摘要
以同源重组、CRISPR等为代表的遗传操作技术是合成生物学的重要支撑技术。高效准确的遗传操作依赖于可准确定位编辑位点和实现序列改造的编辑序列(如引物序列、同源臂序列、sgRNA序列等)。由于遗传改造目标与遗传操作技术丰富多变,加之近年来基于生物铸造厂(BioFoundry)的自动化遗传改造模式对高通量设计的需求日益增加,使得通过计算机辅助设计工具实现精准快速的编辑序列设计愈发重要。本文针对不同阶段实现不同目标的微生物遗传操作技术和相应的编辑序列辅助设计工具的发展进行了综述。按照不同编辑序列类型和遗传操作应用场景,将编辑序列设计工具划分为四种类型:引物设计工具、DNA组装的设计工具、sgRNA设计工具、基因组编辑的全流程设计工具,对各类编辑序列设计工具的应用及存在的问题进行了分析总结并对未来的研究方向进行了展望。编辑序列设计工具的发展将有助于实现合成生物学“设计—构建—测试—学习”(designbuild-test-learn,DBTL)工作循环中上游的基因型“设计”与下游“构建”两个关键环节之间的无缝衔接。
Genetic manipulations such as homologous recombination and CRISPR are basic technologies of synthetic biology aiming to design and construct artificial life. One key factor affecting the efficiency and accuracy of microbial genetic manipulations is the editing sequences(ES), namely the assisting sequences used for precisely locating and editing a target sequence in a genome, such as a primer, a homologous arm or a sgRNA sequence. For different genetic manipulation technologies, diverse manipulation types and multiple ESs are required for different stages of manipulation processes, especially for the high-throughput design with recently developed biofoundries to enable automatic strain modifications. Therefore, it is becoming essential to use computational tools for precise, fast, highthroughput and whole workflow ES design. This article reviews tools for ES design at different stages using various microbial genetic manipulation technologies. The tools are classified into four categories based on the types of ESs and their application scenario: primer design, DNA assembly design, sg RNA design and whole workflow ES design. We first give a brief introduction to the tools used for basic primer design with an emphasis on the widely used open-source tool Primer3. Then we have an extensive discussion on the tools used in the design of ESs for DNA assembly using technologies like Gibson and Golden Gate assembly which are required for linking the editing sequences and/or the inserted sequences together as one big fragment to be transformed into target strains. Various tools for designing sg RNA used in the latest CRISPR technologies for genome and base editing are also evaluated and compared in detail. Moreover, we argue that one-stop ES design tools which integrate various design methods to cover the whole genetic manipulation workflow would be very important in addressing challenges for the high throughput design raised by automatic strain construction biofoundries to enable highly precise and efficient genome editing for different sequence manipulations at any location and in any organism. These ES design tools will seamlessly link the genotype “Design” step and the strain “Build” step in the “design-build-test-learn(DBTL)” working cycle of synthetic biology to facilitate the creation of artificial organisms.
作者
杨毅
毛雨丰
杨春贺
王猛
廖小平
马红武
YANG Yi;MAO Yufeng;YANG Chunhe;WANG Meng;LIAO Xiaoping;MA Hongwu(Key Laboratory of Systems Microbial Biotechnology,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China)
出处
《合成生物学》
CSCD
2023年第1期30-46,共17页
Synthetic Biology Journal
基金
国家重点研发计划“合成生物学”重点专项(2018YFA0902900)
国家自然科学基金(32101186)
中国科学院青年创新促进会
天津市合成生物技术创新能力提升行动项目(TSBICIP-PTJS-001,TSBICIP-KJGG-005)。
