e coli strains neb5 alpha f i q  (New England Biolabs)

Journal: Nature Communications

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

doi: 10.1038/s41467-020-16731-6

Figure Lengend Snippet: a A schematic diagram of transformation of CRISPR-Cas13a and CRISPR-Cas9 with targeting bla IMP-1 into bla IMP-1 -expressing E. coli STBL3. b E. coli STBL3 expressing bla IMP-1 from a plasmid (plasmid-borne bla IMP-1 ) and chromosome (chromosome-borne bla IMP-1 ) were prepared, and transformed with CRISPR-Cas13a or CRISPR-Cas9, both with spacer targeting bla IMP-1 or no spacer (nontargeting). The resulting transformants were plated on an LB plate containing kanamycin (Km) to test sequence-specific bacterial killing by CRISPR-Cas13a and CRISPR-Cas9. Km was used to maintain the plasmids. c The number of bacteria on the plate obtained in the experiment of b was counted. The statistical significance was determined by two-sided Student’s t -test. Each bar represents the mean with standard deviation ( n = 3). d A schematic diagram of the experiment to test CRISPR-Cas13a-dependent cell growth inhibition. Anhydrotetracycline (aTc)-inducible bla IMP-1 - or rfp -expression plasmid (pKLC56) was co-transformed with pKLC21 plasmid expressing CRISPR-Cas13a with spacer targeting bla IMP-1 or rfp into E. coli STBL3. The resulting transformants were then cultured and the aTc induction in the presence of the antibiotics Km and Cm (Km and Cm for maintaining plasmids) was carried out at the indicated time points. Thereafter, the OD values were measured every hour. e Growth curves were plotted. Each line of the growth curves represents the mean with standard deviation. f The number of viable cells was counted to calculate the ratio of cell death caused by bla IMP-1 -targeting CRISPR-Cas13a. The bacterial culture prepared in d was diluted to 1/100, then aTc was added to induce bla IMP-1 . Km and Cm were also added to maintain the CRISPR-Cas13a and the aTc-inducible bla IMP-1 plasmids. Thereafter, the bacterial cultures were sampled at the indicated time points, and the number of surviving bacteria was counted on fresh LB plates. Each bar represents the mean with standard deviation of four biological replicates. p -values were determined by two-sided Student’s t -test. Source data are available in the Source Data file.

Article Snippet: First, we transformed E. coli strains NEB5-alpha F′ I q (New England Biolabs, US) and MC1061 with plasmid pKD46 that carries ARA-inducible Red recombination system .

Techniques: Transformation Assay, CRISPR, Expressing, Plasmid Preparation, Sequencing, Standard Deviation, Inhibition, Cell Culture

Journal: Nature Communications

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

doi: 10.1038/s41467-020-16731-6

Figure Lengend Snippet: a Graphical concept model depicting the mode of action of CapsidCas13a, with bla IMP-1 -targeting CRISPR-Cas13a packaged into E. coli M13 phage capsid (EC-CapsidCas13a- bla IMP-1 ) for delivery into host bacterial cells during the normal course of viral infection. In the presence of the target gene ( E. coli carrying bla IMP-1 ), CRISPR-Cas13a will be activated and the subsequent non-sequence-specific RNase activity will result in host cell death. In the absence of the target gene ( E. coli without bla IMP-1 ), CRISPR-Cas13a will not be activated and there will be no subsequent cell death. b – d Spot assay with generated CapsidCas13a(s) on bacterial lawn of E. coli NEB5α F′ I q to test bactericidal activity. A clear lysis zone of the spotted area represents bacterial killing. NEB5α F′ I q carrying bla IMP-1 expression vector and empty vector (control) were infected with tenfold serial dilutions of EC-CapsidCas13a- bla IMP-1 in the presence or absence of L-arabinose for inducing bla IMP-1 expression ( b ); CapsidCas13a(s) programmed to target different carbapenem resistance genes ( bla IMP-1 , bla OXA-48 , bla VIM-2 , bla NDM-1 , and bla KPC-2 ) and colistin resistance genes ( mcr-1 and mcr-2 ) were spotted on a series of NEB5α F′ I q strains expressing each of the resistance genes ( c ); comparison of bactericidal activity between CRISPR-Cas9 and CRISPR-Cas13a. Nontargeting and bla NDM-1 -targeting CapsidCas13a and CapsidCas9, in tenfold serial dilutions, were spotted on lawn of NEB5α F′ I q with or without expression of bla NDM-1 on plasmid or chromosome ( d ). All assays were replicated three times.

