A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

CRISPR-Cas9 which stands for "clustered regularly interspaced short palindromic repeats-associated protein 9" was first discovered as part of studies on bacterial adaptive immunity^1,2. Today, CRISPR/Cas9 has become the most recognized tool for programmable gene editing and different iterations of the system have been developed to allow transcriptional and epigenetic modulations³. This technology enables the highly precise genetic manipulation of almost any sequence of DNA⁴.

The essential components of any CRISPR gene editing are a customizable guide RNA sequence and the Cas9 nuclease⁵. The RNA guide binds to the target-complementary sequence in the DNA, directing the Cas9 nuclease to perform a double-strand break at a specific point in the genome^3,4. The resulting cleavage site is then repaired by non-homologous end-joining (NHEJ) or homology-directed repair (HDR), with the consequent introduction of changes in the targeted DNA sequence⁵.

CRISPR/Cas9 based gene editing is easy to use, and relatively inexpensive compared to previous gene-editing techniques and it has been proven to be both efficient and robust in a multiplicity of systems^2,4,5. Yet, the system presents some limitations. The constitutive expression of Cas9 has often been shown to result in an increased number of off-targets and high cell toxicity^4,6,7,8. Additionally, the constitutive targeting of essential and cell survival genes by Cas9 takes away from its ability to perform certain types of functional studies such as kinetic studies of cell death⁷.

Different inducible or conditionally controlled CRISPR-Cas9 tools have been developed to address those issues⁶, such as Tet-ON and Tet-Off⁹; site-specific recombination¹⁰; chemically-induced proximity¹¹; intein dependent splicing³; and 4-Hydroxytamoxifen Estrogen Receptor (ER) based nuclear localization systems¹². In general, most of these procedures (intein splicing and chemically induced proximity split systems) do not offer reversible control, present a very slow kinetic response to drug treatment (Tet-On/Off system), or are not amenable to high-throughput manipulation⁶.

To address these limitations we developed a novel toolkit that not only provides fast and robust temporal-controlled gene editing but also ensures traceability, tunability, and amenability to high throughput gene manipulation. This novel technology can be used in cell lines, organoids, and animal models. Our system is based on an engineered domain, when fused to Cas9, it induces its rapid degradation. However, it can be rapidly stabilized with a highly selective, non-toxic, cell-permeable small molecule. More specifically, we engineered the human FKBP12 mutant "destabilizing domain" (DD) to Cas9, marking Cas9 for rapid and constitutive degradation via the ubiquitin-proteasome system when expressed in mammalian cells¹³. The DD synthetic ligand, Shield-1 can stabilize DD conformation, thereby preventing the degradation of proteins fused to DD (such as Cas9) in a very efficient manner, and with a fast kinetic response^14,15. Of note, Shield-1 binds with three orders of magnitude tighter to the mutant FKBP12 than to its wild-type counterpart¹⁴.

The DD-Cas9/Shield-1 pair can be used to study the systematic identification and characterization of essential genes in cultured cells and animal models as we previously showed by conditionally targeting the CypD gene, which plays an important role in the metabolism of mitochondria; EGFR, a key player in oncogenic transformation; and Tp53, a central gene in DNA damage response. In addition to temporally and conditionally controlled gene editing, another advantage of the method is that the stabilization of DD-Cas9 is independent of its transcription. This feature enables co-expression, under the same promoter, of traceable markers as well as recombinases, such as the estrogen receptor-dependent recombinase, CRE^ER. In this work, we show how our method can be successfully used in vitro, to conditionally target for example, DNA replication gene, RPA3.

Protokół

1.The DD-Cas9 vector

1. Obtain DD-Cas9 vector from Addgene (DD-Cas9 with filler sequence and Venus (EDCPV), Plasmid 90085).
   ​NOTE: This is a lentiviral DD-Cas9 plasmid with a U6 promoter that drives the single guide RNA (sgRNA) transcription while the EFS promoter drives the DD-Cas9 transcription. The DYKDDDDK sequence (flag-tag) is present at the C-terminal of Cas9 followed by 2A self-cleaving peptide (P2A) that separates DD-Cas9 and modified fluorescent protein Venus (mVenus).

