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. 2019 Feb 22;13(1):210–221. doi: 10.1111/1751-7915.13382

Figure 1.

Figure 1

A single‐plasmid‐based CRISPRi system in Pseudomonas putida KT2440. (A) The CRISPRi expression plasmid, pSECRi. The CRISPRi system consists of an l‐rhamnose‐inducible SpdCas9 protein and a designed sgRNA chimera encompassing a J23119 constitutive promoter in a low copy (RK2 origin) pSEVA221 plasmid. (B) The architecture of customized sgRNAs with the constitutive J23119 promoter. The sgRNA consists of base‐pairing nucleotides for specific target DNA sequence binding (N20 spacer sequence, 20 bp), a 42 bp SpdCas9‐binding handle and 40 bp Streptococcus pyogenes terminator. A plasmid‐borne green fluorescent protein (GFP) and endogenous GlpR were chosen as target sites. (C) Schematic representation of CRISPRi targeting the gene of interest. A 20 bp sgRNA spacer sequence targeting the gene was designed according to the following criteria; (i) the 3′ end of the target region should contain a proto‐spacer adjacent motif (PAM) sequence (5′‐NGG‐3′), (ii) the sgRNA should bind to the non‐template DNA strand proximal to the ATG translational start codon of the target gene for high repression efficiency.