CRISPR-Cas12a, an RNA-guided nuclease, has been repurposed for genome editing and molecular diagnostics due to its capability of cis-cleavage on target DNA and trans-cleavage on non-target single-strand DNA (ssDNA). However, the mechanisms underlying the activation of trans-cleavage activity of Cas12a, particularly in the context of split DNA activators, remain poorly understood. We elucidate the synergistic effect of these activators and introduce the concepts of induced targeting effect and exon-unwinding to describe the phenomenon. We demonstrate that upon binding of split DNA activators adjacent to the Protospacer Adjacent Motif (PAM) to the Cas12a ribonucleoprotein (Cas12a–RNP), a ternary complex form that can capture and interact with distal split DNA activators to achieve synergistic effects. Notably, if the distal activator is double-strand DNA (dsDNA), the complex initiates exon-unwinding, facilitating the RNA-guide sequence's access. Our findings provide a mechanistic insight into action of Cas12a and propose a model that could significantly advance our understanding of its function.

中文翻译：

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Cas12a 分裂的 DNA 激活剂与外显子解旋和诱导靶向效应的协同作用


CRISPR-Cas12a 是一种 RNA 引导的核酸酶，由于其对靶 DNA 的顺式切割和非靶标单链 DNA （ssDNA） 的反式切割能力，已被重新用于基因组编辑和分子诊断。然而，Cas12a 反式切割活性激活的潜在机制，特别是在分裂的 DNA 激活剂的情况下，仍然知之甚少。我们阐明了这些激活剂的协同作用，并引入了诱导靶向效应和外显子解旋的概念来描述这种现象。我们证明，在与原始间隔区相邻基序 （PAM） 相邻的分裂 DNA 激活剂与 Cas12a 核糖核蛋白 （Cas12a-RNP） 结合后，Cas12a 核糖核蛋白是一种三元复合物形式，可以捕获远端分裂的 DNA 激活剂并与之相互作用以实现协同效应。值得注意的是，如果远端激活剂是双链 DNA （dsDNA），则该复合物会启动外显子解旋，从而促进 RNA 引导序列的进入。我们的研究结果提供了对 Cas12a 作用的机制见解，并提出了一个可以显着促进我们对其功能的理解的模型。