2. Small guide RNA (sgRNA) design

1. Design small guide RNA (sgRNA) that will be cloned into the DD-Cas9 vector by using one of several algorithms, targeting a specific genomic region.
   NOTE: To reduce the off-target effects, select sgRNA sequences with the highest score by using the website: http://crispr.mit.edu.
2. Design and order two oligonucleotides per sgRNA sequence to make a complete sgRNA.
   ​NOTE: Since the plasmid will be digested by the BsmBI enzyme, design the forward and the reverse oligonucleotide by adding BsmBI digestion overhangs to the sgRNA sequence. Use the template from Table 1.

3. Cloning of sgRNA into the lentiviral DD-Cas9 vector

1. Dephosphorylate the 5′-ends of DD-Cas9 plasmid by using alkaline phosphatase (FastAP) in a restriction enzyme digestion reaction.
   NOTE: Vectors can re-circularize during the ligation especially when they are linearized by a single restriction enzyme. To ensure the vector will not re-circularize, de-phosphorylate the plasmid by adding phosphatase directly into the digestion reaction.
   1. Dephosphorylate and digest the plasmid with BsmBI enzyme by preparing a mix of 5 µg of DD-Cas9 plasmid, 6 µL of digest buffer (10x), 0.6 µL of DTT (100 mM), 3 µL of BsmBI, 3 µL of FastAP, add up to 60 µL nuclease-free water to make 60 µL the total reaction volume.
      NOTE: Add the BsmBI enzyme and FastAP to the mixture in the end.
   2. Incubate the mixture for 30 min at 37 °C heat block or thermocycler.
2. Purify the digested DD-Cas9 plasmid.
   1. Prepare an 0.8% DNA agarose gel to remove incompletely digested plasmid and traces of enzymes. Use a wider gel-comb for better separation and run the gel at 90 V.
   2. Visualize bands under 360-365 nm UV light and cut the larger plasmid band out of the gel. Discard the 2 kb fragment which is corresponding to the filler.
   3. Purify the large cut gel-band by using a commercial gel purification kit and follow the manufacturer's instructions.
3. Ligate annealed oligos with digested DD-Cas9 plasmid.
   1. Phosphorylate and anneal the sgRNA oligos.
      1. Prepare the mix of 1 µL of T4 Ligation Buffer (10x), 1 µL of Oligo 1 (100 µM), 1 µL of Oligo 2 (100 µM), 6.5 µL of nuclease-free water. Lastly, add 0.5 µL of T4 PNK. The total volume of this reaction is 10 µL.
      2. Add the reaction-mix to the thermocycler with the following conditions: 37 °C for 30 min, 95 °C for 5 min, and then ramp down to 25 °C with the speed of 5 °C/min.
         ​NOTE: The PNK must be heat-inactivated before the oligonucleotides are put into the ligation otherwise the PNK will phosphorylate the vector. The phosphorylation step and annealing step are performed together in a thermocycler. The phosphorylation step where 5' phosphate is added to the sgRNA oligonucleotides is required for the ligation to occur.
   2. Ligate DD-Cas9 digested plasmid and sgRNA oligos.
      1. Dilute the annealed oligos to 1:200 in nuclease-free water and use 50 ng of plasmid DNA in the ligation reaction.
         ​NOTE: Use the molar ratio of the 6 insert : 1 vector, to promote insert integration or use a ligation calculator to calculate the molar ratio: http://nebiocalculator.neb.com/#!/ligation.
      2. Prepare the ligation reaction mix: 50 ng of digested and purified DD-Cas9 vector, appropriate volume of diluted annealed oligos, 1 µL of T4 Ligation Buffer (10x), 1 µL of T4 DNA Ligase, up to 10 µL nuclease-free water to obtain the total volume of the reaction 10 µL.
      3. If using "high concentration" ligase, incubate the reaction at room temperature for 5 min. Otherwise, incubate at room temperature for 2 h, or to achieve a higher yield of ligation, incubate at 16 °C overnight.
         ​NOTE: Heat-inactivate if using the standard T4 DNA Ligase at 65 °C for 20 minutes. Do not heat-inactivate if you are using ligase master mixes.

