A plethora of CRISPR effectors, such as Cas3, Cas9, and Cas12a, are commonly employed as gene editing tools. Among these, Cas12 effectors developed based on Class II type V proteins exhibit distinct characteristics compared to Class II type VI and type II effectors, such as their ability to generate non-allelic DNA double-strand breaks, their compact structures, and the presence of a single RuvC-like nuclease domain. Capitalizing on these advantages, Cas12 family proteins have been increasingly explored and utilized in recent years. However, the characteristics and applications of different subfamilies within the type V protein family have not been systematically summarized. In this review, we focus on the characteristics of type V effector (CRISPR/Cas12) proteins and the current methods used to discover new effector proteins. We also summarize recent modifications based on engineering of type V effectors. In addition, we introduce the applications of type V effectors for gene editing in animals and plants, including the development of base editors, tools for regulating gene expression, methods for gene targeting, and biosensors. We emphasize the prospects for development and application of CRISPR/Cas12 effectors with the goal of better utilizing toolkits based on this protein family for crop improvement and enhanced agricultural production.

中文翻译：

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CRISPR/Cas介导的植物基因组工程的V型效应子


大量的 CRISPR 效应子，例如 Cas3、Cas9 和 Cas12a，通常用作基因编辑工具。其中，基于II类V型蛋白开发的Cas12效应子与II类VI型和II型效应子相比表现出明显的特征，例如它们产生非等位基因DNA双链断裂的能力、其紧凑的结构以及存在单个 RuvC 样核酸酶结构域。利用这些优势，Cas12家族蛋白近年来得到了越来越多的探索和利用。然而，V型蛋白家族中不同亚家族的特征和应用尚未系统总结。在这篇综述中，我们重点关注 V 型效应蛋白 (CRISPR/Cas12) 的特征以及当前用于发现新效应蛋白的方法。我们还总结了基于 V 型效应器工程的最新修改。此外，我们还介绍了V型效应器在动植物基因编辑中的应用，包括碱基编辑器的开发、调控基因表达的工具、基因打靶方法和生物传感器。我们强调CRISPR/Cas12效应子的开发和应用前景，目标是更好地利用基于该蛋白家族的工具包进行作物改良和提高农业产量。