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. Author manuscript; available in PMC: 2019 Aug 19.
Published in final edited form as: Drug Discov Today Technol. 2018 Feb 4;28:53–61. doi: 10.1016/j.ddtec.2018.01.001

Figure 4. Two Golden Gate assembly-based cloning strategies.

Figure 4.

(A) Schematic of a two-step strategy. Guide sequences are first cloned into single sgRNA plasmids. Each sgRNA expression cassette is amplified by primers carrying type IIs enzyme binding and a series of digesting sequences. Type IIs enzyme digestion produces overhangs that allow for assembly of multiple fragments in a designed order. (B) Schematic of a single-step strategy. Primers carrying guide sequences are used to amplify RNA scaffold and promoter sequences from specially designed vectors (Lenti-multi-Guide or Lenti-multi-CRISPR, Addgene #85401 or 85402). BsmBI digestion produces a series of overhangs that allow for assembly of multiple fragments in a designed order.