4. Bacterial transformation

1. Pre-warm 10 cm LB-ampicillin agar plates at room temperature.
2. Thaw 50 µL of "One shot Stbl3" competent bacterial cells on ice.
3. Add 2-3 µL of ligation mixture to 50 µL of competent bacterial cells and gently mix by flicking the bottom of the tube with a finger 4 times. Incubate on ice for 30 min.
4. Heat-shock the cells at exactly 42 °C for 40 seconds, using a timer. Cool down the bacterial cells on ice for 5 min.
5. Add 500 µL of SOC media without antibiotic to the bacterial cells and grow them in the shaking incubator at 250 rpm for 45 min, at 37 °C.
6. Spread 100 µL of transformed bacteria on pre-warm LB-ampicillin plates and incubate overnight at 37 °C.

5. Mini/maxi-prep of ligated plasmid

1. Prepare a maxi/mini-prep starter culture.
   1. The next day pick a single bacterial clone and inoculate a starter culture with 8 mL of LB media containing ampicillin.
   2. Grow the bacteria in the shaking incubator at 250 rpm, 37 °C for 10-12 h.
   3. Use 3-5 mL of bacteria for mini-prep or use it as a starter culture for maxi-prep. Use the rest of the bacteria as a bacterial glycerol-stock by adding 100% glycerol in the ratio of bacterial culture:glycerol is 1:1.
      ​NOTE: Here, we will describe the mini-prep procedure.
2. Mini-prep procedure
   1. Harvest 3-5 mL of bacterial cells and centrifuge at 12,000 x g for 1 min.
   2. Resuspend the pellet of bacterial cells with 200 µL of P1 buffer, containing RNase A and stored at 4 °C.
   3. Add 200 µL of buffer P2 and mix gently by inverting the tube 10 times until the liquid clarifies.
   4. Add 300 µL of buffer P3 and mix by inverting the tube 10 times. The liquid will become cloudy. Proceed immediately with centrifuging the samples at 12,000 x g for 10 min.
   5. Transfer clear supernatant to the spin column and centrifuge at 12,000 x g for 1 min.
      Discard the flow-through.
      ​NOTE: Make sure the supernatant is clarified. Particles can clog the spin column and reduce the column efficacy.
   6. Add 400 µL of PD buffer and centrifuge at 12,000 x g for 1 min. Discard the flow-through.
   7. Add 600 µL of PW buffer to wash the spin column and centrifuge at 12,000 x g for 1 min. Discard the flow-through.
   8. Centrifuge again the spin column at top speed for 3 min to remove the buffer residues. Discard the flow-through and let it sit for 2 min.
   9. Place the spin column to a fresh tube and add 50 µL of elution buffer. Incubate for 5 min and centrifuge at top speed for 2 min.
   10. Use the nanodrop device to measure the concentration of purified DD-Cas9 plasmid with the sgRNA insert (ng/µL).
3. Validate the sgRNA cloning by DNA sequencing of the DD-Cas9 plasmid. Use the U6 primer sequence in Table 2.

