Principle

Why it works

SpCas9 cuts DNA three base pairs upstream of the PAM. Plant cells repair most of these breaks by non-homologous end joining, which often leaves a small indel. If you PCR-amplify the target from a plant carrying more than one allele and Sanger-sequence the product, the trace is clean up to the cut site and then becomes a superposition of the sequences of each allele, shifted by their indels.

Decomposition tools model that mixed trace as a weighted sum of the wild-type sequence shifted by every possible indel, and report which indels are present and in what proportion. TIDE (Brinkman et al., 2014) and ICE (Conant et al., 2022) both do this from a wild-type control trace and the edited trace. For a diploid plant, those proportions map onto genotypes: one allele at 100%, two at about 50% each, or something messier that signals a chimera.

Safety and biosafety

Before you start

Plants carrying Cas9 transgenes are genetically modified organisms; handle and dispose of them under your institution's approvals. Ethidium bromide or other gel stains need the precautions on their safety data sheets. Otherwise this protocol involves only standard molecular biology hazards.

Materials

Genomic DNA from each edited plant and from at least one wild-type plant of the same genotype. A quick extraction (Edwards et al., 1991) is sufficient; CTAB DNA also works.
PCR primers flanking the target (design rules in step 1)
High-fidelity, proofreading DNA polymerase
PCR clean-up: enzymatic (exonuclease I plus shrimp alkaline phosphatase) or spin columns
Sanger sequencing service accepting purified PCR products
Free web tools: TIDE (tide.nki.nl) or ICE (ice.synthego.com)

Recipe: Edwards quick-extraction buffer (50 mL)

Component	Final	Amount for 50 mL
Tris-HCl pH 7.5 (1 M)	200 mM	10 mL
NaCl (5 M)	250 mM	2.5 mL
EDTA pH 8.0 (0.5 M)	25 mM	2.5 mL
SDS (10%)	0.5%	2.5 mL
Water		to 50 mL

Grind a leaf disc in 400 µL buffer, centrifuge 1 min, precipitate 300 µL of supernatant with 300 µL isopropanol, spin 5 min, air-dry and dissolve in 100 µL TE. Use 1 µL per PCR.

Procedure

CriticalDesign primers that amplify 500–800 bp with the cut site about 200–300 bp from the sequencing primer.Decomposition needs clean sequence before the cut and a readable window after it. Keep the cut site at least 150 bp from either primer. In polyploids, make at least one primer specific to the homoeologue you are genotyping.
Check that the target and primer sites in your accession match the reference.Sequence the wild-type amplicon once. A SNP in the PAM or guide explains "no editing" more often than a bad guide.
Extract DNA from each edited plant and from wild-type controls.
Amplify the target with a high-fidelity polymerase for 28–32 cycles.Proofreading enzymes keep PCR errors from being mistaken for low-frequency edits.
CriticalRun 5 µL on a 1.5% agarose gel. Expect one band of the predicted size.A second, smaller band means a large deletion; such plants need cloning or long-read sequencing, not decomposition.
Purify the remaining product enzymatically or on a column.
Pause pointSend for Sanger sequencing with the primer that sits farther from the cut site, together with the wild-type product.Purified products keep for weeks at −20 °C.
Open each chromatogram and look at the cut site, 3 bp upstream of the PAM.Clean, single peaks throughout: wild type or homozygous. Clean up to the cut site and double peaks after it: more than one allele.
Upload the control and edited trace files (.ab1) to TIDE or ICE, enter the 20-nt guide sequence without the PAM, and set the maximum indel size.Start with a maximum of 10–20 bp. Raise it if the fit is poor and you suspect larger deletions.
CriticalCheck the goodness of fit before reading genotypes.Both tools report an R² for how well the modelled mixture explains the trace. Treat calls with R² below about 0.9 as unreliable and confirm them independently.
Call a genotype for each plant using the table under Expected results.
Confirm chimeric and ambiguous plants by cloning the amplicon and sequencing 8–12 colonies, or by amplicon deep sequencing.
To recover transgene-free edited plants, test progeny of edited plants by PCR for Cas9 and the selection marker, and keep plants that carry the edit but not the transgene.

Expected results: calling genotypes

Decomposition result	Genotype in a diploid	Notes
Wild type ≈ 100%	Unedited	Check the guide and accession sequence if all plants look like this
One indel ≈ 100%	Homozygous	The trace is clean but differs from wild type at the cut site
Two indels ≈ 50% each	Biallelic	Both alleles edited, differently
Wild type ≈ 50%, one indel ≈ 50%	Heterozygous	Expect 1 : 2 : 1 segregation in progeny
Three or more alleles, or uneven proportions	Chimeric	Common in T0. Genotype the progeny, not the parent, to know what is heritable

In T0 plants that still express Cas9, expect a high proportion of chimeras. Genotypes become reliable in the T1 or T2 generation, once the edit has passed through seed. Proportions near 33% or 25% in a diploid are a sign of chimerism or of amplification from more than one locus, not of a stable genotype.

Polyploids

In wheat, potato, oilseed rape and other polyploids, non-specific primers amplify homoeologues together, and the proportions then reflect all copies at once. Use homoeologue-specific primers, or move to amplicon deep sequencing, which reads every allele separately.

Troubleshooting

Problem	Likely cause	What to change
Noisy trace before the cut site	Poor template, primer dimers, or a mixture of products	Re-purify; redesign primers; check the gel for a single band
Low R² in the decomposition	Indels larger than the set range; insertion of vector DNA; mixed homoeologues	Raise the maximum indel size; clone and sequence; use specific primers
Plant has a phenotype but looks wild type	A large deletion removed a primer site, so only the intact allele amplifies	Repeat with primers placed farther out
No editing in any plant	SNP in the PAM or guide in your accession; inactive guide; Cas9 not expressed	Sequence the wild-type target; test the guide in a transient assay; check Cas9 expression
Edits found in T0 but not in T1	The T0 edit was somatic (chimeric) and did not reach the germline	Screen more T1 plants; prefer constructs with germline- or egg-cell-specific Cas9 promoters

References

Brinkman, E. K., Chen, T., Amendola, M., & van Steensel, B. (2014). Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Research, 42(22), e168. doi:10.1093/nar/gku936
Conant, D., Hsiau, T., Rossi, N., et al. (2022). Inference of CRISPR edits from Sanger trace data. The CRISPR Journal, 5(1), 123–130. doi:10.1089/crispr.2021.0113
Edwards, K., Johnstone, C., & Thompson, C. (1991). A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research, 19(6), 1349. doi:10.1093/nar/19.6.1349
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816–821. doi:10.1126/science.1225829

Change log

1.0 (25 October 2026). First version.

How to cite this protocol

Kumar A. Genotyping CRISPR edits in plants by PCR, Sanger sequencing and trace decomposition. plantbiomethods.com, version 1.0, updated 25 October 2026. https://www.plantbiomethods.com/protocols/crispr-edit-genotyping-sanger.html