TITLE: In vivo Nanopool: Pooled sgRNA competition assays to evaluate in vivo cancer dependencies
AUTHORS: Thomas Skipper, Daniel Bondeson, Brenton Paolella, Francisca Vazquez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">sgRNA competition assays with constitutive Cas9 systems allow for rapid de-risking of targets in a more representative in vivo microenvironment. Though not a full in vivo validation, the assay is useful as an “invalidation” strategy to discern artifacts of in vitro cultre from promising bona fide targets. This document describes a protocol to select an appropriate cell line, design and generate an sgRNA library for targets of interest, and perform parallel in vitro/in vivo experiments to assess in vivo dependency of the selected genes.</div></div>

[STEPS]
?. [Develop cell line model]
Choose dependent cell lines using the Cancer Dependency Map portal. Search the literature to determine whether the cell lines proliferate subcutaenously in vivo.
?. [Develop cell line model]
Plate and infect cell lines of interest to assess Cas9 expression.
?. [Develop cell line model]
Infect with 100µL Cas9 nuclease lentivirus and add 4µg/mL polybrene. Add RPMI with 10% FBS (R10) to a final volume of 2mL.
?. [Develop cell line model]
Plate 400,000 cells per well in 6-well plate.
?. [Develop cell line model]
Lift cells the next day and split into two 6-well plates per cell line. Select one with R10 + 10µg/mL blasticidin (10B). We use high concentrations of blasticidin to select for high levels of expression of the integrated transgenes.
?. [Develop cell line model]
Calculate infection efficiency by lifting all cells, counting live cells with a ViCell Coulter counter, and dividing counts in plus puro (selected) by minus puro wells. No Infection Control (NIC, 0µL virus) should be close to zero to pass quality control check.
?. [Develop cell line model]
Expand selected (plus puro) cells and maintain 10B in media. Also expand unselected NIC as a negative control.
?. [Develop cell line model]
Collect pellets from both conditions and rinse twice in cold PBS. Run a western to confirm robust Cas9 expression. As Cas9 is a large protein, it transfers best by wet transfer protocols.
?. [Develop cell line model]
Expand Cas9-expressing cells for in vivo model development in potential dependent cell lines. The exact cell counts will vary according to cell line growth rate. Standard subcutaneous tumor sizes are 4-10 million cells per 100-200 µL injected.
?. [Develop cell line model]
Initiate 2 subcutaneous tumors bilaterally per mouse at variable tumor sizes. Record mouse weight and tumor size every 3 days.
?. [Develop cell line model]
Euthenize mice at humane endpoint. Note tumor latency and initial cell count that leads to tumor growth for 3-4 weeks.
?. [Develop cell line model]
Choose cell line with strong dependency, expression of Cas9, and robust in vivo growth kinetics to carry forward to next steps.
?. [Generate and quality control small library of guides]
Design library of single guide RNAs (sgRNA). See "Guidelines" section for library considerations.
?. [Generate and quality control small library of guides]
Prepare plasmid for each sgRNA and accurately quantify DNA concentration following midi- or maxiprep. Accurate quantification of the plasmid virus is essential to achieve equal representation in the pool. We recommend using fluorescent DNA quantification (like Qubit Broad Range Assay) instead of UV-based spectroscopy (like NanoDrop).
?. [Generate and quality control small library of guides]
Prepare lentivirus in arrayed format using HEK293T cells. Pool to generate about 75mL of virus. Freeze down 4 x 10mL aliquots for future in vivo experiment and the rest in 1mL aliquots at -80C.
?. [Generate and quality control small library of guides]
Quality control and titrate the pooled virus by infecting cell line of interest.
?. [Generate and quality control small library of guides]
Thaw one aliquot to infect one 6-well plate per cell line of interest with a range of viral titers along with 4µg/mL polybrene. Aim for 500x representation, or 500 cells per guide, to ensure adequate representation. We suggest 0µL, 25µL, 50µL, 100µL, 500µL of pooled virus.
?. [Generate and quality control small library of guides]
The next day, split each well into 2 equivalent wells of a plus selection and minus selection 6-well plate. Add RPMI + 2µg/mL puromycin to plus selection plate, normal RPMI to the other.
?. [Generate and quality control small library of guides]
After two days, calculate infection efficiency by lifting all cells, dividing cell counts in plus puro (selected) by minus puro wells. Determine the viral titer at which infection efficiency is 30-50%. At these infection percentages, each cell is likely infected with only one sgRNA. Pellet two or more replicates of cells at 500 times the number of guides in the pool.
?. [Generate and quality control small library of guides]
Extract gDNA from cell lines using QIAGEN DNA Mini Kit.
