Generation of artificial drooping leaf mutants by CRISPR-Cas9 technology in rice.

CRISPR-Cas9 technology, which uses an RNA-guided nuclease, has been developed as an efficient and versatile genome-editing method to induce mutations in genes of interest. To examine the feasibility of this method in developmental studies of a model monocot, rice (Oryza sativa), we introduced the construct gDL-1, which produced a guide RNA targeting the DROOPING LEAF (DL) gene. DL regulates midrib formation in the leaf and carpel specification in the flower. Because loss of function of DL causes the drooping leaf phenotype in regenerated seedlings, the effect of gene disruption should be easily detected. In transgenic plants carrying gDL-1, the DL gene was disrupted at high efficiency: seven out of nine plants examined had bi-allelic mutations. All transgenic plants with the bi-allelic mutation showed the drooping leaf phenotype. Observation of cross sections of the leaf blade clearly indicated that these transgenic plants failed to make midrib structures, and were comparable to the severe dl mutant dl-sup1. Thus, CRISPR-Cas9 technology can be a useful and efficient tool in developmental studies in rice.