Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton

Abstract Background Upland cotton ( Gossypium hirsutum ), harboring a complex allotetraploid genome, consists of A and D sub-genomes. Every gene has multiple copies with high sequence similarity that makes genetic, genomic functional analyses extremely challenging. The recent accessibility CRISPR/Cas9 tool provides the ability to modify targeted locus efficiently in various complicated plant genomes. However, current transformation method targeting one requires complicated, long laborious regeneration process. Hence, optimizing strategy genes is great value genomics genetic engineering. Results To target single experiment, 112 development-related were knocked out via optimized system. We key steps pooled sgRNAs assembly by which 116 together into 4 groups (each group consisted 29 sgRNAs). Each was compiled PCR reaction subsequently went through round vector construction, transformation, identification also transformation. Through mediated Agrobacterium , we successfully generated more than 800 plants. For mutants identification, Next Generation Sequencing technology been used results showed all plants positive covered. Interestingly, among transgenic plants, 85% harbored sgRNA insertion, 9% two insertions, 3% three different 2.5% mutated sgRNAs. These exhibited numerous combinations phenotypes flowering tissues. Conclusion All edited specificity. Our offers simple, fast efficient method/strategy time surely accelerated study function cotton.