CRISPR Screen Sequencing Protocol

Genomic DNA Isolation

1. Clean the genomic DNA station with 10% bleach, followed by water and finally 70% ethanol. Prepare aliquots of required amounts of Wizard Genomic DNA Purification Kit solutions. Prewarm TE buffer (Rehydration Solution) to 65 °C and prepare 70% ethanol (for samples) using UltraPure water.
2. Thaw cell pellets at room temperature for 5–10 min.
3. Resuspend cells in 1 ml PBS and transfer to a 50-ml centrifuge tube. Rinse the original tube with 400 μl PBS and add to the same 50-ml tube.
4. Add 5 ml Nuclei Lysis Solution to the resuspended cells. Mix by pipetting with 10-ml pipette (five times).
5. Add 32 μl RNase A (20 mg/ml stock; final concentration of 100μg/ml) to the nuclear lysate and mix by inverting the tube five times. Incubate at 37 °C for 15 min and then let cool down to room temperature (~10 min).
6. Add 1670 μl Protein Precipitation Solution to the nuclear lysate and vortex vigorously for 20 s.
7. Centrifuge at 4500 × g for 10 min at room temperature.
8. Using a 10-ml pipette, transfer the supernatant to a 50-ml centrifuge tube containing 5 ml isopropanol. Be very careful not to transfer any precipitate.
9. Gently mix the solution ten times by inversion, until the white thread-like strands of DNA form a visible mass.
10. Centrifuge at 4500 × g for 5 min at room temperature. The DNA will be visible as a small white pellet.
11. Carefully remove the supernatant, avoid dislodging the pellet. Add 5 ml 70% ethanol to the DNA. Gently rotate the tube to wash the DNA pellet and the sides of the centrifuge tube.
12. Centrifuge at 4500 × g for 5 min at room temperature.
13. Carefully and thoroughly remove all ethanol, avoid dislodging the pellet. Air-dry the genomic DNA at room temperature for 10–30 min until pellet appears translucent. Do not overdry (before visible cracks appear). Avoid contamination by dust particles.
14. Add 400μl TE buffer (DNA Rehydration Solution) and let DNA dissolve by incubating at 65 °C for 1–4 h. Mix DNA by gently flicking the tube every 15 min until the pellet is completely dissolved. Suspension may be slightly viscous but should not contain any clumps of DNA.
15. Centrifuge at 4500 × g for 1 min at room temperature and transfer genomic DNA to a 1.5-ml low-binding tube.
16. Quantitate DNA using a Qubit fluorometer, and store at −20 °C.

Sequencing Library Preparation

1. Always include the T0 sample to determine library representation in the screen and allow gRNA fold change calculation.
2.Clean the PCR1 and PCR2 work stations with 10% bleach, followed by water and finally 70% ethanol.
3. Prepare a 1% agarose gel containing SYBR Safe DNA stain for checking PCR1 amplified products. PCR1 enriches the gRNA cassette from the genome.
4. In the PCR hood: set up one diagnostic reaction (do not add genomic DNA yet) and one no-template negative control.
5. Check PCR amplification by running 2μl of the PCR product on the prepared agarose gel. Expected product size is ~600 bp.
6. Amplify PCR1 reactions in a thermocycler using the program.
7. Proceed to the PCR2 work station and pool all 14 + 1 (diagnostic) PCR1 reactions in a 1.5-ml microfuge tube.
8. Soak agarose gel equipment (gel tray, comb and gel tank) for purifying amplified PCR2 products with 0.1 N HCl for 10 min prior to casting gel. Rinse 4 times with tap water and once with purified water.
9. Prepare a 2% agarose gel containing SYBR Safe DNA stain for purifying PCR2 amplified products. Use large combs and allow for ample space between samples. PCR2 amplifies the gRNA cassette and adds Illumina TruSeq adapters with i5 and i7 indices.
10. Set up PCR2 reactions (do not add template PCR1 product yet). Prepare no-template controls as desired.
11. Run the complete PCR2 reaction on the prepared 2% agarose gel at low voltage (1.5–2 h run). Visualize the PCR product on a blue light transilluminator, the expected product size is ~200 bp. Multiple bands may be visible; it is important to separate the bands well.
12. Excise the 200 bp band with a new razor blade, and transfer the gel slice to a 1.5-ml tube.
13. Purify DNA from agarose gel slice using a gel extraction kit. We recommend repeating each binding step twice and including intermittent washes with binding buffer if binding volumes are large. We also recommend a double elution to maximize yield.
14. Store gel-purified DNA in a 1.5-ml low-binding tube.
15. Quantify DNA on Qubit fluorometer.

Next-Generation Sequencing

1. Due to differences in sample submission requirements and quality controls applied by next-generation sequencing facilities and providers, we only provide general recommendations in this section.
2. Sequencing libraries can now be pooled. The degree of multiplexing depends on the desired read depth per sample. For dropout screens, we recommended to read each sample at 200-fold library coverage and 500-fold coverage for T0 samples to ensure robustness during data analysis due to the absence of independent replicates at T0. For strong positive selection screens aiming for identification of enriched gRNAs, 50-fold coverage may be sufficient.
3. Sequencing on Illumina HiSeq2500 or NextSeq550 with standard primers for dual indexing.

Reference:

1. Mair B, Aregger M, Tong A H Y, et al. A Method to Map Gene Essentiality of Human Pluripotent Stem Cells by Genome-Scale CRISPR Screens with Inducible Cas9[J]. Essential Genes and Genomes: Methods and Protocols, 2022: 1-27.