Plasmid pET-Cas9-NLS-6xHis responsible for encoding SpCas9 (wild type Cas9 derived from S. pyogenes) containing nuclear localisation signal (NLS) sequence and 6x-Histidine tag fused at the C-terminal, was obtained from Addgene (#62933). Plasmid pET302-6His-dCas9-Halo, the equivalent nuclease-deficient Cas9 was obtained from Addgene (#72269). All plasmids were verified by DNA sequencing.
Protein expression and purification in Escherichia coli
Recombinant constructs were expressed in E.coli BL21 DE3 cells (Invitrogen) in Luria Bertani media. Proteins were purified by affinity chromatography (HisTrap FF, GE Healthcare). The purest fractions were then further purified through a Superdex 200 16/600 column (GE Healthcare).
MCF10a (ATCC CRL-10317) cells were cultured at 37°C and 5% CO2, in 50% Gibco MEM Alpha medium with GlutaMAX (no nucleosides) and 50% Ham’s F-12 nutrient mixture, supplemented with 5% Fetal Bovine Serum (Gibco), 5% horse serum, penicillin-streptomycin mix diluted to 100 units mL-1, 50μg Cholera toxin, 5μg insulin, 20ngmL-1 human EGF and 0.5μgmL-1 hydrocortisone, 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco).
Cisplatin cis-diammineplatinum(II) dichloride was resuspended in a 0.9% NaCl solution to a concentration of 3.3 mM, following manufacturer’s instructions and used at a concentration of 25 μM.
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 (200μM each) prepared by adding the appropriate volume of RNase-free water. Then, the 100μM solution of crRNA:tracrRNA duplex was created by combining 200μM 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. The Cas9 ribonucleoprotein (RNP) complex was created by combining 1.5μM Cas9 protein and 3μM RNA final concentration and kept in ice until mixed with the resuspended cells in Opti-MEM medium.
Electroporation of the Cas9:RNA complex was achieved using a Gene Pulser/MicroPulser Electroporation Cuvettes with 0.2 cm gap cuvettes at in Gene Pulser Xcell Electroporation System and an exponential pulse at 300V and 300μF. Complete cell culture media was then added to the Opti-MEM in 1:1 ratio. Electroporated MCF10A cells were seeded on to coverslips pre-coated with 50 μg/ml poly-D-lysine (Sigma) and incubated at 37°C in 5% CO2.
MCF10a cells were fixed for 15 min at room temperature in 4% (w/v) paraformaldehyde (PFA) in TBS and residual PFA was quenched for 15 min with 50 mM ammonium chloride in TBS. For staining with Halo-TMR (Promega G8252), 10 nM ligand was added for 15 min and washed three times in warm cell culture media before fixation. All subsequent steps were performed at room temperature. Cells were permeabilised and simultaneously blocked for 15 min with 0.1 % (v/v) Triton X-100 and 2 % (w/v) BSA in TBS. Cells were then immuno-stained by 1 h incubation with the indicated primary and subsequently the appropriate fluorophore-conjugated secondary antibody (details below), both diluted in 2 % (w/v) BSA in TBS. The following antibodies were used at the indicated dilutions: Rabbit anti-Cas9 (1:200, Abcam ab204448), Mouse anti-phospho-H2A.X (1:500 Sigma 05-636), Donkey anti-rabbit Alexa Fluor 488-conjugated (1:250, Abcam Ab181346), Donkey anti-mouse Alexa Fluor 647-conjugated (1:250, Abcam Ab150103). For Hoechst staining, coverslips were washed three times in TBS followed by Hoechst 33342 solution for 10 mins at RT in the dark. The coverslips were then washed three times in TBS and once with ddH2O. Coverslips were mounted on microscope slides with Mowiol (10% (w/v) Mowiol 4-88, 25% (w/v) glycerol, 0.2 M Tris-HCl, pH 8.5), supplemented with 2.5% (w/v) of the anti-fading reagent DABCO (Sigma).
Widefield Fluorescent Imaging
Widefield immunofluorescence images were obtained using CytoVision Olympus BX61 microscope equipped with Olympus UPlanFL 100 X/1.30 NA oil objective lens and Hamamatsu Photonics Digital CCD Camera ORCA-R2 C10600-10B-H.
Cells were visualised using the ZEISS LSM 880 confocal microscope. This was equipped with a Plan-Apochromat 63x/1.4 NA oil immersion lens (Carl Zeiss, 420782-9900-000). The built-in dichroic mirrors (Carl Zeiss, MBS-405, MBS-488 and MBS-561) were used to reflect the excitation laser beams on to cell samples. The emission spectral bands for fluorescence collection were 410 nm-524 nm (Hoechst, Thermo Fisher), 493 nm-578 nm (AlexaFluor 488, Thermo Fisher) and 650 nm-697 nm (AlexaFluor 647, Thermo Fisher). The detectors consisted of two multi anode photomultiplier tubes (MA-PMT) and 1 gallium arsenide phosphide (GaAsP) detector. The green channel was imaged using GaAsP detector, while the blue and red channels were imaged using MA-PMTs. ZEN software (Carl Zeiss, ZEN 2.3) was used to acquire and render the confocal images.
