Target Guide Sequence Cloning Protocol

In order to clone the guide sequences into the CRISPR lentiviral vectors the ‘targeted guide sequence cloning protocol’ was used from the Zhang lab. This protocol gives a detailed method of Lentiviral vector digestion, oligo annealing and cloning into digested vector. One amendment was made to the protocol; Step 1: vector digestion at 37°C overnight instead of for 30 minutes as stated.

5 μg of lentiCRISPRv2 or lentiGuide-puro were digested and dephosphorylated, using FastDigest BsmBI, FastAP, 10xFastDigest Buffer, freshly prepared 100 mM DTT and made up to 60 μL with H2O. This reaction was incubated at 37°C overnight, in order to

ensure cuts were made at the BsmBI digestion sites and to dephosphorylate its overhanging complementary ends and inhibit annealing between them. This reaction made two cuts, either side of an 1880bp filler sequence of the vectors, leaving this site available for the target sequences. The digestion products were then separated on a 1% agarose gel. The fragments of interest, lentiCRISPRv2 (12993 bp) and lentiGuide-puro (8297 bp) backbone fragments, were isolated under long-wave UV light using a clean scalpel. The products were gel-purified by using the QIAquick Gel Extraction Kit (Qiagen, Cat no. 28704) according to manufacturer’s instructions. The concentration and quality of extracted DNA were measured with a NanoDrop ND-2000 spectrophotometer.

At the same time each pair of oligonucleotides for the target sequence were annealed and phosphorylated. This was performed by setting up a reaction containing; 100 μM oligo 1 (sense) and 100 μM oligo 2 (antisense), 10x ligation buffer (NEB), T4 Polynucleotide Kinase and ddH2O. (10x Ligation buffer from NEB was used as it contains ATP required

for the phosphorylation reaction). The reaction was put in a thermocycler under the following parameters: 37°C for 30 minutes, up to 95°C for 5 minutes and then down 5°C every 60 seconds until reaching 25°C. Following the thermocycler, the annealed oligos were diluted 1:200 in sterile water.

The ligation reaction performed by mixing; 50 ng of the BsmBI digested plasmid, 1 μL of the diluted oligo duplex, 2x Quick ligase buffer (NEB), ddH2O to make it up to 10 μL and

1 μL of quick ligase (NEB M2200S). The ligation reaction was incubated at room temperature for 10 minutes. A negative control was also performed.

After the incubation period the ligation was transformed into Subcloning Efficiency™ DH5α™ Competent Cells (Thermo Fisher Scientific Cat no. 18265017), following manufacturer’s instructions. 5 μL of the ligation reaction was carefully mixed with 50 μL of bacteria and incubated on ice for 30 minutes. A positive control was also performed by adding 50 ng of undigested plasmid vector to the bacteria for transformation. The bacteria and DNA were heat shocked at 42°C for 20 seconds, then immediately placed on ice for 2 minutes. 950 μL of pre-warmed S.O.C. media (Sigma, Cat no. S1797) was added to the tube and incubated at 37°C for 1 hour at 225 rpm. 200 μL from each transformation, including the negative controls, was spread out on pre-warmed LB agar plates containing 100 μg/mL ampicillin. The plates were incubated overnight at 37°C. After incubation, the plates were checked for bacterial growth, the positive control, which had intact plasmid DNA had high transformation efficiency with many bacterial colonies grown. The negative control contained digested plasmid DNA and the addition of ddH2O, the DNA is therefore

not intact and the bacteria cannot grow as they do not have the ampicillin resistant gene, which comes from the plasmid. The bacteria transformed with the lentiCRISPRv2 backbone fragment and the ATG7 oligos had a low number of colonies, indicating low transformation efficiency. However, 3 single colonies were randomly selected and incubated in 5 mL of LB broth for 4 hours at 37°C with constant agitation; the cultures were then transferred into 250 mL of LB broth and cultured overnight at 37°C with constant agitation.

The plasmid DNA was collected from the bacterial cells 24 hours later by using the Maxi Prep Kit (Qiagen, Cat no. 12163) as described by the manufacturer’s instructions or by the Beatson services. 2mL of each culture were stored as bacterial glycerol stock (30% glycerol/ sterile H2O).

2.3.16 Animal work

2.3.16.1 Ethical issues

All animal work was carried out in accordance with regulations set by the Animals Scientific Procedures Act 1986 and UK Home Office regulations. Animals were housed at the Beatson Institute for Cancer Research or at the Veterinary Research Facility at the University of Glasgow. All experiments were carried out under my personal licence (60/12683) and Dr Alison Michie’s project licence (60/4076). Tumour initiating CML cell transplants were carried out on animals between 8 and 12 weeks. The details of the numbers of animals used and gender for each experiment is provided in each figure legend.

2.3.16.2 Mouse models

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ or more commonly known as the NOD scid gamma (NSG).

