Abstract Several Golden Gate-based DNA assembly techniques have been developed previously with different limitations including the requirement for domestication of sequences with internal sites for the type IIS restriction enzymes used, insertion of persistent scars in assembled DNA and the need for multiple assembly vectors and overhang sequences. We developed UniClo, which overcomes all these problems. Sequences with internal type IIS sites can be assembled, it allows fully scarless hierarchical assembly and requires only three assembly vectors and two universal overhang sequences. This is achieved by three key elements: 1. Recombinant methylases are used in vitro to methylate, and thus inactivate, any sites in fragments to be assembled for the type IIS restriction enzyme used in the assembly as well as the outer sites of the assembly vectors. 2. A CRISPR-dCas9 molecule is used to protect the type IIS restriction enzyme sites required for the assembly from methylation, thus preserving the activity of these sites. 3. A set of engineered vectors is used to trim overhangs that would otherwise generate scars. Here, we present a detailed protocol for performing DNA assembly using UniClo and describe the methylation-protection of the fragments to be assembled, methylation of the scarless vectors, the assembly reaction, and analysis of the final assembled DNA molecule. UniClo offers substantial flexibility in the assembly design and enables the assembly of any DNA molecule regardless of its sequence, nature and application.
Flores‐Fernández et al. (Fri,) studied this question.