Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination

Abstract

Direct cloning represents the most efficient strategy to access the vast number of uncharacterized natural product biosynthetic gene clusters (BGCs) for the discovery of novel bioactive compounds. However, due to their large size, repetitive nature, or high GC-content, large-scale cloning of these BGCs remains an overwhelming challenge. Here, we report a scalable direct cloning method named Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) which consists of Cas12a digestion, a DNA assembly approach termed T4 polymerase exo + fill-in DNA assembly, and Cre-lox in vivo DNA circularization. We apply this method to clone 47 BGCs ranging from 10 to 113 kb from both Actinomycetes and Bacilli with ~100% efficiency. Heterologous expression of cloned BGCs leads to the discovery of 15 previously uncharacterized natural products including six cyclic head-to-tail heterodimers with a unique 5/6/6/6/5 pentacyclic carbon skeleton, designated as bipentaromycins A-F. Four of the bipentaromycins show strong antimicrobial activity to both Gram-positive and Gram-negative bacteria such as methicillin-resistant Staphylococcus aureus, vancomycinresistant Enterococcus faecium, and bioweapon Bacillus anthracis. Due to its robustness and efficiency, our direct cloning method coupled with heterologous expression provides an effective strategy for large-scale discovery of novel natural products.

Fig. 1. Development of the CAPTURE method.

a Overview of the workflow. In the first step, purified genomic DNA is digested by Cas12a enzyme to release the target BGC fragment. In the second step, digestion products are mixed with two DNA receivers containing loxP sites at their ends. The target BGC fragment and DNA receivers are assembled together using T4 DNA polymerase exo + fill-in DNA assembly. In the final step, the assembly mixture is transformed into E. coli cells harboring a circularization helper plasmid. The linear DNA is able to circularize in vivo by Cre-lox recombination. b DNA map of helper plasmid pBE14. tcr: tetracycline resistance marker; araBAD: L-arabinose inducible promoter and its regulator; gam: phage lambda Red gam gene; pSC101: temperature-sensitive origin of replication; recA1: mutated E. coli recA gene to increase transformation efficiency. c Comparison of recombination frequency between Flp (pBE11) and Cre (pBE12) helper plasmids. -: without L-arabinose induction, +: with L-arabinose induction. Recombination frequencies were calculated based on the ratio of white colonies to the total number of acquired colonies. d Linear DNA transformation efficiency for E. coli cells harboring pBE11 (Flp), pBE12 (Cre), pBE14 (Cre and recA1) helper plasmids. Both pBE12 and pBE14 E. coli cells exhibited transformation efficiencies similar to circular DNA. e Comparison of in vitro versus in vivo circularization for two large (50 kb, 73 kb) linear DNA molecules. In vivo circularization showed ~33-fold and 150-fold higher frequency than in vitro circularization for 50 kb and 73 kb molecules, respectively. Circularization frequencies were calculated based on the number of colonies acquired for each circularization experiment in comparison to the number of colonies acquired after transformation of the original circular DNA (see Methods for full description). Each experiment was performed in three biological replicates and data are presented as mean values ± standard deviation (SD). Source data are provided as a Source Data file.

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.

Fig. 3. Metabolic profile and chemical structures of the characterized compounds from heterologous expression of five of the seven positive uncharacterized BGCs.

a HPLC analysis of the crude extract from heterologous expression of BGC #1 from Streptomyces sp. NRRL F-6131 in S. avermitilis SUKA17 and the chemical structures for the 6 heterodimers (1–6). b HPLC analysis of the crude extract from heterologous expression of BGC #11 from Streptomyces alni NRRL B-24611 in S. avermitilis SUKA17 and the chemical structure for the 4 new angucyclines and derivatives (7–10). c Chemical structures of the rest of characterized compounds including two new anthraquinone or naphthoquinone derivatives (11 and 12) containing an acetylcysteine moiety identified from Streptomyces cyaneofuscatus NRRL B-2570 BGC #2, two molecules allenomycin A and allenomycin B (16 and 17) identified from Streptomyces griseofuscus NRRL B-5429 BGC #25, two known citreodiols (14 and 15) and a new derivative (13) identified from S. griseofuscus NRRL B-5429 BGC #38.