Limosilactobacillus reuteri CRISPR-Cas9系統特性分析

Abstract

CRISPR-Cas系統是細菌與古菌對抗外來核酸片段的適應性免疫機制。其中，CRISPR-Cas9屬於type II CRISPR-Cas系統，因能有效辨識並準確切割特定DNA序列，目前已廣泛應用於基因編輯技術。Limosilactobacillus reuteri為常見的益生菌，可為宿主提供多項健康益處，相關益生功能之研究甚多，然而對其內源性CRISPR-Cas9系統的探討仍相當有限。本研究針對L. reuteri菌株的內源性CRISPR-Cas9系統進行分析，包括具備此系統菌株的篩選、基因組成、Cas9 蛋白特性及PAM序列辨識偏好等比較。進一步以L. reuteri Pg4菌株為代表，依據PAM預測結果建構用於功能驗證之干擾質體，探討Pg4 Cas9作為基因編輯工具的可行性。分析結果顯示，64株L. reuteri菌株中，僅13株菌株（含Pg4）具備內源性CRISPR-Cas9系統，且不同菌株間的CRISPR array組成與cas基因分布表現出差異。透過Cas9蛋白質多序列比對發現，L. reuteri菌株Cas9蛋白序列具有高度保守性，雖與目前主要用於基因編輯之spCas9存在明顯差異，但在催化殘基與Bridge helix中的重要精胺酸殘基處仍具高度保守性，顯示其潛在的DNA切割能力。此外，AlphaFold預測之三維結構顯示，Pg4 Cas9與其他12株L. reuteri菌株的Cas9結構高度相似，但與spCas9整體結構有顯著差異，特別是在核酸酶葉的HNH結構與PI domain區域，可能影響其DNA切割能力與PAM序列辨識特性。在PAM序列預測方面，首先以Protein2PAM工具，根據Cas9蛋白質序列，預測L. reuteri Cas9 偏好之 PAM 序列皆為5’-NNAAA-3’；進一步針對Pg4菌株，以CRISPRTarget及WebLogo工具分析其 protospacer 序列，預測其偏好辨識的PAM序列為5’-ACAAA-3’，與Protein2PAM預測結果相互呼應。為驗證Pg4 Cas9對PAM序列之辨識效果，本研究已建構含該預測PAM之干擾質體，預期將轉形至Pg4菌株進行功能性驗證。總結而言，本研究透過整合基因體與蛋白質序列分析、三維結構預測、功能驗證實驗設計與干擾質體建構，建立了L. reuteri Pg4內源性CRISPR-Cas9系統之基礎研究框架。不僅拓展對L. reuteri CRISPR-Cas9系統之了解，也為後續深入探討其Cas9蛋白功能與基因編輯應用提供了重要材料與發展方向。

The CRISPR-Cas system is an adaptive immune mechanism in bacteria and archaea that defends against invading nucleic acids. Specifically, the CRISPR-Cas9 system has been widely applied in genome editing due to its efficient and precise DNA cleavage activity. Limosilactobacillus reuteri is a common probiotic known for its health-promoting effects, however, its endogenous CRISPR-Cas9 system remains largely unexplored. In this study, we analyzed the endogenous CRISPR-Cas9 system of L. reuteri strains, including the presence of the system, gene composition, protein characteristics, and PAM sequence preferences. Among 64 L. reuteri strains, only 13 strains, including Pg4, were found to harbor CRISPR-Cas9 systems, exhibiting diversity in CRISPR arrays and cas gene arrangements. Multiple sequence alignment of Cas9 proteins revealed that the Cas9 sequences among L. reuteri strains are highly conserved. Although they show noticeable differences from spCas9, which is commonly used in genome editing, key catalytic residues and critical arginine residues within the bridge helix remain conserved, suggesting potential DNA cleavage activity. AlphaFold-predicted structures revealed that Pg4 Cas9 shares high similarity with other L. reuteri Cas9 proteins but exhibits notable structural differences from spCas9, particularly in the HNH and PI domains, which may affect DNA binding activity or PAM recognition. PAM prediction using Protein2PAM indicated that L. reuteri Cas9 proteins commonly prefer the PAM sequence 5′-NNAAA-3′. Furthermore, CRISPRTarget and WebLogo analyses based on protospacer sequences predicted that the preferred PAM of the Pg4 strain is 5′-ACAAA-3′, consistent with the Protein2PAM result. An interference plasmid containing the predicted PAM was constructed for future validation in the Pg4 strain. In summary, this study establishes a foundational framework for understanding the CRISPR-Cas9 system in L. reuteri Pg4 and provides valuable insights for the future development of strain-specific genome editing tools.