The pCW-Cas9 plasmid was a gift from Eric Lander &#38; David Sabatini (Addgene plasmid # 50661) and the pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W plasmid from the Yusa lab (Addgene plasmid #67974. We would like to thank the Flow Cytometry core at SBP Medical Discovery Institute for timely help with flow analysis and sorting. We would like to acknowledge the support of the Lady Tata Memorial Foundation to A.D. We would like to also acknowledge the support of the following funding sources: NIH/NCI P30 CA030199 Cancer Center Sponsored Grant, the V-Foundation and the San Diego NCI Cancer Centers (C3) #PTC2017to A.J.D.

1. Generating AML Cell Line Clones with High Expression of Stable and Active Cas9
<ol>
	<li>Production of Cas9 lentivirus
	<ol>
		<li>Day 0: Plate 4 x 106 293T cells in 10 mL of DMEM with 10% fetal bovine serum (FBS) and penicillin and L-glutamine in a 10 cm tissue culture dish in a biosafety Level 2 (BSL2) certified cell culture hood. Place the dish in a 37 &#176;C incubator.</li>
		<li>Day 1: The plated 293T cells should be 70&#8211;80% confluent on day 1. Perform the transfection using the following protocol in the afternoon.</li>
		<li>Warm the transfection medium, culture media and transfection reagent to room temperature. Thaw all the required plasmids for transfection.</li>
		<li>Mix 9 &#181;g of psPAX2, 0.9 &#181;g of pMD2.G and 9 &#181;g of pLenti-Cas9 plasmids with 500 &#181;L of transfection medium in a 5 mL tube.</li>
		<li>Add 1.7 mL of transfection medium to a 14 mL round bottom tube. Add 57 &#181;L of transfection reagent directly into the transfection medium in the tube to avoid touching the wall of the tube.</li>
		<li>Gently add the entire plasmid mix to transfection reagent solution and mix by gently tapping the tube on the side. Incubate the mixture at room temperature for 20&#8211;30 min. Meanwhile, change the medium from the seeded 293T plate.</li>
		<li>Add the transfection mix dropwise to the plate and gently rock the plate sideways for efficient mixing. Place the plate in a 37 &#176;C incubator.</li>
		<li>Day 2: Aspirate the supernatant from the transfection plate gently such that the cells are not disturbed or dislodged from the plate. Discard the supernatant. Add 10 mL of fresh 10% DMEM medium by sliding down gently from the sides of the plate to avoid dislodging the cells.</li>
		<li>Day 3: Collect the virus containing supernatant from the transfection plate slowly by drawing it into a 10 cm sterile syringe. After the supernatant is collected into the syringe, attach a sterile 0.45 &#181;M filter to the syringe and hold the syringe and filter over a fresh, sterile 15 mL polypropylene conical tube. Gently plunge the syringe to filter the virus containing supernatant through the 0.45 &#181;M filter into the 15 mL polypropylene conical tube.</li>
		<li>Store the viral conditioned medium in aliquots of 2 mL each in 2 mL cryovials at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Transduction of AML cell lines
	<ol>
		<li>Day -1: Dilute 1 mg/mL recombinant human fibronectin fragment stock to 10 &#181;g/mL with sterile phosphate-buffered saline (PBS). Coat a non-tissue culture treated 6 well plate with 2 mL of the 10 &#181;g/mL recombinant human fibronectin fragment working solution in a BSL2 approved cell culture hood. Wrap the plate in cling wrap to avoid loss on evaporation and store at room temperature overnight.</li>
		<li>Day 0: Thaw the viral supernatant containing Cas9. Aspirate the recombinant human fibronectin fragment solution completely from the coated plate just before spinfection. Add 2 mL of viral conditioned medium with Cas9 to the plate and spin it at 1,300 x g for 90 min at 35 &#176;C. This helps the viral particles attach to the spinfected well.</li>
		<li>Meanwhile, count the cells to be transduced and spin down 2 x 106 cells in a 15 mL polypropylene conical tube. Aspirate the supernatant and resuspend the pellet in 2 mL of fresh culture media.</li>
		<li>After spinfection, remove all the viral supernatant from the spinfected well and discard. Retroviral particles from the supernatant are attached to the bottom of the spinfected well. To this well, add the 2 x 106 cells resuspended in 2 mL of medium from the step above.</li>
		<li>Spin the plate again at 1,300 x g very briefly (1&#8211;2 min) sufficient to allow the cells to settle down at the bottom. Place the plate back into the incubator and leave overnight for transduction with the spinfected viral particles attached to the well.</li>
