CAREER: Functional and evolutionary analysis of transposon-encoded, RNA-guided nucleases across the three domains

职业：跨三个域的转座子编码、RNA 引导的核酸酶的功能和进化分析

• 批准号: 2239685
• 负责人: Samuel Sternberg
• 金额: $ 96.87万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2239685&HistoricalAwards=false
• 关键词: CAREER; Functional; evolutionary; analysis; transposon

This project will investigate abundant genetic elements known as transposons, or ‘jumping genes,’ and will specifically examine a primordial family of transposon-encoded enzymes that use guide RNAs to target and cut matching DNA sequences. These enzymes are directly related to CRISPR-Cas9 and CRISPR-Cas12, which similarly rely on guide RNAs for targeted DNA cleavage, and which have revolutionized scientists’ ability to perform precise genetic modifications in plants, animals, and humans. By exploring what may be the evolutionary ancestors of these enzymes, this project has the potential to uncover new tools for genome engineering and new knowledge about diverse biological pathways where RNA-guided DNA targeting offers an advantage to host cells and genetic parasites. CRISPR-based tools continue to disrupt biotechnology sectors, posing urgent new questions about how to best harness these developments for societal benefit. Promoting broader public knowledge about CRISPR, genetics, and precision medicine will be of central importance in making informed decisions about how we shepherd CRISPR technology in the future. The broader impacts of this project include a commitment to educating middle- and high-school students from traditionally underrepresented groups in STEM on these topics, thereby inspiring the next generation of young thinkers to pursue careers in science and research.TnpB-family enzymes were recently shown to use transposon-encoded guide RNAs to direct cleavage of complementary DNA target sequences, but the biological and functional importance of this biochemical activity remains unclear. Even more mysteriously, TnpB-family genes are present across all three domains of life in diverse genetic contexts, and in association with distinct families of transposases. This project will investigate the hypothesis that that RNA-guided nucleases play a fundamental role in promoting transposon survival and proliferation, explaining their widespread conservation in bacteria, archaea, and eukarya. This problem will be studied across three major experimental aims, including the determination of how transposases and nucleases coordinate their enzymatic activities; the exploration of RNA-guided nucleases encoded by diverse bacterial transposons; and the discovery of novel nucleases encoded within archaeal and eukaryotic transposable elements. These aims will employ a multidisciplinary approach blending phylogenetics, biochemistry, genetics, and high-throughput sequencing. By elucidating the molecular origins of CRISPR RNA-guided nucleases, this project will fundamentally transform our knowledge and appreciation of transposon domestication during the evolution of host immune systems, and will offer new opportunities to develop next-generation genome editing tools.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

该项目将研究丰富的遗传元件，称为转座子，或“跳跃基因”，并将专门研究转座子编码的酶的原始家族，这些酶使用向导RNA来靶向和切割匹配的DNA序列。这些酶与CRISPR-Cas9和CRISPR-Cas 12直接相关，CRISPR-Cas9和CRISPR-Cas 12同样依赖于引导RNA进行靶向DNA切割，并且彻底改变了科学家在植物，动物和人类中进行精确遗传修饰的能力。通过探索这些酶的进化祖先，该项目有可能发现基因组工程的新工具和关于不同生物途径的新知识，其中RNA引导的DNA靶向为宿主细胞和遗传寄生虫提供了优势。基于CRISPR的工具继续扰乱生物技术领域，提出了关于如何最好地利用这些发展以造福社会的紧迫新问题。促进更广泛的公众对CRISPR、遗传学和精准医学的了解，对于我们未来如何引导CRISPR技术做出明智的决定至关重要。该项目的更广泛影响包括致力于教育传统上在STEM领域代表性不足的初中和高中学生，从而激励下一代年轻思想家追求科学和研究事业。TnpB家族酶最近被证明使用转座子编码的指导RNA来指导互补DNA靶序列的切割，但这种生物化学活性的生物学和功能重要性仍不清楚。更神秘的是，TnpB家族基因在不同的遗传背景下存在于生命的所有三个领域，并与不同的转座酶家族相关。该项目将研究RNA引导的核酸酶在促进转座子存活和增殖方面发挥重要作用的假设，解释它们在细菌，古细菌和真核生物中的广泛保护。这个问题将在三个主要的实验目标进行研究，包括转座酶和核酸酶如何协调其酶活性的测定;探索由不同的细菌转座子编码的RNA引导的核酸酶;以及发现古细菌和真核生物转座因子内编码的新型核酸酶。这些目标将采用多学科的方法，融合生物遗传学，生物化学，遗传学和高通量测序。通过阐明CRISPR RNA引导核酸酶的分子起源，该项目将从根本上改变我们对宿主免疫系统进化过程中转座子驯化的认识和理解，并将为开发下一代基因组编辑工具提供新的机会。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响评审标准进行评估，被认为值得支持。