Recent progress in computational tools for designing editing sequences used in microbial genetic manipulations

YANG Yi; MAO Yufeng; YANG Chunhe; WANG Meng; LIAO Xiaoping; MA Hongwu

doi:10.12211/2096-8280.2022-055

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Recent progress in computational tools for designing editing sequences used in microbial genetic manipulations

Invited Review | 更新时间：2025-03-20

- Recent progress in computational tools for designing editing sequences used in microbial genetic manipulations
- Synthetic Biology Journal Vol. 4, Issue 1, Pages: 30-46(2023)
- 作者机构：
  
  1.中国科学院天津工业生物技术研究所中国科学院系统微生物工程重点实验室，天津　300308
  2.天津科技大学生物工程学院，天津　300457
- 作者简介：
- 基金信息：
- DOI：10.12211/2096-8280.2022-055
  CLC： Q939.97
- Received：03 October 2022，
  
  Revised：2022-11-23，
  
  Published：28 February 2023
- 稿件说明：
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杨毅，毛雨丰，杨春贺，王猛，廖小平，马红武. 面向微生物遗传操作的编辑序列设计工具的研究进展［J］. 合成生物学， 2023， 4（1）： 30-46

YANG Yi， MAO Yufeng， YANG Chunhe， WANG Meng， LIAO Xiaoping， MA Hongwu. Recent progress in computational tools for designing editing sequences used in microbial genetic manipulations［J］. Synthetic Biology Journal， 2023， 4（1）： 30-46
杨毅，毛雨丰，杨春贺，王猛，廖小平，马红武. 面向微生物遗传操作的编辑序列设计工具的研究进展［J］. 合成生物学， 2023， 4（1）： 30-46 DOI： 10.12211/2096-8280.2022-055.

YANG Yi， MAO Yufeng， YANG Chunhe， WANG Meng， LIAO Xiaoping， MA Hongwu. Recent progress in computational tools for designing editing sequences used in microbial genetic manipulations［J］. Synthetic Biology Journal， 2023， 4（1）： 30-46 DOI： 10.12211/2096-8280.2022-055.

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”（design-build-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。

Abstract

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

high-throughput 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

sgRNA 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 sgRNA 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.

关键词

Keywords

references

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