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 BC 3 F 1 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.