6. Lentiviral preparation

1. Prepare HEK293T packaging cells for transfection.
   1. Use healthy HEK293T up to passage 10 and seed them evenly at density 1-2 x 10⁶ cells per 10 cm tissue culture plate. Use 10 mL of Glucose Dulbecco's Modified Eagle Medium (DMEM) with 10% filtered Fetal Bovine Serum (FBS). Incubate cells in the tissue culture incubator, at 5% CO₂ and 37 °C for 20 h.
   2. Next day, confirm that cells reached 70% confluency by brightfield microscopy and that they are evenly dispersed across the plate. Change media to cells 1 h before the transfection with the final volume of 10 mL.
      ​NOTE: The media should be absent of antibiotics and antimycotics.
   3. Prepare the mixture of 2 tubes with 500 µL of warm media (e.g., OptiMEM).
      1. Add 25 µL of transfection reagent to tube 1 containing warm 500 µL of media, and incubate at room temperature for 5 min.
      2. Meanwhile prepare a mix of plasmids to tube 2 containing 3.5 µg of DD-Cas9 containing cloned sgRNA, 6 µg of the packaging plasmid (psPAX2), and 3 µg of the envelope plasmid (pMD2.G) into warm 500 µL of media.
      3. Mix tube 1 with tube 2 to form a transfection mixture and incubate at room temperature for 20 min.
      4. Dropwise the transfection mixture to HEK293T cells and incubate plate in the tissue culture incubator, at 5% CO₂ and 37 °C overnight.
      5. After 18 h, carefully change the media to 10 mL of fresh DMEM with 10% FBS to remove the transfection reagent. Incubate for the next 48 h.
      6. After 48 h, collect the supernatant with a 10 mL syringe and pass it through a 0.45 µm filter.
      7. Aliquot and store the virus supernatant at -80 °C.

7. Determining virus titer and transduction efficacy with flow cytometry

1. Seed the HEK293T cells with 5 x 10⁵ cells/well into a 6-well plate. Seed cells into two 6-well plates. Use one plate for counting the next day.
2. Incubate the plates in the incubator at 37 °C, 95% humidity, 5% CO₂ overnight to reach 50-60% confluency the next day.
3. The next day, use one of the 6-well plates to count the cells in 6 wells.
4. Thaw the virus aliquot that will be used to perform the serial dilution and add 8 µg/mL of polybrene reagent.
5. Prepare DMEM with 10% FBS for serial dilution of the virus and add 8 µg/mL of polybrene reagent.
6. Prepare 2 mL of ten-fold serial dilutions of the lentivirus from 1 x 10^-1 to 1 x 10^-4 in polybrene-containing media.
7. Remove media from 6-well plate and add 1 mL of viral dilutions to wells. Leave one well with media alone as negative control and one well with 100% virus.
8. Incubate cells with the virus in the incubator for 24 h.
9. The next day, remove the media with virus from 6-well plates and change it for 2 mL of fresh DMEM with 10% FBS. Incubate cells for 48-72 h. Observe GFP by using a fluorescent microscope every day.
10. After 48-72 h, detach the cells and resuspend them in MACS buffer.
11. Use a flow cytometer to determine the percentage of GFP expression.
12. Calculate virus titer by using the following formula: TU/mL = (Number of cells transduced x Percent fluorescent x Dilution Factor)/(Transduction Volume in mL)

8. Lentiviral transduction of target cells

1. Plate the cell line of interest to a 10 cm plate and incubate overnight to reach confluency 50-60% the next day.
   NOTE: We used the A549 cell line expressing DD-Cas9 and two independent RPA3-gene sgRNAs. We also used the A549 cell line expressing vector with Renilla control. We seeded those cells at the density of 2 x 10³cells/cm² and incubated them at 37 °C, 95% humidity, 5% CO₂ overnight to reach 50-60% confluency the next day. We used RPMI media with 10% filtered FBS. We proceeded with the next steps as follow:
2. The next day, infect the cells with 500-2000 µL of virus particles in 10 mL total volume of culture medium. Incubate viral media for 24 h.
3. The next day, change the viral media to culture media and determine the percentage of GFP positive cells by using flow cytometry.
4. Select GFP positive cells by FACS cell sorting or by using bleomycin selection.
5. Expand positively selected cells and freeze a stock.

