State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, School of Life Sciences, Hubei University, Wuhan 430062, China.
Biomolecules. 2023 Jun 21;13(7):1022. doi: 10.3390/biom13071022.
Programmable endonucleases, such as Cas (Clustered Regularly-Interspaced Short Repeats-associated proteins) and prokaryotic Argonaute (pAgo), depend on base pairing of the target DNA with the guide RNA or DNA to cleave DNA strands. Therefore, they are capable of recognizing and cleaving DNA sequences at virtually any arbitrary site. The present review focuses on the commonly used in vivo and in vitro recombination-based gene cloning methods and the application of programmable endonucleases in these sequence- and ligation-independent DNA assembly methods. The advantages and shortcomings of the programmable endonucleases utilized as tools for gene cloning are also discussed in this review.
可编程核酸内切酶，如 Cas（成簇规律间隔短回文重复相关蛋白）和原核 Argonaute（pAgo），依赖于靶 DNA 与向导 RNA 或 DNA 的碱基配对来切割 DNA 链。因此，它们能够识别和切割几乎任意位置的 DNA 序列。本综述重点介绍了常用的体内和体外基于重组的基因克隆方法，以及可编程核酸内切酶在这些无需序列和连接的 DNA 组装方法中的应用。本文还讨论了将可编程核酸内切酶用作基因克隆工具的优缺点。
