Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins.
Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins.
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DOI：
10.1038/s41564-022-01265-y
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发表时间：
2023-01
影响因子：
28.3
通讯作者：
中科院分区：
文献类型：
--
作者：
规则间隔短回文重复序列（CRISPR）相关的Cas9蛋白是在原核适应性免疫系统中起主要作用的效应子，通过该效应子可以靶向并切割入侵的DNA以使其失活。Cas9内切核酸酶通过引导RNA（gRNA）引导至靶位点，其中Cas9可以识别外源DNA中的特异性序列（PAM），其然后用作切割相邻RNA匹配DNA区域的锚定点。尽管CRISPR-Cas9系统已被广泛研究并重新用于多种应用（特别是基因组编辑），但其起源和进化仍有待阐明。在这里，我们研究了Cas9从灭绝的厚壁菌门物种中复活的古老核酸酶（anCas）的进化，这些物种的历史可以追溯到2.6年前。令人惊讶的是，我们证明了这些古老的形式在其PAM和gRNA支架要求方面比现代Cas9酶更加灵活。此外，anCas描绘了从切口酶到双链断裂活性的逐渐古酶适应，这表明了一种机制，通过这种机制，古老的CRISPR系统可以在携带具有最少PAM的Cas酶时繁殖。最古老的anCas也表现出对ssDNA和ssRNA靶标的高水平活性，类似于相关系统类型中的Cas核酸酶。最后，我们说明了人类细胞中anCas酶的编辑活性。anCas蛋白的预测和表征揭示了导致功能灵活的古老酶的进化轨迹。
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 protein is an effector that plays a major role in a prokaryotic adaptive immune system, by which invading DNA can be targeted and cut for inactivation. The Cas9 endonuclease is directed to target sites by a guide RNA (gRNA) where Cas9 can recognize specific sequences (PAMs) in foreign DNA, which then serve as an anchoring point for cleavage of the adjacent RNA-matching DNA region. Although the CRISPR-Cas9 system has been widely studied and repurposed for diverse applications (notably, genome editing), its origin and evolution remain to be elucidated. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species as old as 2.6 By to the current day. Surprisingly, we demonstrate that these ancient forms were much more flexible in their PAM and gRNA scaffold requirements compared to modern day Cas9 enzymes. In addition, anCas portrays a gradual paleoenzymatic adaptation from nickase to double-strand break activity, suggesting a mechanism by which ancient CRISPR systems could propagate when harboring Cas enzymes with minimal PAMs. The oldest anCas also exhibit high levels of activity with ssDNA and ssRNA targets, resembling Cas nucleases in related system types. Finally, we illustrate editing activity of the anCas enzymes in human cells. The prediction and characterization of anCas proteins uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.
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影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin
影响因子：
82.9
作者：
Charlesworth, Carsten T.;Deshpande, Priyanka S.;Porteus, Matthew H.
通讯作者：
Porteus, Matthew H.
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
64.8
作者：
Boussau, Bastien;Blanquart, Samuel;Gouy, Manolo
通讯作者：
Gouy, Manolo