How Many Clones to Screen for a Homozygous Knockout?

With per-allele editing efficiency p, the probability that a clone is homozygous is roughly p² (assuming independent editing of the two alleles). To have a 90% chance of finding at least one homozygous clone, screen approximately:

The practical implication: at efficiencies below ~30%, cloning becomes a numbers game, and improving efficiency is cheaper than screening hundreds of clones.

Three corrections matter in practice:

• • Indels are not nulls. A homozygous clone with in-frame indels may retain function; you are screening for homozygous edits, then for null alleles.
• • Allele independence is approximate. Editing of one allele can affect the other (for example, by large deletions spanning both), so the true rate is not exactly p².
• • Clone viability varies. Some genotypes grow poorly, so the clones you can pick are a biased subset.

For a functional claim, plan on finding two independent validated null clones, which roughly doubles the screening burden.

A useful sequence: measure efficiency in the pool by amplicon sequencing first, then compute how many clones you need rather than picking a habitual number.

1. Junction or amplicon PCR on pools of clones (5–10 per pool) to eliminate pools with no editing.
2. Individual PCR on clones from positive pools.
3. Sanger or amplicon NGS to identify indels and estimate zygosity.
4. Digital PCR for definitive zygosity.
5. Protein to confirm absence, not just transcript.

Screening pools first can cut the number of individual assays substantially.

For many questions, a heterozygous clone is sufficient — for example, when testing a dominant variant or when the phenotype is dosage-sensitive. Decide before screening, because the required clone number differs by an order of magnitude.

EDITGENE reports pool-level efficiency before cloning, screens calculated clone numbers, confirms zygosity by digital PCR, and verifies protein absence — with two independent clones per genotype where the claim requires it.

Tell us the gene and the cell line. Contact EDITGENE to request a project consultation or quote.

Clone numbers here follow a simple probabilistic model; actual rates depend on guide activity, locus, cell type, and viability of edited clones.

Homozygous editing is not equivalent to a null allele; protein-level confirmation is required for a knockout claim.

• • Sentmanat MF, et al. A survey of validation strategies for CRISPR-Cas9 editing. Scientific Reports. 2018;8:888.
• • Brinkman EK, et al. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Research. 2014;42:e168.
• • Doench JG. Am I ready for CRISPR? A user's guide to genetic screens. Nature Reviews Genetics. 2018;19:67–80.