抵御病毒感染。细菌有更简单的免疫机制来寻找和破坏病毒DNA。它们使用酶和一种特殊的“向导”来帮助识别和破坏感染病毒的DNA。这些系统的简单性使它们能够重新用于一系列生物技术应用。这些特定的“向导”可以识别DNA，即使它只有部分匹配。这对细菌来说是一个有用的特性，但对大多数生物技术应用来说可能是一个很大的问题。该项目的总体目标是了解这种特性如何使细菌免疫受益，并提高基于这种新兴技术的工具的效率。PI将为理科生和非理科生开设有关CRISPR-Cas技术的科学和伦理的新课程。本课程将帮助培养学生的科学传播能力，并与公众进行非正式的科学讨论。本提案的总体目标是定义和提高2类CRISPR-Cas内切酶的序列特异性。具体的科学目标是：1)利用高通量筛选确定gRNA和靶序列对Cas内切酶特异性的影响；2)确定Cas内切酶特异性如何影响CRISPR-Cas对快速进化的病毒入侵者的免疫；3)利用化学修饰的grna，创建一种辅助工具来提高Cas内切酶的特异性。这项工作的成功完成将揭示CRISPR-Cas保真度对内源性免疫和宿主-病毒共同进化的重要性。此外，高通量特异性筛选的发展将能够快速评估各种Cas内切酶，这些酶可用于生物技术应用。这些筛选的结果将有助于改进grna的设计，以降低非特异性基因组编辑的风险。最后，化学修饰grna的研究将对化学修饰对Cas内切酶活性和特异性的影响进行分类，从而确定最佳修饰位点，并在体内基因组编辑研究中实施这些改进的grna。

Most higher organisms have complex immune systems that help fight off viral infections. Bacteria have simpler immune mechanisms that seek and destroy viral DNA. They use enzymes and a specific 'guide' that help to identify and destroy the infecting virus DNA. The simplicity of these systems allow them to be re-purposed for a range of biotechnological applications. These specific 'guides' can recognize DNA even if it has only a partial match. This turns out to be a useful characteristic for bacteria but can be highly problematic for most biotechnological applications. This project has the overall goals of understanding how this feature benefits bacterial immunity and of improving the efficiency of tools based on this emerging technology. The PI will offer a new course for both science and non-science majors about the science and ethics of CRISPR-Cas technology. This course will help train students in science communication and to engage in informal scientific discussions with the public.The overall goal of this proposal is to define and improve the sequence specificity of Class 2 CRISPR-Cas endonucleases. The specific scientific goals are to: 1) define the effects of gRNA and target sequence on the specificity of Cas endonucleases using high-throughput screens; 2) determine how Cas endonuclease specificity affects CRISPR-Cas immunity against rapidly evolving viral invaders; 3) create a complementary tool for improved Cas endonuclease specificity using chemically modified gRNAs. Successful completion of the proposed work will reveal the importance of CRISPR-Cas fidelity for endogenous immunity and host-virus co-evolution. In addition, development of high-throughput specificity screens will enable rapid assessment of a variety of Cas endonucleases that could be harnessed for biotechnological applications. Results from these screens will facilitate improved design of gRNAs to reduce the risk of non-specific genome editing. Finally, studies of chemically modified gRNAs will catalog the effects of chemical modifications on Cas endonuclease activity and specificity, allowing for the identification of optimal modification sites and implementation of these improved gRNAs in in vivo genome editing studies.