How to Design a CRISPR sgRNA Oligo Pool
Use this page when you need to turn a knockout, CRISPRa/i, or base-editing guide list into an order-ready pool. It covers pool sizing, pre-synthesis QC, cloning adapters, vendor fit, and sequencing thresholdsso you can catch avoidable failures before placing a CRISPR screen order.
Hundreds of thousands of synthesized sgRNAs directing Cas9 nucleases in a pooled genetic screen.
Key Facts
- •Oligo pools are 60-150x cheaper per guide than individual synthesis for library-scale CRISPR
- •Typical oligo length: 80-110 bp (spacer + scaffold + adapters), within all vendor limits
- •QC targets: ≥90% representation, <10% dropout, Gini <0.25, ≥500x NGS depth
- •Pre-synthesis batch QC catches 5-15% problematic guides (poly-T, extreme GC, hairpins)
CRISPR Screen Types & Pool Size Benchmarks
| Screen Type | Genes | Guides/Gene | Pool Size | Est. Cost |
|---|---|---|---|---|
| Genome-Wide KO (Brunello) | 19,114 | 4 | ~77K | Vendor quote |
| Genome-Wide KO (GeCKO v2) | ~19,000 | 6 | ~123K | Vendor quote |
| Genome-Wide CRISPRa/i | ~19,000 | 10 | ~200K | Vendor quote |
| Kinome/Druggable Genome | 500-2,000 | 8-10 | 4K-20K | $200-1.6K |
| Pathway-Focused | 50-500 | 6-10 | 300-5K | $50-400 |
| Tiling (Regulatory) | Loci-based | 1/5-10 bp | 1K-100K | $50-8K |
Add 1,000 non-targeting controls per library. Pricing varies by vendor — contact directly for quotes. Use Coverage Calculator for exact sizing.
5-Step Pre-Order Workflow for CRISPR sgRNA Pools
Select sgRNA Sequences
Use CRISPick (Broad), Benchling, or GPP to generate 4-10 guides/gene. Filter: Doench >0.5, MIT off-target >50.
Use Check GC Content →Screen for Synthesis Problems
Flag poly-T (>3 nt = Pol III termination), extreme GC (<25% or >75%), homopolymers (>4 nt), and hairpins (ΔG < -3 kcal/mol).
Use Batch QC →Add Scaffold & Adapters
Append tracrRNA scaffold (76 bp for SpCas9) + cloning adapters (CACCG for BsmBI). Final: ~105 bp.
Use Format for Vendor →Verify Full-Length Oligos
Re-check assembled oligos for new secondary structures at scaffold-spacer junctions. Verify length uniformity (±5 bp).
Use Check Structures →Submit Synthesis Order
Export as FASTA with unique IDs (gene_guideN). Select vendor based on pool size and budget. Request NGS QC.
Use Compare Vendors →QC Thresholds Before You Clone or Sequence
| Metric | Minimum | Ideal | Why It Matters |
|---|---|---|---|
| Representation | ≥90% | ≥95% | Missing guides = missed phenotypes |
| Dropout Rate | <10% | <5% | High dropout reduces screen power |
| Uniformity (CV) | <5-fold | <3-fold | Skew requires deeper sequencing |
| Gini Coefficient | <0.35 | <0.20 | Lower = more equal distribution |
| NGS Depth | ≥500x/guide | ≥1000x | Needed to detect rare guides |
Use Uniformity Estimator and Error Rate Calculator for pre- and post-synthesis analysis.
Which Vendor Fits Your CRISPR sgRNA Pool?
| Vendor | Best For | Pool Size | CRISPR Price |
|---|---|---|---|
| Twist Bioscience | Most CRISPR libraries | Min 2K, no stated max | NGS QC included |
| Agilent SurePrint | Large libraries, HiFi | Up to 244K/chip | 1:4,000 error (HiFi) |
| IDT oPools | Targeted panels + QC | Up to 20K (1 pmol) | 99.6% coupling |
| GenScript GenTitan | Custom, CMOS chip | Up to 8M/chip, max 170 nt | 3-5 biz day turnaround |
See full vendor comparison and vendor directory.
Frequently Asked Questions
How many oligos do I need in a CRISPR screening pool?▾
Which vendors are best for CRISPR oligo pools?▾
What is the typical oligo length for a CRISPR pool?▾
How do I QC a CRISPR oligo pool?▾
Can I reuse one oligo pool for multiple screens?▾
Next Pages to Open
Design a Custom NGS Panel Oligo Pool
Turn target regions into a hybrid capture or amplicon panel before ordering.
Design a Mutagenesis Oligo Pool
Plan DMS and saturation libraries with explicit variants and balanced coverage.
Assemble Synthetic Genes from an Oligo Pool
Plan fragment overlaps, amplification, and assembly before synthesis.
Design an Oligo Pool Before Ordering
Beginner-friendly guide to oligo pool design.
Read Oligo Pool QC Metrics
Uniformity, error rate, and QC metric deep dive.
Compare Oligo Pool Vendors
Twist vs Agilent vs IDT vs GenScript.