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Fig. 2 | Biotechnology for Biofuels

Fig. 2

From: Development and characterization of a CRISPR/Cas9n-based multiplex genome editing system for Bacillus subtilis

Fig. 2

The CRISPR/Cas-mediated system for iterative genome editing. a The components and procedure of the CRISPR/Cas based system. Two plasmids respectively harboring gRNA and donor DNA are introduced into the cells, after which expression of Cas protein and homologous recombination are implemented. Gene modifications were introduced, allowing the cells to escape CRISPR mediated cleavage by abolishing the protospacer or the PAM sequences. When induced by mannose, gRNA targeting the rep60 gene is expressed to eliminate the donor DNA plasmid. The gRNA plasmid was eliminated by inhibiting the replication of its thermo-sensitive replicon at increased temperature. b Step by step diagram of the iterative genome editing procedure. The time required for each step is shown in red

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