Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA.
Department of Integrative Biology, University of Texas at Austin, Austin, TX, 78712, USA.
Nat Commun. 2024 Aug 6;15(1):6653. doi: 10.1038/s41467-024-50816-w.
CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π - π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.
CAST 利用 CRISPR 相关蛋白和 Tn7 家族转座子进行 RNA 引导的垂直和水平传播。CAST 编码最小的 CRISPR 阵列，但不能获得新的间隔区。在这里，我们报告 CAST 可以利用防御相关的 CRISPR 阵列进行水平传播。生物信息学分析表明，CAST 与防御相关的 CRISPR 系统共同出现，其中 I 型-B 和 V 型 CAST 亚型的出现频率最高。使用大肠杆菌定量转座测定和体外重建，我们表明 CAST 可以使用这些防御系统的 CRISPR RNA。I 型-F CAST-Cascade 与 III-B 型 CRISPR RNA 的高分辨率结构表明 Cas6 通过序列非依赖性 π-π 相互作用识别直接重复序列。除了使用异源 CRISPR 阵列外，V 型 CAST 还可以通过无引导机制转座，即使 S15 辅助因子过表达也是如此。过表达 S15 和转激活 CRISPR RNA 或单个指导 RNA 会降低，但不会消除 V 型 CAST 的非靶整合。我们的研究结果表明，一些 CAST 可能利用防御相关的 CRISPR 阵列，在将 CAST 转移到异源细菌宿主时必须考虑到这一事实。更广泛地说，这项工作将指导进一步努力设计 CAST 在基因编辑应用中的活性和特异性。
