1. Construction of a Heptamer-type sgRNA Library

1. Select a subset of the 16,384 (4⁷) 7-nt sequences unless the full-scale library is to be constructed.
   NOTE: The sequences can be random and/or designed to target specific cellular RNAs. For the latter, find hairpin structures resembling the T-arm of tRNA in a target RNA with the aid of an appropriate computer program and/or visually, and select sequences complementary to 7-nt sequences immediately downstream of the hairpin structures^4,10,17,18.
2. Synthesize each of the selected heptamers as a fully 2′-O-methylated, 5′- and 3′-phosphorylated RNA with a DNA/RNA synthesizer. Use the phosphoramidite method on the controlled-pore glass (CPG) support, and leave the 5′-terminal 4,4′-dimethoxytrityl (DMT) protecting group on in the last cycle (the addition of the 5′ phosphate) of the synthesis²³.
   NOTE: Custom synthetic RNA can be also obtained from oligonucleotide suppliers.
3. After completion of the last synthesis cycle, transfer the CPG with the synthesized oligonucleotide from the column to a glass vial with a Teflon-liner screw cap, add 1 ml of 29% ammonium hydroxide, and incubate it at 55 °C for 8 hr to cleave the oligonucleotide from the CPG and remove the protecting groups from the bases and phosphates.
4. Evaporate ammonium hydroxide with a centrifugal evaporator at 40 °C and re-dissolve the oligonucleotide in 0.2 ml of 0.1 M n-hexylammonium acetate.
5. Purify the synthetic oligonucleotide through reverse-phase high-performance liquid chromatography using a polystyrene-divinylbenzene column with a buffer containing 10-50% acetonitrile/0.1 M n-hexylammonium acetate. Run the column at 2.0 ml/min for 25 min at 80 °C.
6. Add 0.25-0.5 ml of concentrated acetic acid to the fractionated sample (1-2 ml) to make a 20% acetic acid solution, and incubate this solution for 1 hr at room temperature to remove the DMT group.
7. Dry the sample with a centrifugal evaporator at 40 °C, re-dissolve it in 1 ml of 29% ammonium hydroxide, and incubate the solution for 15 min at room temperature to remove the side chain of the 5′ phosphate.
8. Reduce the volume (1 ml) of the sample to about 0.3 ml with a centrifugal evaporator at 40 °C.
9. Dialyze the entire concentrated sample against distilled water (500 ml) using a dialysis tube of molecular weight cut-off 1,000 at 4 °C overnight.
10. Transfer the dialyzed sample from the dialysis tube to a 1.5-ml tube with a pipette, dry it with a centrifugal evaporator at 40 °C, and subsequently re-dissolve it in water-for-injection-grade water, the volume of which should be small enough to make a greater than 100 µM solution.
11. Measure an optical density of the RNA sample at 260 nm with a spectrophotometer and adjust its concentration to 100 µM by adding water-for-injection-grade water. Store the RNA solution at below -20 °C before use.

2. Screening of the sgRNA Library for Cell Viability

1. Prepare 10³ cells/99 μl/well (e.g., HL60 and RPMI-8226) on a 96-well dish in RPMI-1640 media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Prepare wells containing the media only for background subtraction. To eliminate edge effects, add sterile water to empty wells surrounding the sample wells.
2. Add 1 μl of heptamer-type sgRNA (100 μM) dissolved in water-for-injection-grade water to each well. Assay each sgRNA in triplicate. Incubate the plate at 37 °C in a 5% CO₂ humidified incubator for 3 days.
3. Add an appropriate volume of a commercially available WST-8 solution to each well. Incubate the plate at 37 °C in the same incubator for 1 to 4 hr.
4. Measure absorbance at 450 nm with a microplate reader.
   NOTE: In most cases, linearity between absorbance and viable cell number appears to be maintained unless the absorbance value exceeds 1.0.

3. Evaluation of Effective sgRNAs by Flow Cytometry

1. Prepare 10³ cells/99 µl/well (e.g., HL60) on a 96-well dish in RPMI-1640 media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Prepare at least 10 wells for one sgRNA to be tested. To eliminate edge effects, add sterile water to empty wells surrounding the sample wells.
2. Add 1 µl of heptamer-type sgRNA (100 µM) dissolved in water-for-injection-grade water to each well. Incubate the plate at 37 °C in a 5% CO₂ humidified incubator for 3 days.
3. Collect the cells treated with the same sgRNA from at least 10 wells into a 5-ml tube with a pipette. Spin the tube for 5 min in a centrifuge at 300 x g, and discard the media.
4. Add 2 ml of 1x phosphate buffered saline (PBS) to the tube, and re-suspend the cells. Spin the tube for 5 min in a centrifuge at 300 x g, and discard PBS. Add 150 µl of 1x annexin V binding buffer to the tube, and re-suspend the cells.
5. Add 2 µl of phycoerythrin (PE)-conjugated annexin V solution and 1 µl of 7-aminoactinomycin D (7AAD) solution to the tube, mix them well, and incubate the tube for 15 min at room temperature in the dark. Analyze the sample using a flow cytometer²⁴.