Name: investigation of genetic dependencies using crispr-cas9-based competition assays
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 11 min 5 s
Description: Watch this Scientific Journal Video about Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays at JoVE.com

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 2.0x 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

Universidad San Sebastian

Research

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

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

Genetics

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance of its potentially deleterious off-target effects. Several methods have been devised to reduce such effects. Here, we present an Escherichia coli-based directed evolution method called Sniper-screen to obtain a Cas9 variant with optimized specificity and retained on-target activity, called Sniper-Cas9. Using Sniper-screen, positive and negative selection can be performed simultaneously. The screen can also be repeated with other single-guide RNA (sgRNA) sequences to enrich for the true positive hits. By using the CMV-PltetO1 dual promoter to express Cas9 variants, the performance of the pooled library can be quickly checked in mammalian cells. Methods to increase the specificity of Sniper-Cas9 are also described. First, the use of truncated sgRNAs has previously been shown to increase Cas9 specificity. Unlike other engineered Cas9s, Sniper-Cas9 retains a wild-type (WT) level of on-target activity when combined with truncated sgRNAs. Second, the delivery of Sniper-Cas9 in a ribonucleoprotein (RNP) format instead of a plasmid format is possible without affecting its on-target activity.

Here, we present a protocol to optimize CRISPR-Cas9 to achieve a higher specificity without the loss of on-target activity. We use a directed evolution approach called Sniper-screen to find a mutant Cas9 with the desired characteristics. Sniper-Cas9 is compatible with truncated single-guide RNAs and delivery in a ribonucleoprotein format, well-known strategies for achieving higher specificities.

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory tool to introduce accurate and targeted modifications in the genome. Its enormous popularity and rapid spread are attributed to its easy use and accuracy compared to its predecessors. Yet, the constitutive activation of the system has limited applications. In this paper, we describe a new method that allows temporal control of CRISPR/Cas9 activity based on conditional stabilization of the Cas9 protein. Fusing an engineered mutant of the rapamycin-binding protein FKBP12 to Cas9 (DD-Cas9) enables the rapid degradation of Cas9 that in turn can be stabilized by the presence of an FKBP12 synthetic ligand (Shield-1). Unlike other inducible methods, this system can be adapted easily to generate bi-cistronic systems to co-express DD-Cas9 with another gene of interest, without conditional regulation of the second gene. This method enables the generation of traceable systems as well as the parallel, independent manipulation of alleles targeted by Cas9 nuclease. The platform of this method can be used for the systematic identification and characterization of essential genes and the interrogation of the functional interactions of genes in in vitro and in vivo settings.

Here, we describe a genome-editing tool based on the temporal and conditional stabilization of clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (Cas9) under the small molecule, Shield-1. The method can be used for cultured cells and animal models.

The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants

Early development depends on a pool of maternal factors incorporated into the mature oocyte during oogenesis that perform all cellular functions necessary for development until zygotic genome activation. Typically, genetic targeting of these maternal factors requires an additional generation to identify maternal-effect phenotypes, hindering the ability to determine the role of maternally-expressed genes during development. The discovery of the biallelic editing capabilities of CRISPR-Cas9 has allowed screening of embryonic phenotypes in somatic tissues of injected embryos or "crispants," augmenting the understanding of the role zygotically-expressed genes play in developmental programs. This article describes a protocol that is an extension of the crispant method. In this method, the biallelic editing of germ cells allows for the isolation of a maternal-effect phenotype in a single generation, or "maternal crispants." Multiplexing guide RNAs to a single target promotes the efficient production of maternal crispants, while sequence analysis of maternal crispant haploids provides a simple method to corroborate genetic lesions that produce a maternal-effect phenotype. The use of maternal crispants supports the rapid identification of essential maternally-expressed genes, thus facilitating the understanding of early development.

Early development is dependent on maternally-inherited products, and the role of many of these products is currently unknown. Herein, we described a protocol that uses CRISPR-Cas9 to identify maternal-effect phenotypes in a single generation.

Early development depends on a pool of maternal factors incorporated into the mature oocyte during oogenesis that perform all cellular functions necessary ...

Detecting Off-Target CRISPR-Cas9 Cleavage Events with CIRCLE-Seq

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of ...

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

Summary

Explore More Videos

Chapters in this video

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants

CIRCLE-Seq for Interrogation of Off-Target Gene Editing