Fig. 2: Characterization of various genomic DNA digestion/DNA assembly combinations in the CAPTURE method.

a Schematics of T4 DNA polymerase exo + fill-in DNA assembly. In step 1, DNA molecules ends are chewed back by T4 DNA polymerase to create ssDNA overhangs. The reaction mixture’s temperature is increased to 75 °C to inactivate T4 DNA polymerase and potentially remove ssDNA secondary structures. Temperature is then decreased to 50 °C to allow for ssDNA overhang hybridization. In step 2, by addition of fresh T4 DNA polymerase, and dNTPs, DNA gaps in the hybridized DNA molecule are filled. E. coli DNA ligase is then used to ligate the nicks and produce the final assembly product. b Comparison of different digestion/DNA assembly combinations in cloning four high GC-content BGCs from Actinomycetes. The FnCas12a/T4 exo + fill-in strategy showed ~100% cloning efficiency for all four target BGCs. RE: restriction enzymes. For each cloning experiment, at least seven colonies were selected and the purified plasmids from each colony were analyzed by restriction digestion. The cloning efficiencies were calculated as the ratio of correct colonies to the total number of checked colonies. Each experiment was performed in three biological replicates and data are presented as mean values ± standard error (SEM). c Summary of results for cloning uncharacterized BGCs using CAPTURE. BGCs ranging from 10 to 113 kb can be robustly cloned using the CAPTURE method at close to 100% efficiency regardless of their GC-content. Source data are provided as a Source Data file.