| Rice（Oryza sativa）is one of the most important crops and a research model of monocotyledon plant.Investigation of rice gene function and other agronomic trait can provide insights in rice molecular breeding.CRISPR/Cas9 is a novel genome editing system with high efficiency,and low cost to knock out endogeneous gene,and thus is considered as the most significant approach to produce mutants for reverse genetic study of rice gene function nowadays.Micro RNAs（mi RNAs）are a class of small non-coding RNAs that usually contains 20～24 nucleotide（nt）.In plants,mi RNAs usually regulate target genes at post-transcriptional level by m RNA cleavage and translation repression,and involved in diverse biological processes,including plant growth and development as well as adaptation to adverse environments.Recent studies showed that 24-nt rice mi RNA can also modulate targets through DNA methylation at the transcriptional level,suggesting that plant mi RNAs have a complicated regulatory system for target expression.mi R408,a plant conserved mi RNA,has been implicated in positively controling of rice biomass and production,but its action mechanism remains unclear.The precursor of mi R1850 and mi R1876 were shown to be able to produce 24-nt mi RNAs,but their biological relevance is still elusive.In this study,mi R408,mi R1850 and mi R1876 knock-out plants were generated via CRISPR/Cas9 gene editing system in rice,and their phenotypes were recorded in their T1 generation.The main results are as follows:1.Two target sites close to the mature mi RNA sequences were selected for CRISPR/Cas9 sg RNA design and the website tool CRISPR-GE was used to predict sg RNA potential off-target.2.Os U6a and Os U3 promoter drived-sg RNA expression cassettes and Cas9expression backbone vector were used to make transformation constructs.Nipponbare rice were transformed by Agrobacterium-mediated method and the rate of positive transgenic plants was more than 50%.3.Determined by target site sequencing,the mutagenesis frequencies were34.5%,68.9%and 15.4%for mi R408,mi R1850 and mi R1876,respectively.The mutation efficiency by Os U6-sg RNA scaffolds is much higher than that of Os U3a promoter.4.The developmental performances were observed in T1 mi RNA knock out transgenic plants.Compared with wild-type,the growth and germination rate was significantly inhibited in mi R408Cas9 plants,while bigger roots and better vigor were found in mi R1850Cas9 and mi R1876Cas9 mutants,suggesting that mutation of these mi RNAs in rice can affect rice development.Our results indicate that CRISPR/Cas9 is an efficient strategy for generating rice mi RNA loss-of-function mutants,and provide several important genetic materials for mi RNA functional study and molecular breeding. |
