Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Gene. 2020 Aug 30;753:144813. doi: 10.1016/j.gene.2020.144813. Epub 2020 May 26.
Microbial production of bio-based ingredients often requires metabolically engineered bacterial strains with the edited genome. Genome editing tools are also essential for gene identification and investigating genotype-phenotype connections. Currently, one of the most common tools of genome editing is based on a natural bacterial adaptive immune system known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) due to its simple, rapid, and efficient activities. Although successful in some in vitro systems, its application as an approach of metabolic engineering and genome editing is still not so extensive. Here, we discuss existing barriers and challenges of the CRISPR/Cas9 editing tool for in vitro systems. Firstly, we aim to briefly introduce the CRISPR/Cas9 method as an in vitro gene editing tool. Next, we discuss existing obstacles to CRISPR-based editing in bacterial and in vitro model systems and offer guidelines to help achieve editing in an expanded range of in vitro systems.
微生物生产生物基成分通常需要经过基因组编辑的代谢工程化细菌菌株。基因组编辑工具对于基因鉴定和研究基因型-表型关系也是必不可少的。目前，最常用的基因组编辑工具之一是基于一种称为 CRISPR（成簇规律间隔短回文重复）/Cas9（CRISPR 相关蛋白 9）的天然细菌适应性免疫系统，因为它具有简单、快速和高效的活性。尽管在一些体外系统中取得了成功，但它作为代谢工程和基因组编辑方法的应用还不是那么广泛。在这里，我们讨论了 CRISPR/Cas9 编辑工具在体外系统中存在的障碍和挑战。首先，我们旨在简要介绍 CRISPR/Cas9 方法作为体外基因编辑工具。接下来，我们讨论了基于 CRISPR 的编辑在细菌和体外模型系统中存在的障碍，并提供了一些指导方针，以帮助在更广泛的体外系统中实现编辑。
