Progress and prospect of genome editing tools development and molecular breeding in cotton

Genome editing technology can achieve efficient and accurate editing at the biological gene and transcription level, which is a revolutionary technology in the field of life science. The genome editing system based on Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas), has become the most widely used genome editing system in the field of plants with its simple, fast and efficient characteristics. Cotton is an important economic crop and the main cultivated species of upland cotton (G. hirsutum L.) is an allotetraploid plant species with complex genome. The upland cotton genome contains At- and Dt- two subgenomes with multiple gene copy, therefore, the gene functional redundancy is common. Therefore, the conventional T-DNA insertion and chemistry, physical mutagenesis and other technologies to create mutants are very low efficiency in cotton. In recent years, Chinese cotton researchers have established efficient genome editing tools including CRISPR/Cas9/12a/12b, CBE/ABE/ABE8e/CABE base editor, Cas13a/b/c and dCas9-TV transcriptional activation, which can achieve knock out, knock in, knock down, knock up and point mutation of the target genes. In addition, they have established a system to evaluate off-target effects of genome editing based on whole genome re-sequencing strategy. Whether the genome editing system can be effectively delivered to plants is an important step in gene editing, and the genotype of receptor material plays a decisive role in the effective delivery of genome editing system in the genetic transformation mainly mediated by Agrobacterium. Jin668, a cotton material with high genetic transformation efficiency, is a widely used receptor material in the world at present, and is an important receptor material for exploring the function of cotton endogenous genes and conducting gene editing research. Here, the Successive Regeneration Acclimation (SRA) strategy used by cotton researchers to obtain Jin668 with high transformation efficiency and the analysis of its regeneration mechanism is introduced in detail. It is expected to break the genotypic restriction of other cotton varieties during transformation and regeneration. In this review, the establishment process of cotton genome editing tools is introduced in particular. Using the above efficient genetic transformation system and genome editing tools, researchers have created a mutant library of more than 5,000 genes, including cotton fiber quality, resistance to disease, insect, high temperature, salt and alkali, cotton seed quality, haploid induction and other traits, effectively promoting the development of basic research and molecular breeding of cotton. This paper also discusses the limitations of the genome editing system itself and the problems in the delivery process of the genome editing system and the solutions. At the end of the paper, the future development direction of cotton gene editing research is also prospected. The era of breeding 4.0, namely the stage of molecular design breeding, is the trend of crop breeding in the future. For cotton, the strategy of "precise genome+efficient gene editing" is expected to drive the establishment of an efficient molecular design breeding system for cotton in the future.