Generation of a RIP1 Knockout U937 Cell Line Using the CRISPR-Cas9 System

Summary

As CRISPR-related protocols become increasingly useful and accessible, complications and obstacles can still arise under specific experimental conditions. This protocol outlines the creation of a receptor-interacting serine/threonine-protein kinase 1 (RIPK1/RIP1) knockout human cell line using CRISPR/Cas9 and highlights potential challenges encountered during this process.

Abstract

This protocol outlines a procedure for knocking out the RIP1 gene using CRISPR/Cas9 in the human monocyte U937 cell line. The method utilizes designated guide RNA plasmids and lentiviral packaging plasmids to achieve RIP1 gene knockout. The protocol addresses challenges and improvements to traditional CRISPR methods, enabling its replication for future cell death studies. The resulting mutant cells can be used to investigate mechanistic changes in cell death, where functional RIP1 proteins would otherwise play a role. Viability assays demonstrated a significant reduction in cell death in knockout cells following necroptosis induction. Fluorescence microscopy revealed a marked decrease in mitochondrial reactive oxygen species (ROS) in knockout cells under the same conditions. Together, these functional assays confirm the loss of RIP1 protein. Optimized for use with U937 human monocytes, this procedure may also be adapted to target other key cell death regulators, yielding functional, non-lethal mutants. Potential pitfalls are addressed throughout to provide insights into challenges that may arise during mutant generation.

Introduction

The use of the CRISPR/Cas9 gene-editing technology has been rapidly evolving since its discovery^1,2,3. The ability to knock-in or knockout genes within cell lines or bacteria is invaluable to forwarding research and the understanding of intracellular mechanisms^1,2,3,4,5,6. The CRISPR-Cas9 system improves upon previous gene editing methods, such as transcription activator-like effector nuclease (TALEN), by simplifying the engineering of gene specificity. This procedure includes two fundamental components: guide RNA (gRNA) used to locate the intended gene target, and Cas9, which is an endonuclease that modifies the intended genome location with a double-stranded DNA break^3,4. The gRNA will act as a guide for the Cas9 endonuclease to locate and initiate the double-stranded break at the intended genetic sequence through Watson-Crick base pairing. The full process of genome editing by the CRISPR/Cas9 system involves cellular machinery repairing these double-stranded breaks in DNA through non-homologous end joining (NHEJ) or homologous recombination^3,7. It is more likely that NHEJ will occur, effectively creating a mutation in the genome that results in a loss of expression for the target gene^3,4.

Commercial sources have been able to create libraries of gRNA targets that can be expressed through bacterial growth and isolation, which significantly improves their ease of use. However, the main limitation of the CRISPR/Cas9 system is the difficulty in delivering the gRNA and Cas9 complex into target cell lines. These limitations arise in suspension cell lines, as they are generally referred to as hard-to-transfect⁸. Typical transfection methods are not generally efficient in delivering the CRISPR/Cas9 system into suspension cells, which is why viral delivery methods like lentiviral transfection and transduction are better suited for this type of cell line^8,9.

This type of transfection requires a lentiviral vector that encodes the gRNA and Cas9 endonuclease along with added lentiviral packaging plasmids, which are transfected into a cell line that is capable of manufacturing lentiviral particles. A typically chosen cell line for this process is HEK293T cells, as they are easier to transfect and work very efficiently in the assembly of gRNA and Cas9^9,10. These particles are then released as lentiviruses into the supernatant, which can be used to transduce the gRNA and Cas9 into the intended suspension cell line, such as U937 human monocytes. As such, the procedure described here has the following changes compared to established methods: (1) Alternate transfection method for hard-to-transfect cell lines; (2) No need to concentrate CRISPR plasmid DNA or use ultracentrifuge; and (3) It eliminates the need for single-cell cloning.

The direct focus of this article was to knockout the RIP1 gene in U937 human monocytes. The canonical form of the highly inflammatory cell death pathway necroptosis is controlled by RIP1, which serves as a pivotal target for cell death studies.^11,12,13,14 As RIP1 becomes active through autophosphorylation, it then recruits and causes direct phosphorylation and activation of receptor-interacting serine/threonine-protein kinase 3 (RIPK3/RIP3) and mixed lineage kinase domain-like (MLKL) pseudokinase to form the necrosome. Following this formation, the necrosome is free to move throughout the cell to interact with organelles such as the mitochondria^12,13. At the mitochondria, RIP1 potentiates a positive feedback loop with cellular metabolism, directly impacting the production of mitochondrial ROS, which in turn promotes further autophosphorylation of RIP1, necrosome formation, and the downstream execution of necroptosis^11,12,13,14.

