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.

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合成生物学。将进行定向的进化实验，以恢复编码细菌转座酶变体的基因，这些变体变化了特异性或提高换位效率，与由野生型转座酶基因编码的产物恢复的基因。细菌靶位点的同源物将用于恢复编码应在真核细胞中有效发挥作用的变体转酶酶的基因。修改后的辅助和供体矢量也将使用具有优化的密码子偏好的启动子和基因构建在系统生物学的许多方面，用于广泛利用的系统。编码转座酶和辅助蛋白的基因将被诱变，以改变原核生物和真核细胞中插入事件的效率。该平台可以通过允许稳定，精确的插入事件而没有随后的重新启动或在目标站点创建Indels/重排的情况，而不是其他基因转移方法具有优势。以这种方式移动大部分DNA的能力将使许多合成生物学领域受益。该奖项反映了NSF的法定任务，并被认为是值得通过基金会的知识分子优点和更广泛影响的评估标准来评估值得支持的。