CATCH Enables Targeted Isolation And Cloning Of Large Genomic DNA Fragments

Molecular cloning is one of the most defining and important techniques in modern biology and has been widely used in various research areas of biology.However,the cloning of large genomic DNA fragments longer than 50 kb still faces many challenges,such as amplification difficulty,low specificity and high cost.The CRISPR-Cas9 system is one of the type II CRISPR-Cas systems in which the Cas9 protein guided by crRNA and tracrRNA can recognize and cleave foreign genetic elements such as phage DNA and plasmids.Taking advantage of its specificity and versatility,the CRISPR-Cas9 system and has been recently harnessed as a prominent tool for genome editing,transcription regulation and cell imaging.This work takes advantage of the in vitro activity of the CRISPR-Cas9 system to obtain intact,large DNA fragments from bacterial chromosomes,which can be subsequently ligated with cloning vector through Gibson Assembly.This technique is named Cas9-Assisted Targeting of CHromosome segments(CATCH).In this study,different pairs of sgRNAs were designed to target specific regions in the genome of Escherichia coli.The results of pulse field gel electrophoresis(PFGE)showed that the target fragments of different lengths(50-200 kb)could be separated efficiently and the cloning positive rates of large DNA fragments(50-100 kb)could be as high as 20-60%(the longer the target fragment,the lower the cloning efficiency).Target fragments longer than 100 kb are usually hard to be obtained directly.The orientations of the sgRNAs and the target locations in the chromosome have no significant effect on the cloning efficiency.Using the CATCH cloning method,large gene clusters have been cloned from Escherichia coli,Bacillus subtilis,Streptomyces venezuelae and Streptomyce aureofaciens.CATCH cloning,for its low cost and high efficiency,may facilitate the cloning of large gene clusters to produce high added-value biomolecules,and promises for many future applications in synthetic biology and genome sequencing.