While the focus of the current research group is the role of RIP1 in cell death, other reasons to study RIP1 include its roles in inflammation and infection. Upon activation by death receptors such as TNF receptors, RIP1 promotes the activation of the NF-κB signaling pathway, which triggers the transcription of pro-inflammatory cytokines, chemokines, and other molecules essential for immune cell recruitment and the amplification of the inflammatory response¹⁵. In addition to NF-κB activation, RIPK1 can also engage MAPK signaling pathways, further enhancing inflammation^15,16. Regarding its role in responses to infection, RIP1 acts as a pivotal mediator of the host inflammatory response, particularly in response to pathogen-associated molecular patterns (PAMPs) recognized by pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs)¹⁷. Moreover, during sepsis, RIP1 is activated by signaling through death receptors such as the TNF receptor, leading to the initiation of pro-inflammatory cascades. RIPK1 mediates the activation of the NF-κB and MAPK pathways, promoting the production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, which are key drivers of the systemic inflammatory response characteristic of sepsis¹⁸.

Protocol

A schematic representation of the procedure is provided in Figure 1. Guide RNA (gRNA) and the target sequence are given in Table 1. Details of the reagents and the equipment used are listed in the Table of Materials.

1. Harvesting of RIP1 -targeting CRISPR gRNA lentiviral expression vector containing Cas9 endonuclease and puromycin resistance from Escherichia coli

1. Quadrant-streak E. coli harboring lentiviral vector gRNAonto LB agar plates supplemented with 100 µg/mL Ampicillin.
2. Incubate plates at 37°C for 1-2 days until individual colonies are visible in the most dilute area on the plate.
3. Select a single colony with a sterile loop and individually add each colony to 5 mL of LB broth supplemented with 100 µg/mL Ampicillin into a 50 mL conical tube. Ensure to pipette thoroughly up and down after adding the single colony to ensure homogenization.
4. Vent and tape down the cap of the 50 mL conical tube and incubate in an orbital shaker at 37°C and 225 rpm for 8 h.
5. During the 8 h incubation at 37°C, add 40 mL of LB broth supplemented with 100 µg/mL Ampicillin to each of four separate 50 mL conical tubes.
6. Following the 8 h incubation at 37°C, take 40 µL of grown E. coli culture and add it to all four 50 mL conical tubes.
7. Vent and tape down the caps of the 50 mL conical tubes and incubate them in an orbital shaker at 37°C and 225 rpm for 12-16 h.
8. Following this incubation at 37°C, spin down all tubes at 3220 x g in a swinging bucket rotor for 20 min to pellet the grown E. coli bacteria.
9. Decant supernatants to a waste beaker, then combine all four pellets in 10 mL of LB broth supplemented with 100 µg/mL Ampicillin.
10. Spin down the combined pellets again at max speed in a swinging bucket rotor for 20 min to get a singular pellet.
11. Discard as much of the supernatants as possible into the waste beaker, and proceed with one of the following options: (1) Freeze wet pellet as is at -80° for up to 1 month. (2) Proceed with lentiviral vector plasmid purification.
    NOTE: If option 1 is chosen, the protocol may be paused here.

2. Transfection of HEK293T cells with purified CRISPR gRNA lentiviral expression vector targeting RIP1

1. Seed HEK293T cells overnight at a concentration of 3 × 10⁶ to 5 × 10⁶ cells into a 10 cm plate containing DMEM with 4.5 g/L D-glucose, L-glutamine, 110 mg/L sodium pyruvate, and 10 % heat-inactivated FBS.
2. The following day, ensure plates have approximately 70%-90% confluency, then proceed with the protocol.
3. Setup the transfection of the cells with a 1:1:1:1 ratio of plasmids (experimental CRISPR plasmid: pLP1: pLP2: pLP/VSVG) using the transfection reagent in a 3:1 ratio (3 parts reagent: 1 part plasmid DNA), along with reduced serum medium.
   NOTE: The pLP1: pLP2: pLP/VSVG portion of the ratio is a lentiviral packaging mix.
   1. Equilibrate the transfection reagent, reduced serum medium, CRISPR plasmid DNA, and lentiviral packaging mix to room temperature prior to setup of transfection.
   2. Mix together 75 µL of the transfection reagent, 2500 µL of reduced serum medium, and 25 µg total DNA (calculation based on spectrophotometer analysis for CRISPR plasmid DNA and 1:1:1:1 ratio with lentiviral packaging mix).
   3. Allow this mixture to incubate at room temperature for 15-30 min. Carefully pipette the entire volume of this mixture to the confluent HEK293T cell plate right into the media (no need to change media).
4. Gently swirl the plate to ensure adequate homogenization of the transfection mix and plate media. Incubate the plate at 37 °C for 48 h.

