Tissue-Specific CRISPR/Cas9 System of Cotton Pollen withGhPLIMP2bandGhMYB24Promoters

CRISPR/Cas9 technology is a powerful tool for improving crop genetic traits. However, CRISPR/Cas9 system for efficient expression of cotton germ cells has still not been established. In this study, we developed a tissue-specific vectors to driveCas9expression withGhPLIMP2bandGhMYB24promoters and established the effective method to transform cotton pollen byAgrobacteriumvacuum infiltration.GhPLIMP2bandGhMYB24promoters of cotton pollen were cloned into Cas9 expression vectors. The sgRNAs targetting toCLA1,ERA1andGGB(drought-resistant negative regulation genes) were designed and constructed intoGhPLIMP2bandGhMYB24promoter vectors. Cotton pollens were tranformed byAgrobacteriumvacuum infiltration withGhPLIMP2bandGhMYB24promoter vectors. The results of clone sequencing shown that mutation types of the sequence were mainly base substitutions wth the frequency from 3.29 to 6.45%. Eleven potential off-target sites were chosen and two sites were observed. Our results indicated thatGhPLIM2bP::Cas9andGhMYB24P::Cas9editing vectors achieved targeted edition of endogenous genes in cotton pollen, but there were a few off-target effects. This study provides an effective gene-editing system for the rapid acquisition of cotton mutants using pollen as a transgenic receptor.

Automated summary

The study focused on establishing an efficient method for CRISPR/Cas9 system expression in cotton pollen by developing tissue-specific vectors and using Agrobacterium vacuum infiltration for transformation. Results showed successful editing of endogenous genes in cotton pollen using GhPLIMP2bP::Cas9 and GhMYB24P::Cas9 editing vectors, with minimal off-target effects. This provides a rapid and effective gene-editing system for acquiring cotton mutants using pollen as a transgenic receptor.