SBIR Phase I: Directed evolution of site-specific bacterial transposase genes to alter specificity and efficiency of insertion of large DNA segments into restorable gene fusions

SBIR 第一阶段：位点特异性细菌转座酶基因的定向进化，以改变大 DNA 片段插入可恢复基因融合的特异性和效率

• 批准号: 2234291
• 负责人: Verne Luckow
• 金额: $ 27.5万
• 依托单位: SYNTHETIC VECTOR DESIGNS, LLC
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2234291&HistoricalAwards=false
• 关键词: SBIR; Phase; Directed; evolution; site

The broader impact of this Small Business Innovation Research (SBIR) Phase I project will be to develop methods to facilitate the efficient, reproducible insertion of large DNA segments into stable locations on bacterial vectors, viral and non-viral shuttle vectors, and the chromosomes of prokaryotic and eukaryotic host cells comprising novel target sequences plus helper and donor vectors that could impact many areas of synthetic biology. Directed evolution experiments will be carried out to recover genes encoding bacterial transposase variants that have altered specificity or increased efficiency of transposition, compared to those recovered by products encoded by the wild-type transposase genes. Homologues of the bacterial target site will be used to recover genes encoding variant transposases that should function efficiently in eukaryotic cells. Modified helper and donor vectors will also be constructed with promoters and genes having optimized codon preferences to facilitate the efficient, direct generation of composite vectors harbored in eukaryotic cells, and eventually, the efficient, reproducible generation of cells harboring large DNA insertions at one or more specific stable sites within a host cell chromosome.The proposed project will exploit the key properties of the bacterial Tn7 transposon system for much broader utilization in many aspects of systems biology. Genes encoding transposases and accessory proteins will be mutagenized to alter the specificity and enhance the efficiency of insertion events in both prokaryotic and eukaryotic cells. This platform could have advantages over other gene transfer approaches by allowing stable, precise insertion events without the subsequent remobilization or the creation of indels/rearrangements at the target site. The ability to move large segments of DNA in such a manner would benefit many fields of synthetic biology.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.

这个小企业创新研究（SBIR）第一阶段项目的更广泛的影响将是开发方法，以促进大DNA片段有效，可重复地插入到细菌载体，病毒和非病毒穿梭载体，原核和真核宿主细胞的染色体上的稳定位置，包括新的靶序列加上辅助和供体载体，可能会影响合成生物学的许多领域。将进行定向进化实验以回收编码细菌转座酶变体的基因，与野生型转座酶基因编码的产物相比，所述细菌转座酶变体具有改变的特异性或增加的转座效率。细菌靶位点的同源物将用于回收编码变体转座酶的基因，所述变体转座酶应当在真核细胞中有效地发挥功能。修饰的辅助载体和供体载体也将用具有优化密码子偏好的启动子和基因构建，以促进在真核细胞中携带的复合载体的有效、直接产生，并且最终，在宿主细胞染色体内的一个或多个特定稳定位点上可重复地产生携带大DNA插入的细胞。拟议的项目将利用细菌Tn 7转座子的关键特性在系统生物学的许多方面有更广泛的应用。编码转座酶和辅助蛋白的基因将被诱变以改变特异性并提高原核和真核细胞中插入事件的效率。该平台可以通过允许稳定、精确的插入事件而不需要随后的再动员或在靶位点处产生插入缺失/重排而具有优于其他基因转移方法的优势。以这种方式移动大片段DNA的能力将使合成生物学的许多领域受益。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审查标准进行评估，被认为值得支持。