Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus

• DOI: 10.1186/s12934-017-0758-x
• Publication Date: 2017-08-16T08:26:11Z
• Authors: Ying-jie Yang; Ye Wang; Zhi-feng Li; Ya Gong; Peng Zhang; Wen-chao Hu; Duo-hong Sheng; Yue-zhong Li
• Keywords: Myxococcus xanthus; 0303 health sciences; Base Sequence; Research; RNA, Guide, CRISPR-Cas Systems; Spacer sequence; Microbiology; QR1-502; 03 medical and health sciences; RNA, Transfer; Genes, Bacterial; Multigene Family; Deletion of large genome fragments; On-target cleavage efficiency; Free energy; CRISPR-Cas Systems; CRISPR/Cas9; Biosynthetic gene clusters for secondary metabolites; Plasmids; Sequence Deletion

The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer.In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA-sgRNA-tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency.By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content.