Targeted isolation and cloning of 100-kb microbial genomic sequences by Cas9-assisted targeting of chromosome segments

• Published in: Nature protocols Vol. 11; no. 5; pp. 960 - 975
• Main Authors: Jiang, Wenjun, Zhu, Ting F
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.05.2016; Nature Publishing Group
• Subjects: 631/1647/1511; 631/1647/2234; 631/326/41/2173; 631/61/17/1998; Analytical Chemistry; Artificial chromosomes; Bacterial Proteins - genetics; Biological Techniques; Chromosomes; Chromosomes, Bacterial; Cloning; Cloning, Molecular - methods; Computational Biology/Bioinformatics; CRISPR; CRISPR-Associated Protein 9; Efficiency; Endonucleases - genetics; Enzymes; Escherichia coli - genetics; Genetic aspects; Genetic Vectors; Genome, Bacterial - genetics; Genomes; Genomics; Genomics - methods; Life Sciences; Methods; Microarrays; Microorganisms; Molecular biology; Organic Chemistry; Protocol; RNA, Guide, CRISPR-Cas Systems; Synthetic Biology - methods; Vectors (Biology); Workflow; United States
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2016.055

Here the authors provide the procedural details of a new approach to clone large (up to 100 kb) microbial genomic sequences using Cas9-assisted targeting of chromosome segments (CATCH). The Cas9 cleavage is achieved in agarose gel, and the cleaved product can be ligated into a cloning vector. Cloning of long microbial genomic sequences is an essential tool in synthetic biology and genome engineering. Such long sequences are often difficult to obtain directly by traditional PCR or restriction enzyme digestion, and therefore the cloning of these sequences has remained a technical obstacle in molecular biology. Based on the in vitro application of RNA-guided Cas9 nuclease, the method of Cas9-assisted targeting of chromosome segments (CATCH) cleaves target DNA in vitro from intact bacterial chromosomes embedded in agarose plugs, which can be subsequently ligated with cloning vector through Gibson assembly. Here we describe an optimized protocol of CATCH cloning for the targeted cloning of long genomic sequences of up to 100 kb from microorganisms. The protocol uses standard laboratory equipment and takes ∼8 h of bench time over several days, and it may potentially simplify and accelerate efforts to isolate and clone large gene clusters from microorganisms.