A PLYCRISPR/Cas9-Mtdi Vector System For Targeting Multiple Gene Sites In Cotton(Gossypium Arboreum)

Cotton is the world’s leading fiber crop and a model system for studies of many biological processes. As such, a lot of work is going on to improve yields and fiber quality and other traits of importance. Genomics research of cotton has also progressed radically in the past few years. Lately, engineered endonucleases such as Zinc Fingers Nucleases(ZFN), Transcription Activator-Like Nucleases(TALENs) and the most recent Clustered Regularly Interspaced Short Palindromic repeats(CRISPR/cas9) systems have been effectively utilized to edit genes in a range of species. These systems produce double-strand breaks(DSBs) at target sites to cause site-specific DNA sequence modifications. The changes in host genomes include sequence insertion and deletion and other mutations via the error-prone non-homologous end joining(NHEJ) pathway or sequence correction through the error-free homologous recombination(HR) pathway. Many workers have used the CRISPR/cas9 tool to generate mutations and to elucidate and understand gene function in many organisms. However, there has been no report so far of the use of CRISPR/cas9 system in editing genes in cotton. In this paper, we attempt to use CRISPR/Cas9 to target multiple genes in cotton, Gossypium arboreum. We targeted three genes: CENH3 associated with kinetochore assembly; RAD21 which is associated with chromosome condensation and pairing and DMC1, a gene that is required for crossover(CO) formation in most eukaryotes. We designed PCR-based techniques to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation. With this system, we edited 3 target sites in cotton on the selected genes. The developed expression vector was used to transiently transform cotton protoplasts. PCR was used to detect DNA cleavage at the target sites. The successful generation of mutations at the CENH3 locus was verified by small bands on the PAGE gel. We observed mutations in the CENH3 locus but not in the other two loci of RAD21 and DMC. We hypothesize that causing targeted mutations on these three genes will tinker with meiosis. This work will serve as a proof of principle that CRISPR/cas9 can edit genes in cotton. We demonstrate the feasibility of targeted DNA mutation in cotton through CRISPR/Cas9 system. Edited genes in cotton will add to the body of knowledge that cotton genomics is producing.