Why did my CRISPR fail?
Indel / knock-in rate far below the expected efficiency for this guide and delivery. Suspected off-target edits or phenotype not explained by the on-target genotype. No single-cell clones expand after editing / selection / limiting dilution.
Playbooks
- CRISPR: low editing efficiencyIndel / knock-in rate far below the expected efficiency for this guide and delivery.
- CRISPR: off-target concernSuspected off-target edits or phenotype not explained by the on-target genotype.
- CRISPR: no clones recoveredNo single-cell clones expand after editing / selection / limiting dilution.
Calculators
FAQ — CRISPR
- Low editing efficiency
- Indel / knock-in rate far below the expected efficiency for this guide and delivery. Validate guide identity by sequencing the sgRNA / oligo stock — Rules out synthesis or labeling mix-ups before redesign. Increase RNP / plasmid dose or switch delivery (electroporation vs lipid) — Delivery failure is the usual method cause when the control guide works. Re-sequence the target locus in the parental line for SNPs under the guide — A seed/PAM SNP silently blocks cleavage.
- Off-target concern
- Suspected off-target edits or phenotype not explained by the on-target genotype. Re-design with a higher-specificity guide or high-fidelity Cas variant — Addresses poor specificity at the design step. Titrate RNP down while monitoring on-target efficiency — Dose-driven promiscuity is a common reversible cause. Sequence top predicted off-targets (and authenticate the line) — Separates true off-targets from clonal noise or wrong-line genetics.
- No clones recovered
- No single-cell clones expand after editing / selection / limiting dilution. Kill-curve the parental line and relax selection to the minimal effective dose — Over-selection kills edited and unedited cells alike. Use conditioned media / larger starting wells for limiting dilution — Many lines need community factors to expand from single cells. Test for mycoplasma and restart from a healthy authenticated parental stock — Sick parents rarely yield clones.