Previous studies have modified microbial genomes by introducing gene cassettes containing selectable markers and homologous DNA fragments. However, this requires several steps including homologous recombination and excision of unnecessary DNA regions, such as selectable markers from the modified genome. Further, genomic manipulation often leaves scars and traces that interfere with downstream iterative genome engineering. A decade ago, the CRISPR/Cas system (also known as the bacterial adaptive immune system) revolutionized genome editing technology. Among the various CRISPR nucleases of numerous bacteria and archaea, the Cas9 and Cas12a (Cpf1) systems have been largely adopted for genome editing in all living organisms due to their simplicity, as they consist of a single polypeptide nuclease with a target-recognizing RNA. However, accurate and fine-tuned genome editing remains challenging due to mismatch tolerance and protospacer adjacent motif (PAM)-dependent target recognition. Therefore, this review describes how to overcome the aforementioned hurdles, which especially affect genome editing in higher organisms. Additionally, the biological significance of CRISPR-mediated microbial genome editing is discussed and future research and development directions are also proposed.

中文翻译：

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精确微生物基因组编辑 CRISPR 技术的进展。


先前的研究通过引入含有选择性标记和同源 DNA 片段的基因盒来修改微生物基因组。然而，这需要几个步骤，包括同源重组和切除不必要的 DNA 区域，例如来自修饰基因组的选择标记。此外，基因组操作常常会留下干扰下游迭代基因组工程的疤痕和痕迹。十年前，CRISPR/Cas系统（也称为细菌适应性免疫系统）彻底改变了基因组编辑技术。在众多细菌和古细菌的各种 CRISPR 核酸酶中，Cas9 和 Cas12a (Cpf1) 系统由于其简单性而被广泛用于所有生物体的基因组编辑，因为它们由单一多肽核酸酶和目标识别 RNA 组成。然而，由于错配耐受性和原型间隔子相邻基序 (PAM) 依赖性目标识别，准确和微调的基因组编辑仍然具有挑战性。因此，这篇综述描述了如何克服上述障碍，尤其是影响高等生物体基因组编辑的障碍。此外，还讨论了CRISPR介导的微生物基因组编辑的生物学意义，并提出了未来的研究和发展方向。