CRISPR Library Design Guide: sgRNA Selection, Pool Synthesis & QC
How to design a CRISPR screen library: Select 4-10 high-activity sgRNAs per gene targeting constitutive exons, score with Azimuth/Rule Set 2 for on-target activity and CFD for off-target specificity, then synthesize as an oligo pool with amplification handles and cloning adapters. This guide covers library types (CRISPRko/a/i), Cas system selection, oligo architecture, pool synthesis QC, and screen analysis. Use our Coverage Calculator, GC Analyzer, and Batch QC Tool for library design and validation.
Key Takeaways
- •Use 4-10 sgRNAs per gene for knockout screens — more guides increase statistical power and reduce false negatives.
- •Select sgRNAs with GC content 40-70%, avoid TTTT (polymerase III terminator), and target constitutive exons in the first 50% of the coding sequence.
- •Activity scoring (Rule Set 2/Azimuth) and off-target analysis (CFD score >0.9, MIT score >80) are both essential for guide selection.
- •Pool synthesis oligos are typically 73-80 nt for SpCas9: 20 nt spacer + scaffold overlap + amplification primers + restriction sites.
- •Verify library representation by NGS at 500-1000x coverage: >90% of guides detected, Gini <0.25, <10% dropout.
- •Include 500-1000 non-targeting control guides plus 50-100 essential gene positive controls in every library.
Table of Contents
1. CRISPR Library Types
CRISPR libraries are classified by their mechanism of action and by their scope (genome-wide vs targeted). Each type requires different guide design rules, scoring algorithms, and oligo architectures.
| Library Type | Cas Protein | Target Region | Guides/Gene | Typical Library Size |
|---|---|---|---|---|
| CRISPRko (Knockout) | SpCas9 (active) | Constitutive coding exons | 4-6 | ~80K-120K guides |
| CRISPRa (Activation) | dCas9-VP64/p65/Rta | Promoter (-200 to +1 of TSS) | 5-10 | ~100K-200K guides |
| CRISPRi (Interference) | dCas9-KRAB | TSS region (-50 to +300) | 5-10 | ~100K-200K guides |
| Base Editing | CBE4 or ABE8e | Coding exons (edit window pos 4-8) | 4-8 | ~80K-160K guides |
| Tiling | SpCas9 or dCas9 | Every PAM across target region | All possible | 50-500 per region |
Cas System Comparison
| System | PAM | Spacer Length | Cut Type | Advantages |
|---|---|---|---|---|
| SpCas9 | NGG | 20 nt | Blunt (3 bp upstream PAM) | Most validated, best scoring tools |
| SaCas9 | NNGRRT | 21-23 nt | Blunt | Smaller size for AAV delivery |
| AsCas12a | TTTV | 23 nt | Staggered (5' overhang) | AT-rich targets, multiplex from single transcript |
| LbCas12a | TTTV | 23 nt | Staggered (5' overhang) | High activity at 37°C |
| enAsCas12a | TTYN, VTTV, TRTV | 23 nt | Staggered | Expanded PAM flexibility |
2. sgRNA Design Criteria
Effective sgRNA selection combines sequence composition rules, on-target activity prediction, and off-target specificity analysis. Apply these criteria sequentially to filter and rank candidate guides.
| Criterion | Optimal | Filter (Hard) | Rationale |
|---|---|---|---|
| GC Content | 40-70% | Exclude <30% or >80% | Binding stability and activity correlation |
| Poly-T | No TTTT | Exclude any TTTT | Pol III termination signal |
| Homopolymer | ≤3 consecutive | Exclude ≥5 consecutive | Synthesis error and misalignment |
| Target Position | First 50% of CDS | Exclude last 10% of CDS | Earlier frameshifts = stronger KO |
| Exon Targeting | Constitutive exons | Avoid alt-spliced exons | Ensure disruption in all isoforms |
| Activity Score | Rule Set 2 >0.6 | Exclude <0.2 | Predicts cutting efficiency |
| Specificity (CFD) | CFD >0.9 | Exclude <0.5 | Off-target risk assessment |
| Specificity (MIT) | MIT >80 | Exclude <50 | Alternative specificity metric |
Activity Scoring Methods
Rule Set 2 (Doench et al., 2016): Logistic regression model trained on 2,549 sgRNAs from 8 cell lines. Input features include dinucleotide composition, GC content, and position-specific base preferences. Scores range from 0-1, with >0.5 indicating high predicted activity.
Azimuth (Doench et al., 2016): Updated gradient-boosted regression tree model. Generally outperforms Rule Set 1 and gives better discrimination for the top-scoring guides. Available through the Broad GPP portal.
DeepCas9 / CHOPCHOP: Deep learning models trained on larger datasets. Consider these for non-standard applications or when designing guides for organisms with limited training data coverage.
