apply-crispr-design-principles

Apply CRISPR Design Principles

Design a CRISPR genome editing experiment by selecting high-efficiency guide RNAs with minimal off-target potential, choosing appropriate delivery and selection strategies, and building in rigorous editing confirmation assays.

Why This Is Best Practice

Adopted by: NIH, Broad Institute, Wellcome Sanger Institute, and every major research institution conducting genome editing use systematic CRISPR design tools (Benchling, CRISPOR, CHOPCHOP, Cas-OFFinder) rather than ad hoc guide selection. The FDA's guidance on CRISPR-based therapeutics requires comprehensive off-target analysis as a prerequisite for IND filing. Impact: Doench et al. (2016) showed that guide RNA efficiency varies by more than 100-fold depending on sequence context — random guide selection produces high failure rates. Hsu et al. (2013) demonstrated that off-target cleavage occurs at sequences with up to 5 mismatches, particularly in the seed region — making systematic off-target prediction essential. The first human CRISPR clinical trials (Vertex/CRISPR Therapeutics CTX001) validated that systematic design followed by rigorous confirmation produces safe editing outcomes.

Steps

1. Define the editing goal precisely

Before designing any guide RNA:

• Knockout: disrupt protein function by frameshift — cut near the start of the coding sequence (first exon, early coding region)
• Knockin: introduce precise sequence change — requires HDR template design; plan donor DNA alongside the gRNA
• Transcription regulation: activate or repress without cutting — use dCas9-KRAB (repression) or dCas9-VPR (activation)
• Base editing: single base change without DSB — use CBE (C→T) or ABE (A→G) depending on target base

The goal determines which Cas variant (Cas9, Cas12a, base editors, prime editors) is appropriate.

2. Select target site and guide RNA

Rules for standard Cas9 (SpCas9, requires NGG PAM):

1. Identify all NGG PAMs in the target region
2. Extract the 20-nt protospacer immediately 5' of the NGG
3. Score each candidate gRNA using a prediction tool:
   • CRISPOR (crispor.tefor.net): on-target efficiency (Doench 2016 score) + off-target scores
   • Benchling or CHOPCHOP: integrated design and off-target analysis
4. Select gRNAs with:
   • Doench efficiency score ≥ 60%
   • Specificity score: no predicted off-targets with <3 mismatches in coding regions
   • GC content: 40-70%
   • Avoid poly-T runs (TTTT) — cause Pol III termination in U6-driven expression
5. Design 2-3 gRNAs per target — test all; select the best-performing one empirically

3. Assess and mitigate off-target risk

Off-target cleavage is the primary safety concern:

• In silico screening: run Cas-OFFinder or CRISPOR off-target prediction; flag all sites with ≤3 mismatches in coding or regulatory regions
• Seed region priority: mismatches in positions 1-12 (PAM-proximal) are better tolerated by Cas9; avoid guides with perfect match to off-target sites in the seed region
• Mitigation options: high-fidelity Cas9 variants (eSpCas9, HiFi Cas9, HEK4) for therapeutic contexts; paired Cas9 nickases for additional specificity; truncated gRNAs (17-18 nt)

4. Select delivery strategy

Match delivery to cell type and context:

• Plasmid transfection: simple; persistent expression; low cost; risk of random integration
• Ribonucleoprotein (RNP) complex (Cas9 + gRNA): preferred for cells with HDR requirement; transient exposure minimizes off-target; used in clinical applications
• Lentiviral/AAV: for difficult-to-transfect cells (neurons, HSCs); size constraints limit AAV-packaged Cas9 (use SaCas9 or split constructs)
• Electroporation: high efficiency for most cell types; RNP delivery is optimal

5. Design editing confirmation assays

Confirming successful editing is mandatory:

• Indel detection (for knockouts):
  • T7 endonuclease I (T7EI) assay or Surveyor assay: detect mismatch at cut site; semi-quantitative
  • TIDE (Tracking of Indels by DEcomposition): quantitative indel profiling from Sanger sequencing
  • Next-generation sequencing (amplicon-seq): most accurate; required for clinical applications
• HDR confirmation (for knockins):
  • PCR across the junction + Sanger sequencing
  • Allele-specific PCR for precise mutations
• Off-target validation (for therapeutic use):
  • GUIDE-seq, CIRCLE-seq, or DISCOVER-seq: unbiased whole-genome off-target detection

6. Clone or isolate edited cells

For most applications: sort or limit-dilute after transfection to obtain clonal populations; sequence both alleles to confirm bi-allelic editing.

Common Mistakes

• Selecting a gRNA based solely on target proximity, not efficiency score: Guide RNA efficiency varies enormously by sequence context. Use a validated prediction tool.
• Skipping the negative selection step after editing: Mixed populations contain unedited cells. Clonal isolation and confirmation are required for downstream experiments.
• No unedited control: Always compare edited and isogenic unedited cells to attribute phenotype to the edit, not to transfection or selection stress.

When NOT to Use

• Targeting mitochondrial DNA: Cas9 lacks efficient mitochondrial delivery; use mitochondria-targeted base editors specifically designed for mtDNA.