		<li>Day 1: Depending on cell density, add more medium to the transduced cells to avoid overgrowth.</li>
		<li>Day 2: Since the pLenti-Cas9 plasmid has a Blasticidin resistance marker, add Blasticidin at a 10 &#181;g/mL dose to transduced and untransduced (control) MOLM13 or mouse MLL-AF9 leukemia cells for the selection of Cas9 expressing cells. 
		NOTE: Blasticidin selection of the Cas9-transduced MOLM13 or MLL-AF9 leukemia cells is considered complete when all the untransduced control cells have been eliminated. For cell lines other than MOLM13 and MLL-AF9 leukemia, a dose response curve must be performed prior to this experiment so that an optimal dose can be employed. Titration of the viral supernatant can also be performed in case of low-transduction rates.</li>
	</ol>
	</li>
	<li>Clone selection of high-Cas9 expressing cells. 
	NOTE: We have noticed that in some AML cell lines, clone selection might not be necessary: the bulk Cas9-Blasticidin selected population already has high genome-editing efficiency. In this case, it is possible to skip Step 1.3 and move on to Step 1.4 to assess Cas9 expression in the bulk Cas9-blasticidin AML cells by western blotting. It would still be important to evaluate gene-editing efficiency in those bulk Cas9-AML cells (Step 1.5) before proceeding to the competition assays. We surmise that the selection of single high-Cas9 clones reduces the genome-editing variability, which is especially important when testing a number of different single guide RNAs (sgRNAs).
	<ol>
		<li>Perform a single-cell sort of the Cas9-Blasticidin selected MOLM13 and MLL-AF9 leukemia cells henceforth called MOLM13-Cas9 and MLL-AF9-Cas9 cells, respectively, using a FACS sorter contained in a BSL2 approved hood. Sorting can be performed into 5&#8211;10 round bottom non-tissue culture treated 96 well plates.</li>
		<li>Once single MOLM13-Cas9 and MLL-AF9-Cas9 clones have been picked and individually named, expand 10&#8211;20 clones with 10 &#181;g/mL of Blasticidin in culture media to ensure the maintenance of Cas9 expression.</li>
	</ol>
	</li>
	<li>Expression check of stable Cas9 protein by immunoblotting.
	<ol>
		<li>Make nuclear extracts of all the Blasticidin selected clones of MOLM13 and MLL-AF9 leukemia using Nuclear Extraction Kit as per the manufacturer&#8217;s protocol. Check Cas9 protein expression using anti-Flag M2 antibody since the Cas9 in the pLenti-Cas9 plasmid is linked to an N-terminal Flag epitope.</li>
		<li>Load 50 &#181;g of total protein from each nuclear extract onto 10% Bis-Tris gel and run the gel at 120 V till upper bands are well separated.</li>
		<li>Block the membrane with 5% milk solution in TBST buffer for 1 h.</li>
		<li>Incubate the membrane with 1:1,000 dilution of anti-Flag M2 antibody at a final concentration of 1 &#181;g/mL at 4 &#176;C overnight.</li>
		<li>Next day, wash the membrane with TBST buffer and incubate with HRP conjugated anti-mouse secondary antibody at a dilution of 1:5,000 (final concentration of 0.16 &#181;g/mL) at room temperature for 1 h.</li>
		<li>Wash the membrane thoroughly with TBST and develop the blot using ECL substrate.</li>
		<li>Pick 3&#8211;4 clones with the highest protein expression of Cas9 as seen by Western blotting for further functional analysis (Figure 1).</li>
	</ol>
	</li>
	<li>Ensuring high Cas9 activity in selected clones
	<ol>
		<li>Prepare lentiviral supernatant from an sgRNA encoding vector with sgRNAs targeting the human or mouse AAVS1 safe-harbor locus as described in the Step 1.1.</li>
		<li>Transduce the top Cas9-expressing MOLM13 or MLL-AF9 leukemia clones with the anti-AAVS1 sgRNA lentiviral supernatant using the spinfection method described above. Select with 2.5 &#181;g/mL Puromycin for 4 days.</li>
		<li>Purify genomic DNA from the MOLM13-Cas9 or MLL-AF9-Cas9 leukemia clones after Puromycin selection together with respective wild-type controls using the Genomic DNA extraction kit as per manufacturer&#8217;s instructions. Use 50 ng of DNA as a template to perform a PCR with the AAVS1_test_primers using 2x master mix of Taq Polymerase and the PCR program listed in Table 1.</li>
		<li>Run the PCR product on a 2% agarose gel and gel-purify the 268 base pair band using a gel extraction kit as per the manufacturer&#8217;s protocol. Sanger sequence the gel purified PCR product using the AAVS1_target-frag_F primer.</li>
