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

Summary

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.

Abstract

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.

Introduction

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 technologies^1,2,3. 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-dependencies^4,5,6.

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.

Protocol

1. Generating AML Cell Line Clones with High Expression of Stable and Active Cas9

1. Production of Cas9 lentivirus
   1. 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 °C incubator.
   2. Day 1: The plated 293T cells should be 70–80% confluent on day 1. Perform the transfection using the following protocol in the afternoon.
   3. Warm the transfection medium, culture media and transfection reagent to room temperature. Thaw all the required plasmids for transfection.
   4. Mix 9 µg of psPAX2, 0.9 µg of pMD2.G and 9 µg of pLenti-Cas9 plasmids with 500 µL of transfection medium in a 5 mL tube.
   5. Add 1.7 mL of transfection medium to a 14 mL round bottom tube. Add 57 µL of transfection reagent directly into the transfection medium in the tube to avoid touching the wall of the tube.
   6. 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 20–30 min. Meanwhile, change the medium from the seeded 293T plate.
   7. Add the transfection mix dropwise to the plate and gently rock the plate sideways for efficient mixing. Place the plate in a 37 °C incubator.
   8. 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.
   9. 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 µM 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 µM filter into the 15 mL polypropylene conical tube.
   10. Store the viral conditioned medium in aliquots of 2 mL each in 2 mL cryovials at -80 °C.
2. Transduction of AML cell lines
   1. Day -1: Dilute 1 mg/mL recombinant human fibronectin fragment stock to 10 µg/mL with sterile phosphate-buffered saline (PBS). Coat a non-tissue culture treated 6 well plate with 2 mL of the 10 µg/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.
   2. 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 °C. This helps the viral particles attach to the spinfected well.
   3. 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.
   4. 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.
   5. Spin the plate again at 1,300 x g very briefly (1–2 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.
   6. Day 1: Depending on cell density, add more medium to the transduced cells to avoid overgrowth.
   7. Day 2: Since the pLenti-Cas9 plasmid has a Blasticidin resistance marker, add Blasticidin at a 10 µg/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.
3. 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).
   1. 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 5–10 round bottom non-tissue culture treated 96 well plates.
   2. Once single MOLM13-Cas9 and MLL-AF9-Cas9 clones have been picked and individually named, expand 10–20 clones with 10 µg/mL of Blasticidin in culture media to ensure the maintenance of Cas9 expression.
4. Expression check of stable Cas9 protein by immunoblotting.
   1. Make nuclear extracts of all the Blasticidin selected clones of MOLM13 and MLL-AF9 leukemia using Nuclear Extraction Kit as per the manufacturer’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.
   2. Load 50 µg 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.
   3. Block the membrane with 5% milk solution in TBST buffer for 1 h.
   4. Incubate the membrane with 1:1,000 dilution of anti-Flag M2 antibody at a final concentration of 1 µg/mL at 4 °C overnight.
   5. 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 µg/mL) at room temperature for 1 h.
   6. Wash the membrane thoroughly with TBST and develop the blot using ECL substrate.
   7. Pick 3–4 clones with the highest protein expression of Cas9 as seen by Western blotting for further functional analysis (Figure 1).
5. Ensuring high Cas9 activity in selected clones
   1. 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.
   2. 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 µg/mL Puromycin for 4 days.
   3. 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’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.
   4. 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’s protocol. Sanger sequence the gel purified PCR product using the AAVS1_target-frag_F primer.
   5. Assess editing efficiency by the comparison of the AAVS1 edited and wildtype sequences using online web-based tools (Figure 2).

