Meliawati Meliawati, Schilling Christoph, Schmid Jochen
Institute for Molecular Microbiology and Biotechnology, University of Münster, Corrensstrasse 3, 48149, Münster, Germany.
Chair of Chemistry of Biogenic Resources, Campus for Biotechnology and Sustainability, Technical University of Munich, Schulgasse 16, 94315, Straubing, Germany.
Appl Microbiol Biotechnol. 2021 Apr;105(8):2981-2990. doi: 10.1007/s00253-021-11243-9. Epub 2021 Mar 23.
Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome engineering and related technologies have revolutionized biotechnology over the last decade by enhancing the efficiency of sophisticated biological systems. Cas12a (Cpf1) is an RNA-guided endonuclease associated to the CRISPR adaptive immune system found in many prokaryotes. Contrary to its more prominent counterpart Cas9, Cas12a recognizes A/T rich DNA sequences and is able to process its corresponding guide RNA directly, rendering it a versatile tool for multiplex genome editing efforts and other applications in biotechnology. While Cas12a has been extensively used in eukaryotic cell systems, microbial applications are still limited. In this review, we highlight the mechanistic and functional differences between Cas12a and Cas9 and focus on recent advances of applications using Cas12a in bacterial hosts. Furthermore, we discuss advantages as well as current challenges and give a future outlook for this promising alternative CRISPR-Cas system for bacterial genome editing and beyond. KEY POINTS: • Cas12a is a powerful tool for genome engineering and transcriptional perturbation • Cas12a causes less toxic side effects in bacteria than Cas9 • Self-processing of crRNA arrays facilitates multiplexing approaches.
成簇规律间隔短回文重复序列(CRISPR)介导的基因组工程及相关技术在过去十年中通过提高复杂生物系统的效率彻底改变了生物技术。Cas12a(Cpf1)是一种与许多原核生物中发现的CRISPR适应性免疫系统相关的RNA引导的核酸内切酶。与其更为著名的对应物Cas9不同,Cas12a识别富含A/T的DNA序列,并能够直接加工其相应的引导RNA,使其成为用于多重基因组编辑工作及生物技术中其他应用的通用工具。虽然Cas12a已在真核细胞系统中广泛使用,但其在微生物中的应用仍然有限。在本综述中,我们强调了Cas12a与Cas9之间的机制和功能差异,并着重介绍了在细菌宿主中使用Cas12a的应用的最新进展。此外,我们讨论了优势以及当前面临的挑战,并对这种用于细菌基因组编辑及其他领域的有前景的替代性CRISPR-Cas系统给出了未来展望。要点:• Cas12a是用于基因组工程和转录干扰的强大工具• Cas12a在细菌中产生的毒性副作用比Cas9少• crRNA阵列的自我加工促进了多重方法。