		<li>Assess editing efficiency by the comparison of the AAVS1 edited and wildtype sequences using online web-based tools (Figure 2).</li>
	</ol>
	</li>
</ol>
2. Cloning and Transduction of sgRNAs in AML-Cas9 Cells
<ol>
	<li>Medium-throughput cloning of sgRNAs
	<ol>
		<li>Design 4&#8211;6 sgRNAs for the gene of interest using a web-based CRISPR design software. There are a number of CRISPR design tools available online such as http://crispr.mit.edu/ which generate sgRNA sequences based on an input DNA sequence.
		<ol>
			<li>Fill in an appropriate information in fields with asterisks. Click on the target genome for sgRNA design. Paste the target sequence in the Sequence box and click on Submit button.</li>
		</ol>
		</li>
		<li>For cloning in the pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W sgRNA expression plasmid, prepend the nucleotides &#8220;CACC&#8221; to the sense oligo and &#8220;AAAC&#8221; to the reverse complemented antisense oligo before ordering. Order premixed sense and anti-sense oligos in a 96-well plate labelled Oligo-Mix from an oligonucleotide synthesis company. 
		NOTE: We have made use of the pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W plasmid, which has the BbsI restriction site for sgRNA cloning. In case other sgRNA expression plasmids are used, the overhangs used for the sgRNA oligonucleotides need to be changed accordingly.</li>
		<li>Take a separate U bottom 96-well plate labelled Annealing Plate. Make a master mix of 1 &#181;L T4 PNK, 1 &#181;L of 10x T4 DNA ligation buffer and 6 &#181;L of water per annealing reaction.</li>
		<li>Add 8 &#181;L of the master mix to each well of the annealing plate. Add 1 &#181;L of 100 &#181;M each, sense and antisense oligo (or 2 &#181;L of mixed sense-anti-sense oligos) to the master mix. Pipette 2&#8211;3 times gently to mix well, spin the plate briefly to get the mixes to the bottom of wells.</li>
		<li>Use the following annealing program on a PCR machine: 30 min at 37 &#176;C, 2 min 30 s at 95 &#176;C followed by slow cooling to 22 &#176;C at the rate of 0.1 &#176;C/s. After annealing, take a fresh 96-well plate labelled Diluted OligoMix. In this plate, dilute the phosphorylated and annealed oligos from the above reaction with water (1:200) using a multichannel pipette.</li>
		<li>Digest 5 &#181;g of pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W vector with 1.5 &#181;L of BbsI restriction enzyme (10,000 units/mL) using an appropriate buffer at 37 &#176;C for 2 h to linearize it for ligation later.</li>
		<li>Take a fresh 96 well plate labelled Ligation Plate and add 20 ng of the BbsI digested pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W vector to one well per desired sgRNA ligation. To this, add 2 &#181;L of phosphorylated, annealed oligos from the Diluted OligoMix plate.</li>
		<li>Add 1 &#181;L of 10x T4 ligase buffer and 1 &#181;L of the T4 DNA Ligase enzyme. Gently pipette with a multichannel pipette and incubate the ligation mix at room temperature for 2 h. 
		NOTE: Ligation mixes can be stored at -20 &#176;C for the transformation step later.</li>
		<li>Meanwhile, thaw 90 &#181;L of chemically competent E. coli cells on ice 10 min before the end of the ligation step.</li>
		<li>Using a multichannel pipette, make 10 &#181;L aliquots of the competent cells into each well of a separate 96 well plate.</li>
		<li>Add the ligation mixture from Step 2.1.8 into the well containing the competent cells, pipette up and down gently and incubate for 10 min at room temperature.</li>
		<li>Pipette 5 &#181;L of the bacteria-DNA mix directly from the above reaction into a 6 well plate containing LB-agar with 100 &#181;g/mL Ampicillin. Repeat for each of the transformation reactions.</li>
		<li>Plate each transformation reaction into a separately labelled well of a 6-well plate. Add approximately 5&#8211;8 glass beads to each well and shake the whole 6-well plate 8&#8211;10 times in a circular motion.</li>
		<li>Pick 1&#8211;2 single colonies from each well with a sterile 20 &#181;L pipette tip and streak it into a carefully labelled spot on a sterile 10 cm Petri-dish with LB agar and 100 &#956;g/mL Ampicillin. After streaking into the LB plate, simply eject the tip used for clone streaking into 3 mL of LB-Ampicillin containing medium in a 14 mL round bottom tube marked with the corresponding bacterial clone number.</li>
		<li>Send the bacterial plate directly for Sanger sequencing with the Human U6 promoter forward primer.</li>
