Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA; Purdue Institute for Cancer Research, Purdue University, West Lafayette, IN 47907, USA.
Cell. 2023 Sep 14;186(19):4204-4215.e19. doi: 10.1016/j.cell.2023.07.010. Epub 2023 Aug 8.
Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key feature of CASTs is their ability to recruit Tn7-like transposons to nuclease-deficient CRISPR effectors. However, how Tn7-like transposons are recruited by diverse CRISPR effectors remains poorly understood. Here, we present the cryo-EM structure of a recruitment complex comprising the Cascade complex, TniQ, TnsC, and the target DNA in the type I-B CAST from Peltigera membranacea cyanobiont 210A. Target DNA recognition by Cascade induces conformational changes in Cas6 and primes TniQ recruitment through its C-terminal domain. The N-terminal domain of TniQ is bound to the seam region of the TnsC spiral heptamer. Our findings provide insights into the diverse mechanisms for the recruitment of Tn7-like transposons to CRISPR effectors and will aid in the development of CASTs as gene knockin tools.
Tn7 样转座子已被“劫持”CRISPR-Cas 系统来促进其自身 DNA 的移动。这些 CRISPR 相关转座子（CASTs）是可编程基因敲入的有前途的工具。CASTs 的一个关键特征是它们能够招募 Tn7 样转座子到核酸酶缺陷型 CRISPR 效应物。然而，不同的 CRISPR 效应物如何招募 Tn7 样转座子仍知之甚少。在这里，我们展示了一个募集复合物的低温电镜结构，该复合物由 Cascade 复合物、TniQ、TnsC 和 Peltigera membranacea 蓝藻 210A 中 I-B 型 CAST 的靶 DNA 组成。Cascade 通过其 C 端结构域识别靶 DNA 来诱导 Cas6 的构象变化，并通过其 C 端结构域来启动 TniQ 的募集。TniQ 的 N 端结构域与 TnsC 螺旋七聚体的缝隙区域结合。我们的发现为 Tn7 样转座子到 CRISPR 效应物的募集提供了不同的机制见解，并将有助于 CASTs 作为基因敲入工具的发展。