For single particle detection, we used Fiji 21 to split the RGB channels and convert the γH2AX channel to a binary image. The Despeckle function was used to remove background noise from the images. The area of the nucleus was selected by creating a mask from the Hoechst channel. The Analyze Particles function used to calculate the number of foci in a given nucleus. For fluorescence intensity measurements. The binary image for single particle detection was used to create a mask and then the mean pixel intensity was calculated for each particle.
Cells were seeded on pre-cleaned No. 1.5, 25-mm round glass coverslips, placed in 6-well cell culture dishes. Glass coverslips were cleaned by incubating them for 3 hours, in etch solution, made of 5:1:1 ratio of H2O : H2O2 (50 wt. % in H2O, stabilized, Fisher Scientific) : NH4OH (ACS reagent, 28-30% NH3 basis, Sigma), placed in a 70°C water bath. Cleaned coverslips were repeatedly washed in filtered water and then ethanol, dried and used for cell seeding. Cells were fixed in pre-warmed 4% (w/v) Paraformaldehyde (PFA) in PBS and residual PFA was quenched for 15 min with 50 mM ammonium chloride in PBS. Immunofluorescence was performed in filtered sterilised TBS. Cells were permeabilized and simultaneously blocked for 30 min with 3% (w/v) BSA in TBS, supplemented with 0.1 % (v/v) Triton X-100. Permeabilized cells were incubated for 1h with the primary antibody and subsequently the appropriate fluorophore-conjugated secondary antibody, at the desired dilution in 3% (w/v) BSA, 0.1% (v/v) Triton X-100 in TBS. The antibody dilutions used were the same as for the normal IF protocol (see above), except from the secondary antibodies which were used at 1:250 dilution. Following incubation with both primary and secondary antibodies, cells were washed 3 times, for 10 min per wash, with 0.2% (w/v) BSA, 0.05% (v/v) Triton X-100 in TBS. Cells were further washed in PBS and fixed for a second time with pre-warmed 4% (w/v) PFA in PBS for 10 min. Cells were washed in PBS and stored at 4 °C, in the dark, in 0.02% NaN3 in PBS, before proceeding to STORM imaging.
Before imaging, coverslips were assembled into the Attofluor® cell chambers (Invitrogen). Imaging was performed in freshly made STORM buffer consisting of 10 % (w/v) glucose, 10 mM NaCl, 50 mM Tris - pH 8.0, supplemented with 0.1 % (v/v) 2-mercaptoethanol and 0.1 % (v/v) pre-made GLOX solution which was stored at 4 0C for up to a week (5.6 % (w/v) glucose oxidase and 3.4 mg/ml catalase in 50 mM NaCl, 10 mM Tris - pH 8.0). All chemicals were purchased from Sigma. Imaging was undertaken using the Zeiss Elyra PS.1 system. Illumination was from a HR Diode 642 nm (100 mW) lasers where power density on the sample was 7-12 kW/cm2.
Imaging was performed under highly inclined and laminated optical (HILO) illumination to reduce the background fluorescence with a 100x/ 1.46NA oil immersion objective lens (Zeiss alpha Plan-Apochromat) with a BP 420-480/BP495-550/LP 650 filter. The final image was projected on an Andor iXon EMCCD camera with 25 msec exposure for 20000 frames. The focal plane was locked using Definite Focus function in the microscope during image acquisition.
The images were processed through our STORM analysis pipeline using the Zeiss Zen Black software. Single molecule detection and localisation was performed using a 9-pixel mask with a signal to noise ratio of 6 in the “Peak finder” settings while applying the “Account for overlap” function. This function allows multi-object fitting to localise molecules within a dense environment. Molecules were then localised by fitting to a 2D Gaussian.
The render was then subjected to model-based cross-correlation lateral drift correction and detection grouping to remove detections within multiple frames. Typical localisation precision was 20 nm for Alexa-Fluor 647. The final render was then generated at 10 nm/pixel and displayed in Gauss mode where each localisation is presented as a 2D gaussian with a standard deviation based on its precision. The localisation table was exported as a csv for import in to Clus-DoC.
The single molecule positions were exported from Zeiss Zen Black and imported into the Clus-DoC analysis software 22 (https://github.com/PRNicovich/ClusDoC). The region of interest was determined by the nuclear staining. First the Ripley K function was completed to identify the r max. The r max was then assigned for DBSCAN. The MinPts was 3 and a cluster required 10 locations, with smoothing set at 7 nm and epsilon set at the mean localization precision for the dye. All other analyses parameters remained at default settings 22. Data concerning each cluster was exported and graphed using Plots of Data 23.
In silico design of guide RNAs
The crRNAs used in this project were designed using FlashFry developed by McKenna, A. and Shendure, J. 17. FlashFry was downloaded from Github - https://github.com/mckennalab/FlashFry and configured according to the author’s recommendations. The binary database was created based on the latest human genome (hg38 build) in FASTA format from UCSC. The verification of the newly designed crRNA hits across the human genome was done in BLAST/BLAT search from Ensembl with adjusted option to report the maximum number of hits to report to 5000, E-value for alignment report at 1.0, match/mismatch scores equal to 1,-1 with filtering low complexity regions and query sequences options enabled.
Unless stated, data fitting and plotting was performed using Plots of data 23 and GraphPad. Cartoons were generated using the BioRender software.
Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways.
Double-Strand Breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state.
The RNA-guided Cas9 (CRISPR associated protein 9) endonuclease enzyme, is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.