		<li>After sequence confirmation of cloned sgRNAs, purify the DNA from the corresponding 14 mL tube using a mini-prep kit according to the manufacturer&#8217;s instructions.</li>
		<li>Measure the concentration and quality of each of the mini-preps with a Spectrophotometer. Normalize each mini-prep to a concentration of 15 ng/&#181;L and pipette into a 96 well plate labelled sgRNA Clones Plate. 
		NOTE: This plate can be stored at -20 &#176;C or used directly for virus preparation.</li>
	</ol>
	</li>
	<li>Viral production of sgRNA constructs and transduction
	<ol>
		<li>For the production of sgRNA lentiviral particles in the 96-well format, follow the &#8220;shRNA/sgRNA/ORF High Throughput Viral Production (96 well)&#8221; protocol from the Genetic Perturbation Platform (GPP web Portal) of the Broad Institute (URL: https://portals.broadinstitute.org/gpp/public/resources/protocols).</li>
		<li>Transfer 200 &#181;L of viral supernatant to sterile tubes and freeze at -80 &#176;C immediately.</li>
		<li>Day -1: Coat a flat bottom Non-tissue culture treated 96 well plate with 100 &#181;L of recombinant human fibronectin fragment at a concentration of 10 &#181;g/mL in a BSL2 cell culture hood. Wrap the plate using cling wrap to avoid evaporation loss and leave it at bench overnight.</li>
		<li>Day 0: Remove the recombinant human fibronectin fragment from each well. Thaw the viral supernatant of each sgRNA at room temperature. Add 50 &#181;L of viral supernatant from each tube to each coated well of the transduction plate. Spin the transduction plate at 1,300 x g for 90 min at 35 &#176;C.</li>
		<li>Towards the end of spinfection, count MOLM13-Cas9 (clone B3) cells from the culture flask. Use 10,000 cells per well in 100 &#181;L volume. Count the cells for all the transduction wells, taking dead volume into consideration.</li>
		<li>After the 90 min spinfection, remove the supernatant from all the wells using a multichannel pipette adjusted to 50 &#181;L slowly by tilting the plate. Add 100 &#181;L of the culture of MOLM13-Cas9 clone B3 cells to each well slowly sliding down from the rim.</li>
		<li>Spin the plate at 1,300 x g for 2 min at 35 &#176;C to let the cells settle onto the bottom. Transfer the plate to the 37 &#176;C tissue culture grade incubator.</li>
		<li>Day 1: Add 100 &#181;L of fresh medium to each well containing sgRNA transduced Cas9-MOLM13 or Cas9-MLL-AF9 leukemia cells such that the final medium volume is 200 &#181;L.</li>
	</ol>
	</li>
</ol>
3. Competitive Growth Assay
<ol>
	<li>Day 3: 72 h (day 3) post transduction, check the percentage of sgRNA containing BFP positive cells in each well by flow cytometry (FACS). Use a cell viability dye to mark and exclude dead cells from the analysis.</li>
	<li>Continue re-plating a proportion of the cells into new wells with fresh medium after every FACS analysis to avoid overgrowth during the assay.</li>
	<li>Repeat the FACS analysis every 2&#8211;3 days to check the relative proportion of BFP positive (BFP+ve) cells compared to the BFP negative (BFP-ve) counterparts (Figure 3). Analyze the percentage of BFP positive cells for each time point (using the FlowJo or similar FACS analysis software).
	<ol>
		<li>Drag Fcs files for each sample to FlowJo software. Double click on any one sample file and plot a dot blot of forward scatter (FSC) vs. Side scatter (SSC) with FSC on X axis and SSC on Y axis. Gate all the cells.</li>
		<li>Double click on the gated cells and plot them on Viability stain (on Y axis) vs. FSC height (H) or Area (A) dot blot. Gate the viability stain negative (live) cells.</li>
		<li>Double click on gated live cells and plot BFP (on Y axis) vs. FSC (A) on X axis. Gate BFP+ve cells. Apply all these gates to all the samples by dragging the selected sample to All Samples under Group tab.</li>
		<li>Click on Table Editor to make an analysis table of all the samples. Drag BFP+ve count from one sample to the table and click on the Display button in the Table Editor to create a batch report of all the samples with a table displaying the percentage of BFP+ve cells. Save the table as an xls.</li>
	</ol>
	</li>
	<li>Calculate the proportional increase or decrease in % BFP positive cells within the live cell population over time and compare this ratio to the ratio of BFP positive cells in the control sgRNAs (such as luciferase, scrambled or GFP sgRNA).</li>
	<li>Plot the ratio of BFP positive cells for the different sgRNAs including the gene(s) of interest as well as controls using day 3 as the baseline.</li>
</ol>

