On-target assessment methods compared: T7E1, TIDE, and amplicon NGS

This is Arc 1, Part 10 of the CRISPR from Bench to Analysis series.

"Did it work?"

It’s the most common question in any CRISPR lab. You’ve spent weeks designing guides and transfecting cells, and now you have a plate of cells that should be edited. But are they? And more importantly, what is the Percentage Gene Modification (% Indel)?

There is no single "right" way to measure CRISPR efficiency. The method you choose depends on your budget, your timeline, and whether you need to know if it cut or exactly how it cut.

This post compares the three pillars of on-target assessment: T7E1, TIDE, and Amplicon NGS.

What you'll learn

• The Three Pillars: T7E1, Sanger-based analysis (TIDE/ICE), and NGS
• Sensitivity vs. Cost: The trade-offs of each method
• When to use which: A practical decision guide
• Why "Resolution" matters: Detecting 1bp indels vs. complex populations

1. T7E1 / Surveyor (The Quick Screen)

The Mismatch Cleavage Assay is the entry-level tool. It uses enzymes that recognize and cut DNA at spots where the sequences don't match (heteroduplexes).

• How it works: PCR your target, denature/re-anneal, add enzyme, run a gel.
• Pros: Fast (4 hours), cheap, works in any lab with a thermocycler and a gel doc.
• Cons: Not very sensitive (limit ~5%), underestimates high-efficiency editing, and doesn't tell you the sequence of the indels.
• Best for: Initial guide screening. "Is this guide alive?"
• Deep Dive: See CRISPR-11 for the full protocol.

2. TIDE / ICE (The Sanger Solution)

If you have a Sanger sequencing core nearby, you can get quantitative data without the cost of NGS. TIDE (Tracking of Indels by Decomposition) and Synthego's ICE are web-based tools that analyze Sanger chromatograms.

• How it works: PCR your target, Sanger sequence it, and upload the .ab1 file. The algorithm compares your edited chromatogram to a wild-type control.
• Pros: Quantitative, provides an indel size distribution (+1, -3, etc.), and is much more accurate than a gel.
• Cons: Requires high-quality Sanger reads; struggles with large deletions (>50bp) or very complex samples.
• Best for: Routine validation and comparing guide performance.
• Link: tide.nki.nl or ice.synthego.com

3. Amplicon NGS (The Gold Standard)

Next-Generation Sequencing (NGS) of a specific PCR product (amplicon) is the most powerful tool we have. It sequences every single molecule in your sample.

• How it works: PCR with NGS adapters, sequence on an Illumina platform (like MiSeq), and analyze with CRISPResso2.
• Pros: Maximum sensitivity (detects <0.1% editing), identifies every specific mutation, and handles complex base-editing or prime-editing results.
• Cons: Expensive ($50-$100 per sample), slow turnaround (1-2 weeks), and requires bioinformatics knowledge.
• Best for: Final publication data, clinical applications, and base/prime editing where every base matters.

The Comparison Matrix

Practical Recommendation

Don't jump straight to NGS for everything. Follow this tiered approach:

1. Screen with T7E1: Use this to find out which of your 3 guides is the "winner."
2. Validate with TIDE/ICE: Once you have your best guide, use Sanger analysis to get a reliable percentage for your day-to-day experiments.
3. Finish with NGS: For the final figure in your paper or when you need to confirm that a base edit is "pure," run Amplicon NGS.

My Take

Most bench scientists over-rely on T7E1 because it's familiar. But if you have access to Sanger sequencing, TIDE/ICE is a massive upgrade for almost no extra effort. It turns a "smudge on a gel" into a quantitative bar graph that PIs and reviewers actually trust.

If you are doing anything more complex than a standard Cas9 knockout (like Base Editing), skip the first two and go straight to NGS. You need the single-nucleotide resolution that only NGS can provide.

What's your primary method for checking editing? Are you still running T7E1 gels, or have you made the jump to TIDE? Drop a comment below.

Resources