Materials and Reagents

Pipette tips
10 cm bacteriological tissue culture (TC) treated Petri dish (Corning, Falcon^®, catalog number: 353003 )
6-well plates TC treated (Corning, Costar^®, catalog number: 353502 )
15 ml Falcon tube (E&K Scientific Products, catalog number: EK-4020 )
50 ml Falcon tube (E&K Scientific Products, catalog number: EK-4023 )
Cell scraper (SARSTEDT, catalog number: 83.1830 )
0.45 μm syringe filters (Corning, catalog number: 431225 )
T-75 flask (Corning, Falcon^®, catalog number: 353136 )
T-125 flask (Corning, Falcon^®, catalog number: 353112 )
Cryovials (Corning, catalog number: 430659 )
RAW264.7 cells (ATCC, catalog number: TIB-71 )
293T cells (ATCC, catalog number: CRL-3216 )
hCas9 plasmid (Addgene, catalog number: 52962 )
FuGene (Promega, catalog number: E2311 )
VSV-G (Addgene, catalog number: 8454 )
pAdVAntage (Promega, catalog number: E1711 )
△VPR (Addgene; catalog number: 8455 )
Genome-wide Mouse lentiviral CRISPR gRNA library v1 (Addgene, catalog number: 50947 )
Filtered DMEM (Thermo Fisher Scientific, Gibco^TM, catalog number: 11995073 )
Filtered (0.2 μm filter) heat-inactivated (30 min at 37 °C) FBS (Thermo Fisher Scientific, Gibco^TM; stock specific)
Protamine sulphate (Sigma-Aldrich, catalog number: P3369-10G ), keep stock at 8 mg/ml at 4 °C
Blasticidin hydrochloride (MP Biomedicals, catalog number: 02150477-25 mg ), keep aliquoted stock at 10 mg/ml at -20 °C
DMSO (Fisher Scientific, catalog number: BP231-100 )
Puromycin dihydrochloride from Streptomyces alboniger (Sigma-Aldrich, catalog number: P8833 )
RAW264.7 and 293T tissue culture media (see Recipes)
Blasticidin selection media (see Recipes)
Puromycin selection media (see Recipes)
Tissue culture freezing media (see Recipes)

Equipment

Pipettes
Fluorescence microscope that can visualize BFP
Incubator at 37 °C with 5% CO₂
Centrifuge

Procedure

Making hCas9-expressing RAW264.7 cells
1. D0: Plate 5 x 10⁶ 293T cells in a tissue culture (TC) treated 10 cm plate in 10 ml DMEM + 10% FBS, and incubate overnight at 37 °C with 5% CO₂. We aim for 30-40% confluency.
2. D1: Create hCas9-expressing retrovirus in 293T cells.
3. D2:
4. D3: Harvest virus and transduce RAW cells
5. D4: Aspirate media off RAW264.7 cells and add new 37 °C DMEM + 10% FBS media to transduced RAW cells.
6. D5: Transfer transduced RAW264.7 cells from 6-well plate to TC treated 10 cm dish. This split should yield an ideal 30% confluence for antibiotic selection the next day. At this time, plate non-transduced RAW264.7 cells in 10 cm at a similar confluency as a negative control for your antibiotic selection (~1 x 10⁶ cells per 10 cm dish).
7. D6: Antibiotic selection
8. D7-8:
9. D8-9:
Amplifying CRISPR gRNA library
1. Follow protocol previously published (Koike-Yusa et al., 2014; https://media.addgene.org/data/67/85/9641c46c-8523-11e3-b6c5-000c298a5150.pdf). Use the ‘on-plate culture’ method of amplifying the library, as the ‘liquid culture’ method can lead to exclusion of some of the plasmids in the library (Figure 1).
2. Freeze back amplified CRISPR gRNA plasmid library.
  
  Figure 1. Graphical abstract of Koike-Yusa et al. (2014) amplification protocol of the gRNA library
Creating CRISPR gRNA lentiviral library
1. Before starting, see Note 5.
2. D0: Plate 1 x 10⁶ 293T cells per T-175 in 25 ml of DMEM + 10% FBS.
3. D1: Creating CRISPR gRNA lentiviral library.
4. D2:
5. D3: Harvest virus and transduce RAW264.7 cells.
6. D4: Plate 5 x 10⁶ RAW264.7-hCas9 expressing cells in a T-175 flask in 25 ml of 37 °C DMEM + 10% FBS for antibiotic selection negative control.
7. D6: Check transfection efficiency using fluorescence microscope and antibiotic selection.
8. D9: Split and expand RAW264.7 cells transfected with the CRISPR gRNA lentiviral library.
9. D13: Pool and freeze RAW264.7 cells transfected with the CRISPR gRNA lentiviral library.

Data analysis

For sequencing your library and analyzing the coverage of your library or your library results, please refer to our original publication (Napier et al., 2016).

Notes

Late in the day, you may start to see syncytia. Syncytia are a sign that the viruses are being produced.
Retroviruses more easily infect mitotic cells, thus by harvesting twice from the same well in the AM and PM, you will double your chances of catching the RAW264.7 cells dividing.
You can store the rest in 4 °C for re-infection later for up to 7 days. However, freezing decreases titers significantly.
In our hands, transduction of RAW264.7 cells do not change the immunological profile of the cell line, however, if another cell line is being used in this protocol, it is imperative to use a retroviral control with a non-coding sequence to ensure that the phenotype of interest is not altered after transduction.
This protocol has been standardized for cell transfection efficiency of 30% by lentivirus produced by 293T cells. This lower transfection efficiency is used to decrease the frequency of double or triple transfection events happening the same cell. You will be transfecting 1,000-fold the gRNA library in cells. For this protocol, there are 87,897 gRNAs (Koike-Yusa et al., 2014); therefore, we transfect (88 million RAW246.7 cells) x (30%) = 264 million RAW246.7 cells to maintain 1,000-fold coverage with 30% transfection efficiency.

Recipes

RAW264.7 and 293T tissue culture media
37 °C DMEM
10% FBS
Blasticidin selection media
37 °C DMEM
10% FBS
10 μg/ml blasticidin
Puromycin selection media
37 °C DMEM
10% FBS
5 μg/ml puromycin
Tissue culture freezing media
50% DMEM
40% FBS
10% DMSO

Acknowledgments

This research was supported by the National Institute of Allergy and Infectious Diseases grants 1F32AI115959-01 (to B.A. Napier) and AI095396-05 (to D.M. Monack), and Defense Advanced Research Projects Agency (DARPA) grant DARPA-15-21-ThoR-FP-006 (to D.M. Monack).

References

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