Shahid Bahonar University of Kerman and Iranian Biotechnology Society Agricultural Biotechnology Journal 2228-6705 17 3 2025 08 23 The effect of the intrinsic resistance of Shigella flexneri 2a to spectinomycin on the efficiency of the CRISPR/Cas9 system The effect of the intrinsic resistance of Shigella flexneri 2a to spectinomycin on the efficiency of the CRISPR/Cas9 system 177 200 5003 10.22103/jab.2025.25382.1717 FA Elaheh Kazemipour Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran Hosseinali Sasan Associate Professor, Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran Mohammadreza Mohammadabadi Professor, Department of Animal Science, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran Journal Article 2025 06 03 Objective<br />Targeted gene editing in pathogenic bacteria such as Shigella flexneri 2a using the advanced CRISPR/Cas9 system is a significant step in developing genetic tools and novel therapeutic strategies. However, the success of this process heavily relies on the precise selection of recombinant clones, typically through suitable antibiotic markers. In the current study, we evaluated the efficacy of antibiotic markers (spectinomycin and kanamycin) in pTargetF:gRNA-pic and pEcCas plasmids in the clinical strain of Shigella flexneri 2a.<br />Materials and methods<br />First, the target region of the pic gene (a crucial virulence factor in Shigella) was identified. Based on this sequence, gRNA guide sequences, specific primers, and homologous recombination arms (HDR arms) were meticulously designed. Subsequently, targeted sgRNA cloning to the pic gene in the pTargetF plasmid was performed, and the plasmid’s structural integrity was confirmed using PCR and sequencing methods. Next, the successful introduction of the pEcCas plasmid into Shigella flexneri 2a cells was achieved via heat shock. Following this, the recombinant pTargetF plasmid, along with HDR arms, was transferred into bacteria containing the pEcCas plasmid using electroporation. Initial selection of transformed clones during the cloning stage on LB media containing kanamycin was successful. To investigate potential antibiotic alternatives, the wild-type Shigella flexneri 2a strain was also cultured on media containing chloramphenicol, ampicillin, and streptomycin.<br />Results<br />After electroporation and culturing of recombinant and wild-type (control) strains on media containing spectinomycin and kanamycin, the entire surface of the plates was covered with uniform bacterial growth, making the differentiation of recombinant clones impossible. This inability to differentiate resistant clones clearly indicates the intrinsic or widespread resistance of the Shigella flexneri 2a strain to spectinomycin. Furthermore, indiscriminate and widespread bacterial growth was observed in the wild-type strain when cultured on media containing chloramphenicol, ampicillin, and streptomycin, confirming the intrinsic resistance of this strain to several common antibiotics.<br />Conclusions<br />These findings highlight that, in this particular strain, the use of spectinomycin as a selection marker is inappropriate. For successful editing of pathogenic genes in similarly resistant strains, it is recommended to employ alternative selection markers to which the bacterium is sensitive (such as kanamycin), or to utilize markerless systems. Moreover, conducting preliminary antibiotic susceptibility testing (even in a simplified form) is essential prior to any molecular cloning or genome editing procedures. Objective<br />Targeted gene editing in pathogenic bacteria such as Shigella flexneri 2a using the advanced CRISPR/Cas9 system is a significant step in developing genetic tools and novel therapeutic strategies. However, the success of this process heavily relies on the precise selection of recombinant clones, typically through suitable antibiotic markers. In the current study, we evaluated the efficacy of antibiotic markers (spectinomycin and kanamycin) in pTargetF:gRNA-pic and pEcCas plasmids in the clinical strain of Shigella flexneri 2a.<br />Materials and methods<br />First, the target region of the pic gene (a crucial virulence factor in Shigella) was identified. Based on this sequence, gRNA guide sequences, specific primers, and homologous recombination arms (HDR arms) were meticulously designed. Subsequently, targeted sgRNA cloning to the pic gene in the pTargetF plasmid was performed, and the plasmid’s structural integrity was confirmed using PCR and sequencing methods. Next, the successful introduction of the pEcCas plasmid into Shigella flexneri 2a cells was achieved via heat shock. Following this, the recombinant pTargetF plasmid, along with HDR arms, was transferred into bacteria containing the pEcCas plasmid using electroporation. Initial selection of transformed clones during the cloning stage on LB media containing kanamycin was successful. To investigate potential antibiotic alternatives, the wild-type Shigella flexneri 2a strain was also cultured on media containing chloramphenicol, ampicillin, and streptomycin.<br />Results<br />After electroporation and culturing of recombinant and wild-type (control) strains on media containing spectinomycin and kanamycin, the entire surface of the plates was covered with uniform bacterial growth, making the differentiation of recombinant clones impossible. This inability to differentiate resistant clones clearly indicates the intrinsic or widespread resistance of the Shigella flexneri 2a strain to spectinomycin. Furthermore, indiscriminate and widespread bacterial growth was observed in the wild-type strain when cultured on media containing chloramphenicol, ampicillin, and streptomycin, confirming the intrinsic resistance of this strain to several common antibiotics.<br />Conclusions<br />These findings highlight that, in this particular strain, the use of spectinomycin as a selection marker is inappropriate. For successful editing of pathogenic genes in similarly resistant strains, it is recommended to employ alternative selection markers to which the bacterium is sensitive (such as kanamycin), or to utilize markerless systems. Moreover, conducting preliminary antibiotic susceptibility testing (even in a simplified form) is essential prior to any molecular cloning or genome editing procedures. https://jab.uk.ac.ir/article_5003_4edcb8c42621b1fdb4cb3ae236d19abe.pdf