Abstract
BackgroundUpland 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 and functional analyses extremely challenging. The recent accessibility of CRISPR/Cas9 tool provides the ability to modify targeted locus efficiently in various complicated plant genomes. However, current cotton transformation method targeting one gene requires a complicated, long and laborious regeneration process. Hence, optimizing strategy that targeting multiple genes is of great value in cotton functional genomics and genetic engineering.ResultsTo target multiple genes in a single experiment, 112 plant development-related genes were knocked out via optimized CRISPR/Cas9 system. We optimized the key steps of pooled sgRNAs assembly method by which 116 sgRNAs pooled together into 4 groups (each group consisted of 29 sgRNAs). Each group of sgRNAs was compiled in one PCR reaction which subsequently went through one round of vector construction, transformation, sgRNAs identification and also one round of genetic transformation. Through the genetic transformation mediated Agrobacterium, we successfully generated more than 800 plants. For mutants identification, Next Generation Sequencing technology has been used and results showed that all generated plants were positive and all targeted genes were covered. Interestingly, among all the transgenic plants, 85% harbored a single sgRNA insertion, 9% two insertions, 3% three different sgRNAs insertions, 2.5% mutated sgRNAs. These plants with different targeted sgRNAs exhibited numerous combinations of phenotypes in plant flowering tissues.ConclusionAll targeted genes were successfully edited with high specificity. Our pooled sgRNAs assembly offers a simple, fast and efficient method/strategy to target multiple genes in one time and surely accelerated the study of genes function in cotton.
Highlights
Upland cotton (Gossypium hirsutum), harboring a complex allotetraploid genome, consists of A and D sub-genomes
Every selected single guided‐RNAs (sgRNAs) was synthesized as a DNA oligonucleotide fused to 16 bp sequences homolog to the linear ends of pRGEB32GhU6.9 vector as following: to construct our pooled sgRNA assemblies, plasmid was digested using BSAI restriction enzyme for 5 h at 37 °C, 29 of oligo sgRNAs were separately pooled in equal amount amplified by PCR
Selection of single guided‐RNAs to target genes related to anther response towards high temperature stress A total of 112 genes were selected from our reported transcriptome data [40] that were suggested to play a vital role in the response of male reproductive organs to high temperature stress in cotton
Summary
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 and functional analyses extremely challenging. The recent accessibility of CRISPR/Cas tool provides the ability to modify targeted locus efficiently in various complicated plant genomes. Current cotton transformation method targeting one gene requires a complicated, long and laborious regeneration process. Optimizing strategy that targeting multiple genes is of great value in cotton functional genomics and genetic engineering. Male sterility is the main challenge that decreases cotton fiber yield [6]. In recent decades, accumulating transcriptomic and proteomic studies of plant species have identified several genes that play roles in the development of male reproductive organs [8,9,10]. There is still lack of information about the molecular machinery of genes regulating male sterility in cotton. We crucially eager to deeply understand how male sterility occurs and how the regulatory mechanism functions during anther development
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