PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage
PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage
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DOI：
10.1093/nar/gkaa208
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发表时间：
2020-05-21
影响因子：
14.9
通讯作者：
Siksnys, Virginijus
中科院分区：
文献类型：
--
作者：
Karvelis, Tautvydas;Bigelyte, Greta;Siksnys, Virginijus
近年来，CRISPR相关(CAS)核酸酶给基因组编辑领域带来了革命性的变化。Cas9和Cas12在RNA的引导下，在短序列附近切割双链(DS)DNA靶标，称为原间隔相邻基序(PAM)，提供了前所未有的灵活性，然而，更紧凑的版本将简化交付和扩展应用。在这里，我们提出了10个特别紧密的(422-603个氨基酸)CRISPR-Cas12f核酸酶的集合，它们以PAM依赖的方式识别和切割dsDNA。它们被归类为2型V-F，起源于先前在细菌中发现的Cas14家族和远亲的V-U3型Cas蛋白。利用生化方法，我们证明了富含5‘T或C的PAM序列可以触发dsDNA靶标切割。基于这一发现，我们评估了它们是否能够保护大肠杆菌中的dsDNA免受入侵，并发现一些但不是所有的都可以。总之，我们的发现表明，小型的Cas12f核酸酶可以保护dsDNA免受入侵，就像更大的2类CRISPR效应器一样，并有可能被用作可编程核酸酶进行基因组编辑。
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 pro-tospacer 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 likemuch larger class 2 CRISPR effectors and have the potential to be harnessed as programmable nucleases for genome editing.