A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster

Author:

Guo Yujing1,Cai Guang1,Li Huiying1,Lin Zhenquan1,Shi Shuobo1ORCID,Jin Jin1ORCID,Liu Zihe1

Affiliation:

1. State Key Laboratory of Chemical Resource Engineering, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China

Abstract

The ability to clone large DNA fragments from genomes is valuable for both basic and applied research, such as the construction of synthetic genomes, and the expression of biosynthetic gene clusters (BGCs) for natural product discovery. Here, we report a fast and efficient platform for the direct capture of genome DNAs, by combining CRISPR and Gibson assembly. We demonstrate this method with the ability of cloning large DNA fragments ranging from 30 to 77 kb from various host genomes, achieving a near 100% cloning fidelity for DNA fragments below 50 kb. We next demonstrate this method by the cloning of a 40 kb fragment from Streptomyces ceruleus A3(2), which is rich in BGCs for natural products; and used this method cloning the 40 kb fengycin synthetic gene cluster from B. subtilis 168, encoding for a class of peptides with bioactivity. This method provides efficient and simple opportunities for assembling large DNA constructs from distant sources.

Funder

National Key Research and Development Program of China

National Natural Science Foundation of China

Fundamental Research Funds for the Central Universities

Publisher

MDPI AG

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