Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea.
J Microbiol Biotechnol. 2021 Jul 28;31(7):903-911. doi: 10.4014/jmb.2106.06056.
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.
先前的研究通过引入含有选择性标记和同源 DNA 片段的基因盒来修饰微生物基因组。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，如修饰基因组中的选择性标记。此外，基因组操作经常留下疤痕和痕迹，干扰下游的迭代基因组工程。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。在众多细菌和古菌的各种 CRISPR 内切酶中，Cas9 和 Cas12a（Cpf1）系统由于其简单性而被广泛应用于所有生物的基因组编辑，因为它们由单个多肽核酸酶和靶标识别 RNA 组成。然而，由于错配容忍性和依赖于前导序列相邻基序（PAM）的靶标识别，精确和精细的基因组编辑仍然具有挑战性。因此，本文描述了如何克服这些特别影响高等生物基因组编辑的障碍。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。
