Engineering Biology Research Center, Kobe University, Japan; Graduate School of Science, Technology and Innovation, Kobe University, Japan.
Engineering Biology Research Center, Kobe University, Japan; Graduate School of Science, Technology and Innovation, Kobe University, Japan.
Metab Eng. 2021 Jan;63:141-147. doi: 10.1016/j.ymben.2020.12.002. Epub 2020 Dec 8.
In metabolic engineering, genome editing tools make it much easier to discover and evaluate relevant genes and pathways and construct strains. Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) systems now have become the first choice for genome engineering in many organisms includingindustrially relevant ones. Targeted DNA cleavage by CRISPR-Cas provides variousgenome engineering modes such as indels, replacements, large deletions, knock-in and chromosomal rearrangements, while host-dependent differences in repair pathways need to be considered. The versatility of the CRISPR system has given rise to derivative technologies that complement nuclease-based editing, which causes cytotoxicity especially in microorganisms. Deaminase-mediated base editing installs targeted point mutations with much less toxicity. CRISPRi and CRISPRa can temporarily control gene expression without changing the genomic sequence. Multiplex, combinatorial and large scale editing are made possible by streamlined design and construction of gRNA libraries to further accelerates comprehensive discovery, evaluation and building of metabolic pathways. This review summarizes the technical basis and recent advances in CRISPR-related genome editing tools applied for metabolic engineering purposes, with representative examples of industrially relevant eukaryotic and prokaryotic organisms.
在代谢工程中，基因组编辑工具使得发现和评估相关基因和途径以及构建菌株变得更加容易。CRISPR 相关（Cas）系统现在已经成为许多生物体（包括工业相关生物体）基因组工程的首选。CRISPR-Cas 靶向 DNA 切割提供了多种基因组工程模式，如插入缺失、替换、大片段缺失、基因敲入和染色体重排，而需要考虑宿主依赖性修复途径的差异。CRISPR 系统的多功能性催生了衍生技术，这些技术补充了基于核酸酶的编辑，特别是在微生物中会引起细胞毒性。脱氨酶介导的碱基编辑以较小的毒性引入靶向点突变。CRISPRi 和 CRISPRa 可以在不改变基因组序列的情况下暂时控制基因表达。通过简化 gRNA 文库的设计和构建，可以实现多重、组合和大规模编辑，进一步加速代谢途径的全面发现、评估和构建。本文综述了用于代谢工程的 CRISPR 相关基因组编辑工具的技术基础和最新进展，列举了具有代表性的工业相关真核和原核生物的实例。
