CRISPR/Cas genome editing in nonregenerative cotton using sexual hybridization

Sexual transfer of the CRISPR/Cas genome-editing system to targeted cotton cultivars could bypass their recalcitrance to regeneration from tissue culture. We used sexual hybridization to transmit a CRISPR/LbCas12a system from a regenerable Gossypium hirsutum donor to a nonregenerable G. barbadense recipient. We knocked out the GbCLA and GbPGF genes in the recipient, generating respectively albino and glandless phenotypes. Focusing on GbPGF, we detected novel mutations in the progeny across generations, and developed a set of nearly isogenic lines. The average editing efficiency of the target gene at crRNA1 exceeded 70% in the BC₃F₁ generation, yielding plants with agronomic traits or fiber quality nearly identical to those of the recurrent parent but lacking glands or gossypol. We introduced the CRISPR/LbCas12a system into three other nonregenerable G. hirsutum genotypes and one diploid cotton by hybridization and edited three more genes in two recipients.