2. Cloning and Transduction of sgRNAs in AML-Cas9 Cells

1. Medium-throughput cloning of sgRNAs
   1. Design 4–6 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.
      1. 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.
   2. For cloning in the pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W sgRNA expression plasmid, prepend the nucleotides “CACC” to the sense oligo and “AAAC” 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.
   3. Take a separate U bottom 96-well plate labelled Annealing Plate. Make a master mix of 1 µL T4 PNK, 1 µL of 10x T4 DNA ligation buffer and 6 µL of water per annealing reaction.
   4. Add 8 µL of the master mix to each well of the annealing plate. Add 1 µL of 100 µM each, sense and antisense oligo (or 2 µL of mixed sense-anti-sense oligos) to the master mix. Pipette 2–3 times gently to mix well, spin the plate briefly to get the mixes to the bottom of wells.
   5. Use the following annealing program on a PCR machine: 30 min at 37 °C, 2 min 30 s at 95 °C followed by slow cooling to 22 °C at the rate of 0.1 °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.
   6. Digest 5 µg of pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W vector with 1.5 µL of BbsI restriction enzyme (10,000 units/mL) using an appropriate buffer at 37 °C for 2 h to linearize it for ligation later.
   7. 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 µL of phosphorylated, annealed oligos from the Diluted OligoMix plate.
   8. Add 1 µL of 10x T4 ligase buffer and 1 µL 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 °C for the transformation step later.
   9. Meanwhile, thaw 90 µL of chemically competent E. coli cells on ice 10 min before the end of the ligation step.
   10. Using a multichannel pipette, make 10 µL aliquots of the competent cells into each well of a separate 96 well plate.
   11. 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.
   12. Pipette 5 µL of the bacteria-DNA mix directly from the above reaction into a 6 well plate containing LB-agar with 100 µg/mL Ampicillin. Repeat for each of the transformation reactions.
   13. Plate each transformation reaction into a separately labelled well of a 6-well plate. Add approximately 5–8 glass beads to each well and shake the whole 6-well plate 8–10 times in a circular motion.
   14. Pick 1–2 single colonies from each well with a sterile 20 µL pipette tip and streak it into a carefully labelled spot on a sterile 10 cm Petri-dish with LB agar and 100 μg/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.
   15. Send the bacterial plate directly for Sanger sequencing with the Human U6 promoter forward primer.
   16. After sequence confirmation of cloned sgRNAs, purify the DNA from the corresponding 14 mL tube using a mini-prep kit according to the manufacturer’s instructions.
   17. Measure the concentration and quality of each of the mini-preps with a Spectrophotometer. Normalize each mini-prep to a concentration of 15 ng/µL and pipette into a 96 well plate labelled sgRNA Clones Plate.
       NOTE: This plate can be stored at -20 °C or used directly for virus preparation.
2. Viral production of sgRNA constructs and transduction
   1. For the production of sgRNA lentiviral particles in the 96-well format, follow the “shRNA/sgRNA/ORF High Throughput Viral Production (96 well)” protocol from the Genetic Perturbation Platform (GPP web Portal) of the Broad Institute (URL: https://portals.broadinstitute.org/gpp/public/resources/protocols).
   2. Transfer 200 µL of viral supernatant to sterile tubes and freeze at -80 °C immediately.
   3. Day -1: Coat a flat bottom Non-tissue culture treated 96 well plate with 100 µL of recombinant human fibronectin fragment at a concentration of 10 µg/mL in a BSL2 cell culture hood. Wrap the plate using cling wrap to avoid evaporation loss and leave it at bench overnight.
   4. Day 0: Remove the recombinant human fibronectin fragment from each well. Thaw the viral supernatant of each sgRNA at room temperature. Add 50 µL 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 °C.
   5. Towards the end of spinfection, count MOLM13-Cas9 (clone B3) cells from the culture flask. Use 10,000 cells per well in 100 µL volume. Count the cells for all the transduction wells, taking dead volume into consideration.
   6. After the 90 min spinfection, remove the supernatant from all the wells using a multichannel pipette adjusted to 50 µL slowly by tilting the plate. Add 100 µL of the culture of MOLM13-Cas9 clone B3 cells to each well slowly sliding down from the rim.
   7. Spin the plate at 1,300 x g for 2 min at 35 °C to let the cells settle onto the bottom. Transfer the plate to the 37 °C tissue culture grade incubator.
   8. Day 1: Add 100 µL 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 µL.

3. Competitive Growth Assay

1. 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.
2. Continue re-plating a proportion of the cells into new wells with fresh medium after every FACS analysis to avoid overgrowth during the assay.
3. Repeat the FACS analysis every 2–3 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).
   1. 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.
   2. 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.
   3. 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.
   4. 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.
4. 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).
5. 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.

Results

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 ...

Discussion

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...

Materials

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