?. [Generate and quality control small library of guides]
PCR-amplify 250 ng (100ng minimum - 10µg maximum) gDNA in a 100µL reaction. Lower concentrations of gDNA will benefit from additional cycles, up to 32. P5 and P7 primers amplify barcode regions for Illumina sequencing.
?. [Generate and quality control small library of guides]
Per reaction, assemble the following on ice: 10µL 10x reaction buffer, 8µL dNTP, 0.5µL P5 primer and P7 primer (100µM), 1.5µL Takara ExTaq polymerase (Clontech). Up to 100µL with water.
?. [Generate and quality control small library of guides]
Run PCR with following thermocycler conditions: (i) 95°C for 1 minute, (ii) 95°C for 30s, (iii) 58°C for 30s, (iv) 72°C for 30s. (v) Repeat steps ii-iv for 28 cycles. (vi) 72°C, 10 minutes, (vii) 4°C hold.
?. [Generate and quality control small library of guides]
For troubleshooting, try a range of annealing temperatures 54-62°C, more cycles, or a range of input gDNA concentrations.
?. [Generate and quality control small library of guides]
Run 10µL of 100µL PCR product on DNA gel to confirm amplification. Visualize by agarose gel electrophoresis, looking for crisp single band of about 400 nucleotides.
?. [Generate and quality control small library of guides]
Clean up PCR products with QIAGEN PCR Purification kit, elute into 50µL water or EB.
?. [Generate and quality control small library of guides]
Submit DNA for CRISPR Illumina sequencing.
?. [Generate and quality control small library of guides]
Analyze data from read counts.
?. [Generate and quality control small library of guides]
Extract read counts from Fastq files using open source MaGECK software.
?. [Generate and quality control small library of guides]
Analyze representation of each guide in the pool, checking for representation of about 50% negative control guides, 20% positive controls, and 30% experimental guides. This will prevent excessive tumor regression early in the in vivo arm.
?. [Generate and quality control small library of guides]
If the library passes this quality control check, proceed to the next section with the infected cell line of interest.
?. [Parallel in vitro/in vivo experiments]
Spinfect cells following Broad's standard protocol. This puromycin optimization step requires about 30 million cells of interest, confirmed to be mycoplasma-free. Puromycin concentration optimization is designed to minimize time in tissue culture before inoculation, as the constitutive CRISPR/Cas9 system will start cutting DNA immediately upon infection with lentiviral guides.
?. [Parallel in vitro/in vivo experiments]
After expansion and spinfection in 6-well plates, add 2mL R10 plus 0µg/mL (0P), 4µg/mL (4P), or 8µg/mL (8P) puromycin on top of existing 2mL R10. A total of five 6-well plates will be necessary from spinfection for 3 0P control plates harvested at 24, 48, and 72 hours from selection.
?. [Parallel in vitro/in vivo experiments]
After 24 hours, visually inspect plates. When selection is complete, lift and assess infection efficiency by comparing cells counts of 2P and 4P to minus puro condition. Overselection is likely if infection efficiency is much lower for higher concentrations of puromycin at constant viral titer. Aim for the viral titer and puromycin concentration that confers 30-50% infection efficiency.
?. [Parallel in vitro/in vivo experiments]
Calculate how many cells need to be infected to have enough for in vitro/in vivo experiments at this infection efficiency. Infection efficiency and desired cell count will be known (for example, 7 million x 10 tumors + 30 million for in vitro = 100 million cells). Solve for total initial cells.
?. [Parallel in vitro/in vivo experiments]
Expand cell line of interest expressing Cas9 to "total initial cells" value calculated above.
?. [Parallel in vitro/in vivo experiments]
Lift and pool all cells together. Concentrate or dilute into 200 mL of media, along with 100µL polybrene and titrated amount of virus from 10.3. We typically use about 20mL pooled virus.
?. [Parallel in vitro/in vivo experiments]
Distribute 3 million cells/2mL media in each well of 12-well plates (about four plates for 150 million cells) for infection.
?. [Parallel in vitro/in vivo experiments]
Spinfect for 2 hours at 931g.
?. [Parallel in vitro/in vivo experiments]
After spin, add 2 mL R10 and incubate for 4-6 hours.
?. [Parallel in vitro/in vivo experiments]
Distribute into maximum-volume flasks or plates. For example, 30 x 15cm plates of 5 million cells and 12 mL media.
?. [Parallel in vitro/in vivo experiments]
Add selection reagent at optimized concentration. To avoid disturbing cells, we add 3x concentrated stock, dissolved in medium, to the cells. For example, adding 6 mL of R10 + 18 ug/mL puromycin to 12 mL of medium yields 18mL media at a final concentration of 3 ug/mL.