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A.J.D is a consultant to A2A pharmaceuticals (New Jersey) and Salgomed Therapeutics (San Diego). Other authors have no conflicts to declare.

In this manuscript, we describe a detailed protocol for conducting a CRISPR-Cas9-based competitive growth assay to investigate the role of candidate genes in AML cell lines using flow-cytometry in human/murine AML cells (Figure 5). The goal of the assay is to identify the effect of gene deletion on maintenance of AML cell proliferation over two to three weeks on a medium-throughput scale. Some critical steps need to be followed carefully to facilitate the scaling up of the described protocol...

The past few decades have seen numerous research efforts focused on identifying the contribution of key molecular pathways in acute myeloid leukemia (AML) pathogenesis. Traditionally, gene-disruption in AML cells has been performed using conditional knockout mice or short-hairpin RNA (shRNA). While knockout mice offer a sophisticated system for spatio-temporal control of gene-deletion, generating gene knockout mice is labor-intensive, time-consuming and expensive. Furthermore, gene-knockouts using recombination strategies is not easily scalable; these strategies do not lend themselves well to the interrogation of several genes in parallel. After the discovery of RNA interference methods to knock-down endogenous mRNAs using small interfering RNA (siRNA) or shRNA, many groups started using RNA interference techniques to investigate the role of specific genes in AML. Since both murine and human AML cells are notoriously difficult to transfect using traditional lipid-based transfection methods, most studies employed lentivirally or retrovirally-encoded shRNA for studying gene function in AML cells. The recent discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and the associated Cas nucleases (CRISPR-Cas9) has revolutionized gene-targeting technologies1,2,3. Using CRISPR-Cas9, specific genes or genomic regions can be deleted, edited or tagged with efficiency and ease. CRISPR-Cas9-based gene-editing is now emerging as the method of choice for investigating genotype-to-phenotype relationships in diverse cell types due to the simplicity, effectiveness, and broad applicability of this technique. CRISPR-Cas9-based methods are also becoming the method of choice in AML, not only for interrogating individual genes, but also as a way to target multiple genes in arrayed or pooled genetic screens aimed at investigating several genes in parallel as potential AML-dependencies4,5,6.
In this manuscript, we describe a simple competitive growth assay for measuring the impact of gene-disruption on the growth of AML cells, based on stable CRISPR-Cas9-mediated gene-editing followed by high-throughput flow cytometry. This method is simple, efficient, and scalable to medium-throughput experiments for investigating the role of several genes in parallel in AML cells.

