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代谢工程改造的细菌菌株，并具有经过编辑的基因组。基因组编辑工具对于基因鉴定和研究基因型-表型联系也是必不可少的。当前，基因组编辑的最常用工具之一是基于天然细菌适应性免疫系统，即CRISPR（簇规则间隔短回文重复序列）/ Cas9（CRISPR相关蛋白9），因为它具有简单，快速和有效的活性。尽管它在某些体外系统中很成功，但它作为代谢工程和基因组编辑方法的应用仍然不那么广泛。在这里，我们讨论了CRISPR / Cas9编辑工具在体外存在的障碍和挑战系统。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，我们讨论细菌和体外模型系统中基于CRISPR的编辑存在的障碍，并提供指导以帮助在扩展的体外系统中实现编辑。