Article Snippet: First, we transformed E. coli strains NEB5-alpha F′ I q (New England Biolabs, US) and MC1061 with plasmid pKD46 that carries ARA-inducible Red recombination system .

Techniques: CRISPR, Infection, Sequencing, Activity Assay, Spot Test, Generated, Lysis, Expressing, Plasmid Preparation, Bla VIM Assay

Journal: Nature Communications

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

doi: 10.1038/s41467-020-16731-6

Figure Lengend Snippet: Programmed CapsidCas13a altered the composition of bacterial population. A mixed cell population was prepared by mixing E. coli NEB5α F′ I q (control) with equal numbers of NEB5α F′ I q expressing bla IMP-1 and mcr-2 , respectively. The cell mixtures were then independently treated with bla IMP-1 -targeting, mcr-2 -targeting, and nontargeting CapsidCas13a. Note that each AMR gene-targeting CapsidCas13a reduced the corresponding target cell population. The percentage represents the mean of three biological replicates. Source data are available in the Source Data file.

Article Snippet: First, we transformed E. coli strains NEB5-alpha F′ I q (New England Biolabs, US) and MC1061 with plasmid pKD46 that carries ARA-inducible Red recombination system .

Techniques: Expressing

Journal: Nature Communications

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

doi: 10.1038/s41467-020-16731-6

Figure Lengend Snippet: Examination of the therapeutic effect of EC-CapsidCas13a- bla IMP-1 using a Galleria mellonella infection model. Administration of EC-CapsidCas13a- bla IMP-1 (MOI 100) into G. mellonella larvae infected with R10-61 (carbapenem-resistant clinical isolates of E. coli carrying bla IMP-1 ) significantly improved host survival compared to controls, EC-CapsidCas13a-nontargeting ( p = 0.044), and phosphate-buffered saline (PBS; p = 0.0016). The p -value between two groups infected with R10-61, and treated with EC-CapsidCas13a-nontargeting and PBS was 0.30. The p -values are calculated by log-rank test. The results are presented as the aggregate values of three independent experiments performed using ten larvae per group. Source data are available in the Source Data file.

Article Snippet: First, we transformed E. coli strains NEB5-alpha F′ I q (New England Biolabs, US) and MC1061 with plasmid pKD46 that carries ARA-inducible Red recombination system .

Techniques: Infection

Journal: Nature Communications

Article Title: Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

doi: 10.1038/s41467-020-16731-6

Figure Lengend Snippet: a A schematic illustration of generation of PICI-based EC-CapsidCas13a targeting bla IMP-1 . Mitomycin C induction promotes the packaging of PICImid carrying a CRISPR-Cas13a system and kanamycin (Km) resistance gene into the capsid of helper phage Φ80. b – d Bactericidal activity test of PICI-based EC-CapsidCas13a::KanR- bla IMP-1 (in tenfold serial dilutions) against E. coli MC1061 with or without the expression of target gene was carried out on LB agar plates ( b ); and the test results were judged by observing bacterial growth on LB bottom agar plates supplemented with Km ( c ), or observation of cell lysis on drug-free LB bottom agar plates ( d ); noting that the former assay had an enhanced sensitivity by about three orders of magnitude against the bacteria carrying target gene. e – j The PICI-based EC-CapsidCas13a(s) were applicable to detect various carbapenem resistance genes ( bla IMP-1 , bla OXA-48 , and bla VIM-2 ) regardless of their location on either the plasmid or chromosome ( e , f ), whereas EC-CapsidCas9 could detect genes located on the chromosome but not on the plasmid ( g ); and the PICI-based EC-CapsidCas13a(s) also effectively detected toxin-encoded genes ( h ), differentiated different genes located on the same plasmid ( i ), and were also applicable to clinical isolates ( j , left panel) as being verified by PCR ( j , right panel). k SA-CapsidCas13a::TetR- mecA generated by packaging mecA -targeting CRISPR-Cas13a into capsid of S. aureus phage 80α exhibited mecA -specific bactericidal activity against MRSA, but not S. aureus strains deficient in mecA . All assays were replicated three times. Uncropped images of the gels are available in the Supplementary Information.

Article Snippet: First, we transformed E. coli strains NEB5-alpha F′ I q (New England Biolabs, US) and MC1061 with plasmid pKD46 that carries ARA-inducible Red recombination system .

Techniques: CRISPR, Activity Assay, Expressing, Lysis, Bla VIM Assay, Plasmid Preparation, Generated