Background: This animal model combines the characteristics of the NOD/ShiLtJ

background (deficiencies in innate immunity), severe combined immune deficiency mutation, scid, and an IL2 receptor gamma chain deficiency, which disables cytokine signalling. As a result, these mice lack mature T cells, B cells, functional NK cells, and are also deficient in cytokine signalling, making these mice very good transplant models.

2.3.16.3 Bioluminescent in vivo imaging of tumour initiating CML cells

KCL22 cells were labelled with lentiviral firefly luciferase, by G. V. Helgason, and selected based on antibiotic resistance, hygromycin. The cells were cultured in RPMI at a cell concentration of 0.25x106/mL. Cells were untreated or treated with 100 nM ponatinib, 10 µM HCQ, 100 nM NVP-Bez235 or the combination of 10 µM HCQ and 100 nM NVP- Bez235. Cells were cultured for 72 hours. In a previous cell titration study performed by Dr. L. Mukherjee, 1x106 cells per mouse was the lowest number of cells to transplant to maintain efficacy. Cells were counted and based on the combination treatment; the volume of cell suspension required for 1x106 cells per mouse was harvested. Therefore, different number of cells will be transplanted per mouse depending on the drug cytotoxicity. Cells were centrifuged at 300 x g for 5 minutes and washed in PBS/ 0.5% FBS and centrifuged a second time at 300 x g for 5 minutes. Each cell sample was resuspended in 200 µL of PBS/ 0.5% FBS and filtered through a 30 µM filter cup, before being transplanted via tail vein into an 8-12 week old female NSG mouse. In order to activate the bioluminescent reaction, the chemical substrate D-Luciferin (Perkin Elmer, Cat no. 122799) must be injected into

the mice. The luciferin oxidises under catalytic effects of the firefly luciferase and ATP and emits light, which is captured by the IVIS spectral range between 550 nm and 620 nm. D-Luciferin was dissolved in PBS and injected subcutaneously at a dose of 3 mg per mouse 5 minutes before measuring the light emission. 30 minutes after the transplantation of the cells the mice were injected subcutaneously with D-luciferin and analysed by luciferase bioimaging via an IVIS Spectrum In Vivo Imaging System (PerkinElmer, Cat no. 124262), to measure the efficiency of transplantation. The engraftment of these cells was subsequently measured weekly, until tumour burden reached the threshold and the animals were sacrificed using appropriate schedule 1 methods.

2.3.16.4 In vivo drug treatment experiments

NSG mice were transplanted with 1×106 KCL22Pon-Res cells per mouse, the cells were transplanted via tail vein into 8–12-week-old female NSG mice. The mice were monitored as previously described in 2.3.16.3. After 1 week, the mice were treated with vehicle control (citrate buffer or NMP/PEG300 (1:10)), ponatinib (5 mg/kg, oral gavage once daily), HCQ (60mgkg, intraperitoneally once daily), NVP-Bez235 (40mg/kg, oral gavage once daily) or the combination of NVP-Bez235 and HCQ for 4-5 weeks. 5 mice were assigned per drug arm per experiment. Results presented represent data from both experiments.

2.3.16.5 Drug formulation

40mg/kg NVP-Bez235 (LC Labs, Cat no. N-4288) 10ml/kg = 54.67mg/kg BEZ235 salt

For 7 days of treatment a stock solution of 54.67 mg/mL of N-Methyl-2-pyrrolidone (NMP) was prepared.

Using a small glass beaker, 109.4 mg of NVP-BEZ235 was dissolved in 2 ml NMP by heating the solution in a water bath 80°C for 20 minutes followed by sonication. The solution was made into 0.25mL aliquots and frozen at -20°C. For each day of treatment, one aliquot was defrosted and resuspended in 2.25 mL of Polyethylene glycol 300 (PEG 300). Each mouse was treated 10 mL/kg, each mouse weighs on average 20g therefore each mouse will be treated with ~ 200 µL via oral gavage.

200 mg vial was dissolved in 40 mL of PBS making a stock solution of 5 mg /mL. Each mouse weighs on average 20g, therefore each mouse will be treated with ~ 240 µL of HCQ intraperitoneally.

2.3.16.6 Dissection

Animals were sacrificed using appropriate schedule 1 methods. Femur, tibia, hip bones, spleen and tumours were dissected and stored in PBS/2%FBS on ice. Peripheral blood was taken from femoral vein post sacrifice and collected into tubes containing EDTA to ensure anti-coagulation, stored at RT and analysed within several hours.

2.3.16.7 Cell extraction

Bones from femur, tibia and hips were crushed in PBS/2% FBS using a mortar and pestle and made into a single cells suspension through filtering through a sterile 0.2µm filter. Spleen and tumours were homogenised in PBS/2% FCS using a sterile plunger and filtered. Peripheral blood (PB) was analysed neat for cellularity analyses.

2.3.16.8 Analysis

Cell suspensions from each tissue were spun and resuspended in appropriate antibodies and analysed by flow cytometry. Tumour and spleen sizes were measure and photographed.

3 Results (I): Identification and validation of suitable TKI-