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 drive Cas9 expression with GhPLIMP2b and GhMYB24 promoters and established the effective method to transform cotton pollen by Agrobacterium vacuum infiltration. GhPLIMP2b and GhMYB24 promoters of cotton pollen were cloned into Cas9 expression vectors. The sgRNAs targetting to CLA1, ERA1 and GGB (drought-resistant negative regulation genes) were designed and constructed into GhPLIMP2b and GhMYB24 promoter vectors. Cotton pollens were tranformed by Agrobacterium vacuum infiltration with GhPLIMP2b and GhMYB24 promoter 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 that GhPLIM2bP::Cas9 and GhMYB24P::Cas9 editing 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.

CRISPR / Cas9技术是改善作物遗传性状的有力工具。然而，尚未建立用于棉胚细胞高效表达的CRISPR / Cas9系统。在这项研究中，我们开发了一种组织特异性载体，用GhPLIMP2b和GhMYB24启动子驱动Cas9表达，并建立了通​​过农杆菌真空渗入法转化棉花花粉的有效方法。将棉花花粉的GhPLIMP2b和GhMYB24启动子克隆到Cas9表达载体中。靶向CLA1，ERA1和GGB的sgRNA（抗逆性负调控基因）被设计并构建到GhPLIMP2b和GhMYB24启动子载体中。通过农杆菌真空渗透用GhPLIMP2b和GhMYB24启动子载体转化棉花花粉。克隆测序的结果表明，该序列的突变类型主要是碱基取代，频率从3.29到6.45％。选择了11个潜在的脱靶站点，并观察到两个站点。我们的结果表明，GhPLIM2bP :: Cas9和GhMYB24P :: Cas9编辑载体实现了棉花花粉中内源基因的靶向编辑，但是有一些脱靶效应。这项研究为使用花粉作为转基因受体快速获得棉花突变体提供了有效的基因编辑系统。