Tae buffer

The presence of virulence genes eaeA, hlyA, stx₁, and stx₂ was verified by multiplex PCR according to methods described by Paton and Paton (1998 (link)). Template DNA was prepared by the boiling extraction method described above. DNA lysate (1 μl) from each isolate was amplified with TopTaq DNA Polymerase (Qiagen) in 25 μl reaction mixtures containing 1X Buffer Solution, 1X Coral Dye, 50 μM dNTP's (Invitrogen), 0.625 U/rxn TopTaq DNA Polymerase, 5 μl Q-solution, and 1 μM of eae and hlyA primers, and 0.4 μM of stx₁ and stx₂ primers. The PCR reaction was carried out under the following conditions in a thermal cycler (Biorad): 95°C for 3 min, followed by 35 cycles each consisting of a denaturation step at 95°C for 1 min; an annealing step at 65°C for 2 min for the first 10 cycles, decrementing 1°C per cycle to 60°C by cycle 15; and an elongation step at 72°C for 1.5 min, incrementing to 2.5 min from cycles 25–35; and a final extension step at 72°C for 5 min. PCR products were visualized in SYBR^®; Safe (Invitrogen) stained 2% agarose gels following electrophoresis using 1X TAE buffer (BioRad) at 80 V for 45 min. The clinical E. coli O157:H7 isolate (Strain ID, BC Centre for Disease Control, stx₁, stx₂, eaeA, hlyA positive)was used to verify PCR assay performance.

All chemicals were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands) unless otherwise specified. spCas9 and spCas9-GFP protein were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands). Modified sgRNA sequences were obtained from Sigma-Aldrich (Haverhill, UK, sequences given in Table S1) and stored in DNAse and RNAse-free water from IDT (Integrated DNA Technologies Inc. Leuven Belgium). DNA oligonucleotides (Table S2), HDR template (Table S3), ATTO-550-labeled tracRNA, non-labeled crRNA (Table S4) and Alexa-fluor-647-labeled HDR template (Table S3) were purchased from IDT. Lipofectamine CRISPRMAX and Lipofectamine3000™ were obtained from Thermo Fisher Scientific (Waltham, MA, USA, ABD). T7 endonuclease and Q5 high-fidelity 2X master mix were purchased from New England Biolabs (Ipswich, MA, USA). The 6His-CM18-PTD4 [19 (link)], S10 [23 (link)], L17E [33 (link)] and LAH5: KKALLALALHHLAHLAHHLALALKKA peptides were purchased from Synpeptide (Shanghai, China). DNA extraction and PCR purification kits were purchased from QIAGEN Benelux B.V. (Venlo, The Netherlands). TAE buffer was purchased from Biorad (Lunteren, The Netherlands).

A T7E1 assay was performed to detect the insertion/deletion (indel) frequency after gene editing [36 (link)]. Genomic DNA was extracted from the cells 48 h after transfection with LAH5 peptide/RNP nanocomplexes using the Qiagen DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s instructions. PCR was performed using the primers designed for the sgRNA target locus (Table S2) using Q5^® Hot Start High-Fidelity 2X Master Mix (New England Biolabs, MA, USA). Afterward, PCR products were purified using the QIAquick PCR Purification kit (Qiagen GmbH, Hilden, Germany). PCR products were denatured at 95 °C for 10 min in the presence of NEBuffer 2 (New England Biolabs), and they were annealed by slowly lowering the temperature (95–85 °C at 2 °C per second and 85–25 °C at 1 °C per second). Subsequently, re-annealed PCR products were incubated with 5U T7E1 enzyme (New England Biolabs, MA, USA) at 37 °C for 18 min to cut heteroduplexes. DNA products were run on a 2% agarose gel in TAE buffer with a pH of 8.3 (Biorad). The indel frequency was calculated by determining the intensities of cleaved and uncleaved bands based on a densitometry analysis using ImageJ version 1.53t.