Efficient Crispr-cas9 Mediated Pooled-sgRNAs Assembly Accelerates Targeting Multiple Genes Related to Male Sterility in Cotton 

Background: Upland cotton (Gossypium hirsutum), harboring a complex allotetraploid genome consists of A and D subgenomes. The genes in have multiple copies with high sequence similarity that makes genetic, genomic and functional analysis extremely challenging. The recent accessibility of CRISPR/Cas9 tool offers 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 to target multiple genes is of great value in cotton functional genomics and practical applications of genetic engineering.Results: To 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. Conclusion: All targeted genes were successfully edited with high specificity which makes our pooled sgRNAs assembly a simple, fast and efficient method/strategy to target multiple genes in one time and surely accelerated the study of genes function in cotton.