Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system.
Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system.
复制标题
Cas4/1 双核酸酶活性可实现 I-G 型 CRISPR-Cas 系统中预间隔区的成熟和定向整合。
DOI：
10.1101/2023.06.05.543779
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Sashital,DipaliG
中科院分区：
文献类型：
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作者：
Dhingra,Yukti;Sashital,DipaliG
CRISPR-Cas适应性免疫系统从外来DNA中吸收短“间隔区”序列，并将其整合到宿主基因组中，作为CRISPR RNA的模板，指导针对未来感染的干扰。CRISPR系统中的适应是由Cas 1-Cas 2复合物介导的，该复合物催化前空间底物整合到CRISPR阵列中。许多DNA靶向系统还需要Cas 4内切核酸酶用于功能性间隔区获取。Cas 4选择含有原型间隔区邻近基序（PAM）的预间隔区，并在整合之前去除PAM，这两者都是确保宿主免疫所需的。Cas 1也被证明在某些系统中起核酸酶的作用，但这种核酸酶活性在适应中的作用尚未得到证实。我们鉴定了I-G型Cas 4/1融合体，其具有可直接参与预间隔加工的溶核活性Cas 1结构域。Cas 1结构域既是整合酶又是序列独立性核酸酶，其切割前间隔子的非PAM末端，产生最佳突出端长度，使得能够在前导侧整合。Cas 4结构域序列特异性切割预间隔区的PAM末端，确保PAM末端在间隔区侧的整合。这两个域具有不同的金属离子要求。虽然Cas 4活性是Mn 2+依赖性的，但Cas 1优先使用Mg 2+而不是Mn 2+。Cas 4/1的双重核酸酶活性消除了在预空间加工中对额外因子的需要，使得适应模块对于预空间成熟和定向整合是独立的。
CRISPR-Cas adaptive immune systems uptake short “spacer” sequences from foreign DNA and incorporate them into the host genome to serve as templates for CRISPR RNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn2+dependent, Cas1 preferentially uses Mg2+over Mn2+. The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing making the adaptation module self-reliant for prespacer maturation and directional integration.