?. [Parallel in vitro/in vivo experiments]
After 24 hours, start early time point pellet (ETP) collection, in vitro plating, and in vivo inoculation.
?. [Parallel in vitro/in vivo experiments]
Trypsinize and pool all cells together. Accurately count the number of cells via duplicate measurements using the Vicell.
?. [Parallel in vitro/in vivo experiments]
Freeze two cell pellets of 1000x sgRNA representation for downstream determination of the “initial representation” of each sgRNA.
?. [Parallel in vitro/in vivo experiments]
Using standard tissue culture seeding densities, etc., passage duplicate “in vitro screens” at 1000x sgRNA representation, typically two T125 flasks. Passage as needed and collect timepoints at days 7 and 14 from infection. For this in vitro arm, use plain R10 without antibiotics to mimic lack of selection markers in vivo.
?. [Parallel in vitro/in vivo experiments]
Pellet 10 x desired cells/tumor from model development experiments. Should be in the range of 5-10 million cells. For example, 10 million cells per xenograft for slow-growing cell lines would require 100 million cells.
?. [Parallel in vitro/in vivo experiments]
Resuspend in 500µL R10. Add 500µL matrigel. Keep 10 x 100µL aliquots on ice.
?. [Parallel in vitro/in vivo experiments]
On the same day, inoculate mice with bilateral subcutaneous tumors.
?. [Parallel in vitro/in vivo experiments]
Check isoflurane levels and charcoal. Set up anaesthesia for tank and nose cone in vivarium procedure room.
?. [Parallel in vitro/in vivo experiments]
Anaesthetize mice. One at a time, transfer mice to the nose cone in the hood and check depth of consciousness with a toe pinch.
?. [Parallel in vitro/in vivo experiments]
Moving quickly, vortex cell/matrigel suspension and draw 100µL into syringe. Be careful to avoid bubbles and keep the cell/matrigel suspensions cold.
?. [Parallel in vitro/in vivo experiments]
Inject on mouse flank, keeping bevel of the needle up and lifting skin with forceps to create a pocket. Repeat on opposite flank.
?. [Parallel in vitro/in vivo experiments]
After injection, transfer mouse back to cage. Ensure mouse returns to alert and active consciousness.
?. [Parallel in vitro/in vivo experiments]
Repeat for each of the 5 mice.
?. [Parallel in vitro/in vivo experiments]
Complete health checks and tumor measurements every 3 days.
?. [Parallel in vitro/in vivo experiments]
Visually check health of the mice.
?. [Parallel in vitro/in vivo experiments]
Weigh mice with scale, record on ‘tumor-tracking’ spreadsheet.
?. [Parallel in vitro/in vivo experiments]
Measure width and length of tumors consistently using calipers.
?. [Parallel in vitro/in vivo experiments]
To capture maximum guide dropout, collect in vitro pellets (2 x 7 million cells) on days 7 and 14. Time points should be adjusted for dependencies that confer viability effects at a different rate.
?. [Parallel in vitro/in vivo experiments]
On days 14, 21 and 28, harvest tumors from randomly selected mouse and collect in vitro pellets:
?. [Parallel in vitro/in vivo experiments]
Transfer all mice except the mouse to be sacked into a new cage.
?. [Parallel in vitro/in vivo experiments]
Sack mouse in home cage using CO2, verify with cervical dislocation.
?. [Parallel in vitro/in vivo experiments]
Harvest both tumours, removing as much skin as possible. Dispose of carcass appropriately.
?. [Parallel in vitro/in vivo experiments]
Weigh and wash tumors in PBS.
?. [Parallel in vitro/in vivo experiments]
Flash freeze tissue sample in Liquid N2 until ready to isolate genomic DNA for sequencing.
?. [Parallel in vitro/in vivo experiments]
Homogenize all tumor tissue samples by mincing then lysing overnight in ATL buffer
?. [Parallel in vitro/in vivo experiments]
Repeat steps 11-13 to extract gDNA from each tissue and pellet sample using QIAGEN DNEasy Blood and Tissue kit, amplify barcode, and submit samples fro sequencing.
?. [Parallel in vitro/in vivo experiments]
Repeat steps 14-15 to submit samples then use MAGeCK to extract read counts from Fastq files.
?. [Parallel in vitro/in vivo experiments]
Suggested normalization steps to determine maximum efficacy/acceptable leakiness include:
?. [Parallel in vitro/in vivo experiments]
Normalize read counts to total “mapped” counts.
?. [Parallel in vitro/in vivo experiments]
Assess leakiness by normalizing “no dox” to early time point counts.
?. [Parallel in vitro/in vivo experiments]
Assess efficacy by normalizing “dox” to “dox” negative control counts at each time point.
?. [Parallel in vitro/in vivo experiments]