Screen your designed spacer sequences with our GC Content Analyzer (batch mode) and Batch Sequence QC to identify sequences with extreme GC, TTTT motifs, or problematic homopolymers before synthesis.
3. Library Sizing & Coverage
Library size and screening coverage determine the statistical power of your CRISPR screen. Insufficient coverage leads to high guide-level noise and missed hits.
| Screen Type | Guides/Gene | Total Library Size | Cell Coverage | Cells Needed |
|---|---|---|---|---|
| Genome-wide KO | 4 | ~80K | 500x per guide | 40M cells |
| Genome-wide CRISPRa/i | 5-10 | ~100-200K | 500x per guide | 50-100M cells |
| Focused sublibrary | 6-10 | 1K-10K | 1000x per guide | 1-10M cells |
| Tiling screen | All available | 5K-50K | 500x per guide | 2.5-25M cells |
Use our Coverage Calculator to determine the minimum cell number, sequencing depth, and replicate count for your screen design. The tool accounts for library complexity, infection efficiency (MOI), and desired statistical power.
4. Oligo Architecture & Cloning
Each oligo in a CRISPR library encodes the sgRNA spacer sequence flanked by elements required for amplification, cloning, and expression. The exact architecture depends on your vector system.
Typical SpCas9 Library Oligo (lentiGuide-Puro)
| Element | Length | Function |
|---|---|---|
| Forward primer | 18-22 nt | PCR amplification handle |
| BsmBI site + overhang | 10 nt | Golden Gate cloning into vector |
| Spacer sequence | 20 nt | sgRNA targeting sequence (variable) |
| BsmBI site + overhang | 10 nt | Golden Gate cloning into vector |
| Reverse primer | 18-22 nt | PCR amplification handle |
| Total oligo | 76-84 nt | Well within array synthesis limits |
Verify your full oligo sequences (spacer + constant regions) through our Batch Sequence QC tool before ordering synthesis. This checks for unintended restriction sites that would interfere with cloning, as well as secondary structures that could impair PCR amplification of the pool.
5. Sequence QC for CRISPR Libraries
Pre-synthesis sequence screening prevents costly design errors. Screen all oligo sequences (full-length, including constant regions) for the following issues:
TTTT Motif Check
The most critical filter: any spacer containing TTTT will produce a truncated, non-functional sgRNA. This is a hard filter — no exceptions.
Use Batch QC →GC Content Analysis
Spacer GC 40-70%. Extreme GC causes synthesis bias (dropout) and reduced Cas9 activity. Use batch mode for genome-wide libraries.
Use GC Analyzer →Secondary Structures
Strong hairpins in the spacer (ΔG < -3 kcal/mol) can block Cas9 loading. Check the full oligo for structures that impair PCR amplification.
Use Structure Predictor →Tm Uniformity
For PCR amplification handles, ensure consistent Tm (58-62°C). The spacer region contributes to overall oligo Tm — check for outliers.
Use Tm Calculator →6. Post-Synthesis QC & Screen Analysis
| QC Stage | Metric | Target | If Failed |
|---|---|---|---|
| Plasmid Library | Representation (NGS) | >90% guides detected | Sub-clone at higher ratio |
| Plasmid Library | Uniformity (Gini) | <0.25 | Re-amplify with fewer cycles |
| Plasmid Library | Dropout rate | <10% | Redesign/resynthesize dropped guides |
| Viral Production | Titer (TU/mL) | >10^7 for lenti | Concentrate by ultracentrifugation |
| Transduction | MOI | 0.3-0.5 (30-50% infection) | Titrate virus on target cells |
| Screen Coverage | Cells per guide | ≥500x | Scale up cell culture |
| Screen Results | Replicate correlation | Pearson r >0.7 | Add technical replicates |
Use our Uniformity Estimator to predict expected representation from your sequencing depth, and our Coverage Calculator to determine minimum sequencing depth for adequate power.
Frequently Asked Questions
How many sgRNAs per gene do I need for a CRISPR screen?▾
What is the difference between CRISPRko, CRISPRa, and CRISPRi libraries?▾
Why should I avoid TTTT sequences in sgRNAs?▾
What Cas systems can I use for CRISPR screens?▾
How do I analyze CRISPR screen results?▾
What controls should I include in my CRISPR library?▾
Related Tools
Coverage Calculator
Determine required library size, cell coverage, and sequencing depth for CRISPR screens.
GC Content Analyzer
Batch GC analysis for sgRNA spacer sequences. Filter guides outside 40-70% range.
Batch Sequence QC
Screen library oligos for TTTT motifs, homopolymers, and synthesis-problematic sequences.
Secondary Structure Predictor
Check spacer sequences for hairpins that block Cas9 loading.
Uniformity Estimator
Predict library representation uniformity from pool size and sequencing depth.
Error Rate Calculator
Estimate synthesis error rates and full-length oligo percentage for your library.