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Fig. 4 | Microbial Cell Factories

Fig. 4

From: Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system

Fig. 4

The two-step strategy of single nucleotide mutation for PAM unavailability sites using CRISPR–Cas9 system. a The N20 sequence from pSEVA-NicA20 was used as Cas9 cutting site in KT2440, and the Gln139 in nicC gene was targeted as mutation site by mutating CAA to CTA. Yellow star means the target nucleotide. b After the first step genome editing, an added artificial N20 sequence (A20 sequence) and single nucleotide mutation (At Gln139 by mutating CAA to CTA) in pSEVA-NicA20 homologous arm were inserted into KT2440 genome. c Through the first step, single nucleotide mutation was applied into target locus. Then, we attempted to eliminate A20 sequence by curing pSEVA-NicA20 and using pSEVA-NicA21 for next genome editing step. In pSEVA-NicA21, the target site was changed from N20 sequence to A20 sequence. In the homologous arm, A20 sequence was eliminated and single nucleotide mutation was retained

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