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Fig. 2 | BMC Microbiology

Fig. 2

From: Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni

Fig. 2

Design of sgRNA target sequences for integration into an sgRNA scaffold to combine with dcas9 to form a CRISPRi homologous recombination vector. An sgRNA target generator script was used to create a list of sgRNA targets using the astA gene sequence (A). Targets were screened for off-target effects using the genome sequences of 81–176 and M1Cam. Target sequences were then screened to detect any hairpin formation with the sgRNA scaffold backbone and selected for a GC content between 30–60%. The final list of 12 sgRNA target sequences are shown in (B), with their relative locations along the astA gene shown in (C). To express sgRNAs, a scaffold construct with a pPorA promoter and an sgRNA backbone was synthesized (D) and inserted into pRC1 in the opposite orientation to dcas9 driven by pCat (E)

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