CRISPR: no clones recovered

1. Ranked causes

   1. Target gene is essential — homozygous KO is lethalHeterozygous edits appear transiently; no homozygous clones expand.
   2. Selection antibiotic / sorting gate too harsh for this lineUnedited controls also die under the same selection.
   3. Single-cell clone density / conditioned media insufficientClones expand in bulk but not after limiting dilution without feeder/conditioned media.
   4. Mycoplasma or poor parental viability before cloningParental culture already low viability; mycoplasma test missing/overdue.

2. Recovery steps

   1. Kill-curve the parental line and relax selection to the minimal effective doseOver-selection kills edited and unedited cells alike.
   2. Use conditioned media / larger starting wells for limiting dilutionMany lines need community factors to expand from single cells.
   3. Test for mycoplasma and restart from a healthy authenticated parental stockSick parents rarely yield clones.

3. Controls first

   • Did unedited parental negative-control cells survive the same selection / sort gate?If no: Selection is too harsh — rebuild the kill curve before blaming the edit.
   • Do heterozygous (or bulk) edited cells expand while homozygous candidates do not?If yes: Essential-gene lethality is plausible — consider knockdown or conditional alleles.

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