A CRISPR/Cas12a-derived DNA Assembly Platform For Efficient-direct Cloning Of High GC And Large Biosynthetic Gene Clusters

Natural products(NPs)are an important source of lead compounds and clinical drugs,and NPs derived from microorganisms account for up to 1/3.After microbial drug discovery experienced a golden period around the 1960s,it has almost stagnated in recent decades,leading to a shortage of new drugs.People once thought that microbial drug resources have been exhausted.With the development of sequencing technology and bioinformatics technology,the whole genome of microorganisms has been continuously revealed,and massive dark matter-like silent gene clusters(BGC)have been discovered.These "dark matter" contain a large number of novel bioactive small molecules(BSMs).Therefore,heterologous expression of target BGCs in a chassis strain has been widely used for discovering novel NPs.One critical step of BGC heterologous expression is gene cluster cloning.However,the cloning of BGCs in Streptomyces is very difficult because of the high GC and large size of those BGCs.Simple,fast and efficient direct cloning of large BGCs,especially for high GC content samples,remains a challenge.We have developed a simple,fast,and direct cloning platform for large gene clusters based on CRISPR/Cas12a,designated CAT-FISHING(CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning platform),suitable for cloning high GC and large DNA fragments.In this study,the CAT-FISHING cloning strategy was first designed,and the conditions for CRSIPR/Cas12a to cleave large DNA fragments in vitro were investigated and optimized.CAT-FISHING was demonstrated by the efficient direct cloning of 50 kb and 80 kb DNA fragments from bacterial artificial chromosomes(GC=66%),with success rates reaching 95%and 50%.In the end,by combining the DNA cleavage activity of CRISPR/Cas12a with the unique features of a BAC library construction,we successfully cloned Paulomycin gene cluster(49 kb,GC=71%)and Surugamides gene cluster(87 kb,GC=76%),the cloning positive rate was 4?5%and 2?4%.Moreover,the NPs encoded by the cloned BGC were successfully produced in an Actinomycetes chassis strain.These results indicate that CAT-FISHING is a promising method for the direct cloning of large BGCs with high GC.The simple protocol as well as the high efficiency of CAT-FISHING should be important assets for novel NPs discovery.Despite previous study reported the CRISPR/Cas12a-based DNA assembly method,but it does not unleash the potential of this tool on large DNA cloning.By combining with BAC library construction,this study expanded the application of CRISPR/Cas12 to direct cloning of high GC and large DNA fragments in vitro.This innovation of a fundamental platform technology for use in genome mining through application of the CRISPR/Cas12 system would facilitate the discovery of novel BSMs from microbial sources.