Flusche Tamara, Rajan Rakhi
Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman, OK, USA.
Adv Exp Med Biol. 2023;1414:27-43. doi: 10.1007/5584_2022_730.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in bacteria and archaea, where immunological memory is retained in the CRISPR locus as short pieces of the intruding nucleic acid, termed spacers. The adaptation to new infections occurs through the integration of a new spacer into the CRISPR array. For immune protection, spacers are transcribed into CRISPR RNAs (crRNA) that are used to guide the effector nuclease of the system in sequence-dependent target cleavage. Spacers originate as a prespacer from either DNA or RNA depending on the CRISPR-Cas system being observed, and the nearly universal Cas proteins, Cas1 and Cas2, insert the prespacer into the CRISPR locus during adaptation in all systems that contain them. The mechanism of site-specific prespacer integration varies across CRISPR classes and types, and distinct differences can even be found within the same subtype. In this review, the current knowledge on the mechanisms of prespacer integration in type II-A CRISPR-Cas systems will be described. Comparisons of the currently characterized type II-A systems show that distinct mechanisms exist within different members of this subtype and are correlated to sequence-specific interactions of Cas proteins and the DNA elements present in the CRISPR array. These observations indicate that nature has fine-tuned the mechanistic details while performing the basic step of DNA integration by Cas proteins, which offers unique advantages to develop Cas1-Cas2-based biotechnology.
成簇规律间隔短回文重复序列（CRISPR）及其相关（Cas）蛋白构成了细菌和古生菌中的一种适应性免疫系统，其中免疫记忆以短片段的入侵核酸（称为间隔序列）的形式保留在CRISPR基因座中。对新感染的适应通过将新的间隔序列整合到CRISPR阵列中来实现。为了实现免疫保护，间隔序列被转录为CRISPR RNA（crRNA），用于引导系统的效应核酸酶进行序列依赖性的靶标切割。根据所观察的CRISPR-Cas系统，间隔序列起源于DNA或RNA中的前间隔序列，并且几乎普遍存在的Cas蛋白Cas1和Cas2在所有包含它们的系统的适应过程中将前间隔序列插入到CRISPR基因座中。位点特异性前间隔序列整合的机制在不同的CRISPR类别和类型中有所不同，甚至在同一亚型内也能发现明显差异。在本综述中，将描述关于II-A型CRISPR-Cas系统中前间隔序列整合机制的当前知识。对目前已表征的II-A型系统的比较表明，该亚型的不同成员中存在不同的机制，并且这些机制与Cas蛋白和CRISPR阵列中存在的DNA元件的序列特异性相互作用相关。这些观察结果表明，自然界在通过Cas蛋白进行DNA整合的基本步骤时对机制细节进行了微调，这为开发基于Cas1-Cas2的生物技术提供了独特优势。
