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. 2018 Jul 17;9:1569. doi: 10.3389/fmicb.2018.01569

Figure 2.

Figure 2

CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994. An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic DNA. Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) PCR evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.