Tracrrna

MCF10A cells were harvested with 0.25% trypsin/EDTA centrifugation at 500 rpm, 4 °C. The cells were then resuspended in 37 °C Opti-MEM Reduced Serum Medium (Gibco).
Both crRNA (Dharmacon) and tracrRNA (Dharmacon Edit-R CRISPR-Cas9 Synthetic tracrRNA-U-002005-20) stock solutions (200 μM each) were prepared by adding the appropriate volume of RNase-free water. Then, the 100 μM solution of crRNA:tracrRNA duplex was created by combining 200 μM stock solutions in a 1:1 ratio. The solution was gently mixed for 10 min and stored at −20 °C for future experiments. The project also utilised HS17 crRNA (5′-CAGACAGGCCCAGATTGAGG-3′) from Berg et al. [16 (link)]. The Cas9 ribonucleoprotein (RNP) complex was created by combining 1.5 μM Cas9 protein and 3 μM RNA final concentration and kept on ice until being mixed with the resuspended cells in Opti-MEM medium.
Electroporation of the Cas9:RNA complex was achieved using Gene Pulser/MicroPulser Electroporation Cuvettes with 0.2 cm gap cuvettes at in the Gene Pulser Xcell Electroporation System and an exponential pulse at 300 V and 300 μF. Complete cell culture media was then added to the Opti-MEM in a 1:1 ratio. Electroporated MCF10A cells were seeded on to coverslips pre-coated with 50 μg/mL poly-D-lysine (Sigma, St. Louis, MO, USA) and incubated at 37 °C in 5% CO₂.

GLI2 and SPP1 knockouts in KP4 cells were generated using the RNP-electroporation method as previously described (Liang et al., 2015 (link)). One million KP4 cells were used per electroporation using the Amaxa 4D Nucleofector kit (V4XC-9064, Lonza). Guide RNA and Cas9 complexes were formed using 160 μM crRNA annealed to 160 μM tracrRNA (Dharmacon) and incubated with 40 μM Cas9 protein (purchased from University of California, Berkeley). Cutting efficiency was assessed 48 hr post-electroporation using PCR and sanger sequencing. GLI2 and SPP1 knockout was confirmed using quantitative RT-PCR, immunoblotting and ELISA after clonal expansion of single cells.
Guide RNA sequences 5’−3’:
GLI2 exon 2 – TTTGGCTTCTTGCTTCTCGGSPP1 exon 2 – GTATGGCACAGGTGATGCCTPCR primer sequences 5’−3’:
GLI2 exon 2 FW – GTGAAGGAGTGAGCGAACATGCGLI2 exon 2 RV – TCTTCGCCCTCCATAAACCCAGSPP1 exon 2 FW – GCAAAATTTCCCTTTCCCTTGCCSPP1 exon 2 RV – ACTGTGCTTGGTACTGGCCTAG

Bacterial strains were cultured for 12–16 h in LB broth supplemented with ampicillin (30 µg/mL) and plasmids were extracted using a NucleoBond Xtra Midi Kit (Macherey-Nagel GmbH, Düren, Germany), according to the manufacturer’s instructions. The eluted DNA was precipitated with 70% isopropanol and reconstituted with TE buffer (Sigma-Aldrich, St. Louis, MO, USA). The plasmids were treated by either Cas9 (PNA Bio, Thousand Oaks, CA, USA) with tracrRNA (Dharmacon Inc., Lafayette, CO, USA) and crRNA (Dharmacon Inc., Lafayette, CO, USA), targeting specific antibiotic resistance genes (bla_CTX-M-14 or bla_CTX-M-15), or just the tracrRNA, without crRNA as control, as shown in the proof-of-concept experiments in Section 3.4. The sequences of the RNA targeting the bla_CTX-M-14 and bla_CTX-M-15 genes were 5′AGAGAGCCGCCGCGATGTGC3′ and 5′CCGTCGCGATGTATTAGCGT3′, respectively. After the Cas9 reaction, the plasmids were mixed with λ-DNA as internal size reference and then fluorescently stained with YOYO-1 and netropsin. The molar ratio of YOYO-1 to DNA was 1:2, while that of netropsin to YOYO-1 was 70:1; the staining was performed in 0.5x TBE buffer for 30 min at 50 °C. The samples were then diluted to 0.05x TBE to optimize the DNA stretching in the nanochannels [13 (link)]. Β-mercaptoethanol was added at 3% (v/v) to suppress photonicking and photobleaching.

CRISPR RNAs (crRNAs) targeting CPSF6 (UUCAGAUCCAACACCAACAA), NTC (GAUACGUCGGUACCGGACCG), and trans-activating crRNA (tracrRNA; proprietary sequence), as well as Cas9-NLS protein, were purchased from Dharmacon. Details of crRNP complex formation were as described previously (81 (link)). Briefly, to form 20 μM crRNP complex, 2.5 μl each of 160 μM tracrRNA and 160 μM crRNA were mixed and incubated at 37°C for 30 min, followed by addition of 5 μl of 40 μM Cas9-NLS and incubation at 37°C for 15 min.
Jurkat T cells (1 × 10⁶) resuspended with nucleofection buffer containing the required supplement from the Cell Line Nucleofector kit V (Lonza) were mixed with 20 μM crRNP and electroporated by nucleofector I according to the manufacturer’s instructions. After electroporation, cells were plated in 6-well plates containing 2 ml prewarmed RPMI and incubated for minimally 3 days to allow recovery prior to immunoblotting and virus infection.

The first step of the CRISPR-Cas9 reaction was the creation of a sequence-specific guide-RNA (gRNA). The 20-nucleotide CRISPR-RNA (crRNA) corresponding to the bla_NDM, bla_KPC and bla_CTX-M group 1 genes and a decoy crRNA (control, with no potential target sites on bacterial plasmids) were designed and synthesized via Dharmacon (Horizon Discovery Ltd.). The target sequences of crRNAs for different genes were:
bla_NDM: 5′ CCGCTGCATTGATGCTGAGC 3′
bla_KPC: 5′ CAACCACCGCATCCGCGCGG 3′
bla_CTX-M: 5′ CCGTCGCGATGTATTAGCGT 3′
decoy: 5′ GGTCCTTGTAACCATCGGTG 3′
The gRNA was created by mixing 0.5 nmol crRNA and 0.5 nmol trans-activating CRISPR RNA (tracrRNA, Dharmacon) in 1 × CutSmart^® Buffer (50 mM Potassium Acetate, 20 mM Tris–acetate, 10 mM Magnesium Acetate, 100 µg/ml BSA, pH 7.9 at 25 °C, New England Biolabs) into a 0.5 ml Eppendorf tube. The final volume was adjusted with milli-Q water to 15 µl. The mixture was incubated for 30 min at 4 °C. Next, 10 µM (0.05 nmol) gRNA, 600 ng Cas9 protein (Sigma-Aldrich), and 1 × CutSmart^® Buffer were added to a new Eppendorf tube and pre-incubated for 10 min at room temperature (25 °C). Finally, 60 ng DNA of plasmid sample and milli-Q water were added to the mixture to a final volume of 15 µl before incubation for 15 min at 37 °C.