Hu Kuang, Chia-Wei Chou, Wilke Claus O, Finkelstein Ilya J
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.
bioRxiv. 2023 Jul 11:2023.03.03.531003. doi: 10.1101/2023.03.03.531003.
CRISPR-associated transposons (CASTs) co-opt CRISPR-Cas 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 show that CASTs instead co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that all CAST sub-types co-occur with defense-associated CRISPR-Cas systems. Using an quantitative transposition assay, we show that CASTs use CRISPR RNAs (crRNAs) from these defense systems for horizontal gene transfer. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B crRNA reveals that Cas6 recognizes direct repeats via sequence-independent - interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via a crRNA-independent unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA (tracrRNA) or a single guide RNA (sgRNA) reduces, but does not abrogate, off-target integration for type V CASTs. Exploiting new spacers in defense-associated CRISPR arrays explains how CASTs horizontally transfer to new hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.
CRISPR 相关转座子（CASTs）利用 CRISPR-Cas 蛋白和 Tn7 家族转座子进行 RNA 引导的垂直和水平传播。CASTs 编码最小的 CRISPR 阵列，但无法获取新的间隔序列。在这里，我们表明 CASTs 反而利用与防御相关的 CRISPR 阵列进行水平传播。生物信息学分析表明，所有 CAST 亚型都与防御相关的 CRISPR-Cas 系统共同出现。使用定量转座分析，我们表明 CASTs 利用来自这些防御系统的 CRISPR RNA（crRNAs）进行水平基因转移。I-F 型 CAST-Cascade 与 III-B 型 crRNA 复合物的高分辨率结构表明，Cas6 通过不依赖序列的相互作用识别直接重复序列。除了使用异源 CRISPR 阵列外，V 型 CASTs 还可以通过不依赖 crRNA 的无引导机制进行转座，即使 S15 辅助因子过表达时也是如此。过表达 S15 和反式激活 CRISPR RNA（tracrRNA）或单导向 RNA（sgRNA）可减少但不能消除 V 型 CASTs 的脱靶整合。利用防御相关 CRISPR 阵列中的新间隔序列解释了 CASTs 如何水平转移到新宿主。更广泛地说，这项工作将指导进一步努力设计 CASTs 的活性和特异性以用于基因编辑应用。
