In recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery and extend application. Here, we present a collection of 10 exceptionally compact (422-603 amino acids) CRISPR-Cas12f nucleases that recognize and cleave dsDNA in a PAM dependent manner. Categorized as class 2 type V-F, they originate from the previously identified Cas14 family and distantly related type V-U3 Cas proteins found in bacteria. Using biochemical methods, we demonstrate that a 5' T- or C-rich PAM sequence triggers dsDNA target cleavage. Based on this discovery, we evaluated whether they can protect against invading dsDNA in Escherichia coli and find that some but not all can. Altogether, our findings show that miniature Cas12f nucleases can protect against invading dsDNA like much larger class 2 CRISPR effectors and have the potential to be harnessed as programmable nucleases for genome editing.

中文翻译：

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微型CRISPR-Cas12f核酸酶对PAM的识别会触发可编程的双链DNA靶裂解。

近年来，CRISPR相关（Cas）核酸酶彻底改变了基因组编辑领域。Cas9和Cas12在RNA的指导下在称为原间隔子相邻基序（PAM）的短序列附近切割双链（ds）DNA靶标，提供了前所未有的灵活性，但是，更紧凑的版本将简化交货并扩展应用范围。在这里，我们介绍了10种非常紧凑的（422-603个氨基酸）CRISPR-Cas12f核酸酶的集合，它们以PAM依赖性方式识别和切割dsDNA。它们归类为2类VF，它们源自先前鉴定的Cas14家族和细菌中发现的远缘相关的V-U3型Cas蛋白。使用生化方法，我们证明了富含5'T或C的PAM序列可触发dsDNA靶标裂解。基于这一发现，我们评估了它们是否可以抵御dsDNA在大肠杆菌中的侵袭，并发现其中一些（但不是全部）可以。总而言之，我们的研究结果表明，微型Cas12f核酸酶可以像更大的2类CRISPR效应子一样防御dsDNA的入侵，并有可能被用作可编程核酸酶进行基因组编辑。