Targeted Genome Editing On Auxin Synthetic Genes OsYUCCA Using CRISPR/Cas9 In Rice

CRISPR-Cas（clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins）systems are the adaptive immunological system observed in bacteria and archaea that defend against infectious viruses and plasmids.The immunity mediated by Cas nucleases employs small RNA guides（the cr RNAs）to specifically break the genome of invading nucleic acids.In typeⅡCRISPR-Cas systems,the DNA-cleaving activity is conducted by a single enzyme Cas9 directed by an RNA duplex.The CRISPR associated protein Cas9 is an endonuclease that employs a guide sequence within an RNA duplex,tracr RNA:cr RNA,to form pairs with DNA target sequences,enabling Cas9 to introduce a site-specific double-strand break in the targeted DNA.Plant auxin plays a critical role in various developmental processes,but its biosynthesis and regulation have not been clearly elucidated in model monocot rice（Oryza sativa）.There are 14 homologous genes of YUCCAs in rice.In order to investigate the function of auxin biosynthesis during rice growth,we made twenty-eight CRISPR-Cas9 gene knockout vectors for these 14 genes,and each gene with two vectors containing one specific guided RNA region of individual Os YUCCA.The twenty-eight targeted sequences of Os YUCCA gene family were individually synthesized and inserted into the vector called hsp Cas9 and then linked to the binary vector of p CAMBIA1300.The resultant plasmids were transformed into rice through Agrobacterium-mediated transformation,and a total of 519 transgenic plants were obtained.Among 519 transgenic plants,we identified the mutations within Os YUCCA1 and Os YUCCA3,respectively.The mutations of Os YUCCA1 occur for A or T insertion at260^th base on the exon and A insertion at 447^th,C replaced by T at 444^thand G replaced by C at 445^th on the exon,respectively.The mutations of Os YUCCA3 contained T insertion at 494^th base on the exon.osyucca1displayed reduced number of tillers and delayed heading date.The knockout mutants of Os YUCCA1 and Os YUCCA3 created by CRISPR/Cas9 provide a genetic basis for further study on the role of Os YUCCAs in rice.