Precursor (pre)-CRISPR RNA (crRNA) processing can occur in both the repeat and spacer regions, leading to the removal of specific segments from the repeat and spacer sequences, thereby facilitating crRNA maturation. The processing of pre-crRNA repeat by Cas effector and ribonuclease has been observed in CRISPR-Cas9 and CRISPR-Cas12a systems. However, no evidence of pre-crRNA spacer cleavage by any enzyme has been reported in these systems. In this study, we demonstrate that DNA target binding triggers efficient cleavage of pre-crRNA spacers by type II and V Cas effectors such as Cas12a, Cas12b, Cas12i, Cas12j and Cas9. We show that the pre-crRNA spacer cleavage catalyzed by Cas12a and Cas9 has distinct characteristics. Activation of the cleavage activity in Cas12a is induced by both single-stranded DNA (ssDNA) and double-stranded DNA target binding, whereas only ssDNA target binding triggers cleavage in Cas9 toward the pre-crRNA spacer. We present a series of structures elucidating the underlying mechanisms governing conformational activation in both Cas12a and Cas9. Furthermore, leveraging the trans-cutting activity of the pre-crRNA spacer, we develop a one-step DNA detection method characterized by its simplicity, high sensitivity, and excellent specificity.

中文翻译：

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DNA 靶标结合诱导的 II 型和 V 型 CRISPR-Cas 系统中的前 crRNA 加工。


前体 （pre）-CRISPR RNA （crRNA） 加工可以发生在重复区和间隔区，导致从重复序列和间隔区序列中去除特定片段，从而促进 crRNA 成熟。已在 CRISPR-Cas9 和 CRISPR-Cas12a 系统中观察到 Cas 效应子和核糖核酸酶对 pre-crRNA 重复序列的加工。然而，在这些系统中没有报道任何酶切割前 crRNA 间隔区的证据。在这项研究中，我们证明了 DNA 靶标结合触发 II 型和 V 型 Cas 效应子（如 Cas12a、Cas12b、Cas12i、Cas12j 和 Cas9）对前 crRNA 间隔区的有效切割。我们表明，Cas12a 和 Cas9 催化的前 crRNA 间隔区切割具有明显的特征。Cas12a 中切割活性的激活由单链 DNA （ssDNA） 和双链 DNA 靶标结合诱导，而只有 ssDNA 靶标结合触发 Cas9 中朝前 crRNA 间隔区的切割。我们提出了一系列结构，阐明了控制 Cas12a 和 Cas9 构象激活的潜在机制。此外，利用 pre-crRNA 间隔区的反切割活性，我们开发了一种一步法 DNA 检测方法，其特点是简单、高灵敏度和出色的特异性。