Reversing anther thermotolerance by manipulating the cis-elements in the promoter of a high-temperature upregulated gene Casein Kinase I in upland cotton.

High temperature (HT) stress causes male sterility, leading to reduced upland cotton yield. Previously, we identified a key gene, Casein Kinase I (GhCKI), that negatively regulates male fertility in upland cotton under HT. However, conventional genetic manipulations of GhCKI would result in male sterility, hindering its utilization in breeding programs. Here, we engineered quantitative variation for anther thermotolerance-related traits in upland cotton by creating weak promoter alleles of GhCKI genes, using CRISPR/Cas9 and CRISPR/Cpf1 genome editing. Then, we screened and identified two new upland cotton plant lines exhibiting a HT-tolerant phenotype with edited GhCKI promoters, and characterized their corresponding heat-tolerant allelic genotypes. Further research revealed that the primary reason for the HT tolerance of the GhCKI promoter editing mutants is that the trans-acting factors GhMYB73 and GhMYB4, which positively regulate GhCKI expression under HT, failed to bind and activate the expression of GhCKI. Overall, our study not only provides a rapid strategy to generate new beneficial alleles but also offers novel germplasm resources and molecular insights for crop HT tolerance breeding.