9. Conditional induction of Cas9 mediated gene editing

1. Plate positively selected cells 24 h after infection as well as untransduced cells to 12-well plate separately. Wait until the cells attach and change the media to cell culture media containing 200 nM of Shield-1. Replace the media in the plates with transduced and untransduced-cells, leaving 2 wells per plate with regular media as a negative control.
2. Incubate and extract proteins from each well at different time points: time 0, 2 h, 6 h, 12 h, 24 h, 48 h, and 72 h after adding the Shield-1 to the wells.
3. Then remove the media with Shield-1 from the rest of the wells and change it for regular cell media.
4. Collect the proteins from those wells 2 h, 6 h, and 12 h after changing the media.
5. Visualize by western blot analysis the reversibility and rapidity of destabilized DD-Cas9 protein regulation after the addition and the withdrawal of its ligand, Shield-1. Use antibody directed towards DYKDDDDK Tag to visualize proteins and a control antibody targeting for example beta-tubulin.
6. Determine the optimal dose of Shield-1 by dose-response curve observed by Western Blot analysis.

10. Validation of gene editing

NOTE: The GFP expression assays, such as flow cytometry analysis and bleomycin selection marker only confirm successful CRISPR reagent delivery but they do not determine if the desired sequence was successfully targeted. The most common assays to confirm successful gene targeting by the CRISPR experiment are Sanger DNA sequencing, Next-generation sequencing, the Surveyor Nuclease Assay, the Tracking of Indels by Decomposition (TIDE) Assay, or western blot analysis^16,17,18.

1. Plate positively selected cells and change media to 200 nM of Shield-1 containing media. Incubate cells for 5 days, changing media with Shield-1 every 3 days.
2. Use the above-mentioned validation techniques 5 days after DD-Cas9 induction by Shield-1.

Wyniki

To enable the conditional expression of Cas9, we developed a dual lentiviral vector construct consisting of a U6-driven promoter to constitutively express sgRNA, and an EF-1α core promoter to drive the expression of the DD-Cas9 fusion protein (Figure 1A)¹⁹. As a paradigm to illustrate the robustness and efficiency of the system, we transduced the lung carcinomatous A549 cell line with the lentiviral construct. The levels of Cas9 i...

Dyskusje

The CRISPR/Cas9 technology has revolutionized the capability of functionally interrogate genomes². However, the inactivation of genes often results in cell lethality, functional deficits, and developmental defects, limiting the utility of such approaches for studying gene functions⁷. Additionally, constitutive expression of Cas9 may result in toxicity and the generation of off-target effects⁶. Different approaches have been developed to temporally co...

Materiały

Name	Company	Catalog Number	Comments
100mM DTT	Thermosfisher
10X FastDigest buffer	Thermosfisher	B64
10X T4 Ligation Buffer	NEB	M0202S
colorimetric BCA kit	Pierce	23225
DMEM, high glucose, glutaMax	Thermo Fisher	10566024
FastAP	Thermosfisher	EF0654
FastDigest BsmBI	Thermosfisher	FD0454
Flag [M2] mouse mAb	Sigma	F1804-50UG
Genomic DNA extraction kit	Macherey Nagel	740952.1
lipofectamine 2000	Invitrogen	11668019
Phusion High-Fidelity DNA Polymerase	NEB	M0530S
oligonucleotides	Sigma Aldrich
pMD2.G	Addgene	12259
polybrene	Sigma Aldrich	TR-1003-G
psPAX2	Addgene	12260
QIAquick PCR & Gel Cleanup Kit	Qiagen	28506
secondary antibodies	LICOR
Shield-1	Cheminpharma
Stbl3 competent bacterial cells	Thermofisher	C737303
SURVEYOR Mutation Detection Kit	Transgenomic/IDT
T4 PNK	NEB	M0201S
Taq DNA Polymerase	NEB	M0273S
α-tubulin [DM1A] mouse mAb	Millipore	CP06-100UG