3. Lentiviral transduction of U937 cells or target cells

1. Once the 48-h incubation at 37^°C is complete, transfer the media from the producer HEK293T cell plate into a 15 mL conical tube.
2. Centrifuge the volume at 800 x g for 5 min at room temperature to pellet any remaining HEK293T cells. Remove all the virus-containing supernatant, taking care not to disturb the pelleted HEK293T cells, and keep it in a separate 15 mL conical tube. Then, decontaminate and discard the pellet-containing tube in the appropriate biohazard waste container.
   NOTE: HIV-based lentivirus supernatants can be stored at -80°C; however, doing so carries a risk of up to 55% loss of virus stability after the first freeze/thaw cycle¹⁹.
3. Count and obtain a pellet of 2 × 10⁶ U937 cells in a 15 mL conical tube by centrifuging an appropriate volume at 400 x g for 10 min at room temperature and decant the supernatant, leaving the cell pellet in the tube.
4. Resuspend the U937 cell pellet with all the virus-containing supernatant and then centrifuge the tube at 290 x g for 60 min.
5. Following centrifugation, use a pipette to resuspend the pellet with the virus-containing supernatant already in the tube.
6. Place the tube on an end-over-end rotator (or similar rotating device) for 60 min.
   1. Following this incubation, centrifuge the tubes at 400 x g for 10 min at room temperature to pellet the cells.
   2. Resuspend the cell pellet with a 1:1 ratio media mixture of both virus-containing supernatants and complete RPMI-1640 media supplemented with 10% heat-inactivated FBS.
7. Transfer the cell mixture to a 10 cm tissue culture plate and incubate at 37°C for 48 h.
8. Following this incubation, centrifuge the U937 cells at 400 x g for 10 min and remove the supernatant.
   1. Resuspend the pellet with complete RPMI-1640 media supplemented with 10% heat-inactivated FBS and 5 µg/mL of puromycin and transfer the cells to a T25 flask.
   2. Incubate cells at 37 °C untouched for 2-3 weeks, ensuring to check on them every 1-2 days for signs of cell growth.
      NOTE: Once cells become confluent and have a healthy turnover time following an initial passage, they can be tested for the effectiveness of the completed protocol.

4. Testing effectiveness of the completed protocol in the creation of RIP1 CRISPR mutant cells using Western blot analysis

1. Generate cellular protein lysates of wild-type (WT) U937 monocytes, non-targeting control (NTC) CRISPR cells, and the RIP1 CRISPR mutant cells^11,12,13.
2. Run these lysates on an SDS-PAGE gel and proceed with Western blot analysis¹⁴.
   1. Normalize samples to a housekeeping protein first.
   2. Following normalization, analyze samples for RIP1 protein expression levels to adequately determine the success of creating a RIP1 CRISPR mutant cell line¹⁴.
3. Following western blot analysis conformation, cells can be used for experimentation purposes.

Results

Following the production of a confluent population of RIP1 CRISPR mutant U937 cells, SDS-PAGE and Western blot analysis were performed. The Western blot analysis was used to determine the successful creation of a RIP1 CRISPR mutant cell line by assessing the loss of RIP1 protein expression levels. This determination was made based on the comparative result of WT U937 monocytes and NTC cells. In Figure 2, the expression of RIP1 was not detected for the 5 µg/mL puromycin...

Discussion

This protocol aims to provide detailed instruction and analysis of potential pitfalls in the efficiency and reliability of lentiviral transfection and transduction to create an RIP1 knockout U937 cell line. Although this method of transfection and transduction is labor and time-intensive, it is generally considered to be an efficient way to incorporate the chosen gRNA and Cas9 endonuclease into hard-to-transfect cell lines^8,9,

Materials

Name	Company	Catalog Number	Comments
Adjusted DMEM Medium	Gibco	11995-040
Ampicillin	Sigma	A1593
bisBenzimide Hoechst 33342 trihydrochloride	Sigma	B2261
Complete RPMI-1640 Medium	Sigma	R6504
CRISPR NTC gRNA E.coli Strain	transOMIC	TELA1011
CRISPR RIP1 gRNA E.coli Strain	transOMIC	TEVH-1162203
End-over-end Rotator	Thermo Scientific
EVOS FL Fluorescence Microscope	Life Technologies
GenElute Plasmid Maxiprep Kit	Sigma	PLX15
Goat Anti-Rabbit IgG Antibody, (H+L) HRP conjugate	Sigma	AP307P
HEK293T Cells	ATCC
Incubator Shaker	New Brunswick Scientific
LB Agar	BD	244520
LB Broth	BD	244610
LV-MAX Lentiviral Packaging Mix	Gibco	A43237
MitoSOX Red	MedChemExpress	HY-D1055
NanoDrop Spectrophotometer	Thermo Scientific
Necrostatin-1	MedChemExpress	HY-14622A
OPTI-MEM	Gibco	31985-062
Puromycin	Sigma	P7255
Rabbit anti-human RIP1 mAb	Cell Signaling Technology	3493
SDS-PAGE and western blot equipment	BioRad
TNF-α	MedChemExpress	HY-P7058
U937 Human Monocytes	ATCC
WST-1 Cell Proliferation Assay System	TaKaRa	MK400
X-tremeGENE 9 DNA Transfection Reagent	Roche Diagnostics	6365779001
z-VAD-FMK	APExBIO	A1902