A User’s Guide to Golden Gate Cloning Methods and Standards

[Image: see text] The continual demand for specialized molecular cloning techniques that suit a broad range of applications has driven the development of many different cloning strategies. One method that has gained significant traction is Golden Gate assembly, which achieves hierarchical assembly of DNA parts by utilizing Type IIS restriction enzymes to produce user-specified sticky ends on cut DNA fragments. This technique has been modularized and standardized, and includes different subfamilies of methods, the most widely adopted of which are the MoClo and Golden Braid standards. Moreover, specialized toolboxes tailored to specific applications or organisms are also available. Still, the quantity and range of assembly methods can constitute a barrier to adoption for new users, and even experienced scientists might find it difficult to discern which tools are best suited toward their goals. In this review, we provide a beginner-friendly guide to Golden Gate assembly, compare the different available standards, and detail the specific features and quirks of commonly used toolboxes. We also provide an update on the state-of-the-art in Golden Gate technology, discussing recent advances and challenges to inform existing users and promote standard practices.