<table>
	<tbody>
		<tr>
			<td>FLAG-M2 Antibody</td>
			<td>sigma-aldrich</td>
			<td>F3165, lot # SLBS3530V</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Antibody</td>
			<td>Invitrogen</td>
			<td>31446, lot # TA2514341</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Femto Maximum Sensitivity Substrate</td>
			<td>Thermo Fisher</td>
			<td>34095</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Imaging System</td>
			<td>BIO RAD</td>
			<td>17001401</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend RT centrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Thermo Fisher</td>
			<td>R21001</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Red</td>
			<td>Thermo Fisher</td>
			<td>S34859</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Thermo Fisher</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>SAFC</td>
			<td>12303C</td>
			<td></td>
		</tr>
		<tr>
			<td>single gRNA vector</td>
			<td>Addgene #67974</td>
			<td>pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W</td>
			<td></td>
		</tr>
		<tr>
			<td>CelLytic Nuclear extraction kit</td>
			<td>sigma-alorich</td>
			<td>NXTRACT</td>
			<td></td>
		</tr>
		<tr>
			<td>XtremeGENE 9</td>
			<td>sigma-alorich</td>
			<td>6365787001</td>
			<td></td>
		</tr>
		<tr>
			<td>Retronectin</td>
			<td>Takara</td>
			<td>T100B</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK</td>
			<td>NEBioLabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligation buffer</td>
			<td>NEBioLabs</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase enzyme</td>
			<td>NEBioLabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher scientific</td>
			<td>BP1760-25</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar</td>
			<td>Fisher scientific</td>
			<td>BP9724-500</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Fisher scientific</td>
			<td>BP9731-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen mini-prep kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop Spectrophotometer</td>
			<td>Thermo Fisher</td>
			<td>NanoDrop One</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine IL-3</td>
			<td>Peprotech</td>
			<td>213-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine IL-6</td>
			<td>Peprotech</td>
			<td>216-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine M-CSF</td>
			<td>Peprotech</td>
			<td>315-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Stable competent cells</td>
			<td>NEBiolabs</td>
			<td>C3040I</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Tissue Culture dishes</td>
			<td>Fisher Scientific</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lysis solution</td>
			<td>Qiagen</td>
			<td>158906</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein precipitation solution</td>
			<td>Qiagen</td>
			<td>158910</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA hydration solution</td>
			<td>Qiagen</td>
			<td>158914</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>BbSI</td>
			<td>New England BioLabs</td>
			<td>R0539S</td>
			<td></td>
		</tr>
		<tr>
			<td>APEX 2.0 X Taq Red Master Mix Kit</td>
			<td>Genessee Scientific</td>
			<td>42-138</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Fisher scientific</td>
			<td>BP2956100</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL polypropylene conical tubes</td>
			<td>Fisher scientific</td>
			<td>1495949A</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL polypropylene conical tubes</td>
			<td>Fisher scientific</td>
			<td>1495970C</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigation of genetic dependencies using crispr-cas9-based competition assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

In our study, we first transduced the MOLM13 human AML cell line that bears the MLL-AF9 translocation with high-titer virus encoding the Cas9-blasticidin lentiviral plasmid. In our hands, bulk unsorted MOLM13-Cas9 cells did not display high level Cas9 expression by Western blotting and also did not perform well when assayed for efficient gene editing-using the method described previously7. Therefore, we proceeded to establish single cell clones and only select the ...

Watch this Scientific Journal Video about Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays at JoVE.com

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

investigation-genetic-dependencies-using-crispr-cas9-based

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JoVE Journal

Cancer Research

9.4K Views.  Sanford Burnham Prebys Medical Discovery Institute. This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Video: Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.