Kabin Xie

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA-gRNA architecture were efficiently and precisely processed into gRNAs with desired 5' targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.

Transcription factors play essential roles in the developmental processes of plants. Many such factors are regulated by microRNAs (miRNAs). SQUAMOSA (SQUA) promoter-binding-like (SPL) genes encode plant-specific transcription factors, some of which contain complementary sequences of miRNA156. In this study, 19 rice (Oryza sativa) SPL (OsSPL) genes and 12 rice miRNA156 (OsmiR156) precursors were identified in the rice genome. Sequence and experimental analysis suggested that 11 OsSPL genes were putative targets of OsmiR156. Plant SPL proteins were classified into six subgroups based on the phylogenetic analysis of SQUA promoter-binding protein domain. Diverse exon-intron structures and distinct organizations of putative motifs beyond the SQUA promoter-binding protein domains were identified in the OsSPL gene family. Transcript level analysis of OsSPL genes in various rice tissues and organs revealed different tempospatial expression patterns. More than half of the OsSPL genes including most OsmiR156-targeted genes are predominantly expressed in the young panicles, whereas OsmiR156 genes are predominantly expressed in the young shoots and leaves of rice. Overexpression of two OsmiR156 genes (OsmiR156b and OsmiR156h) in rice resulted in severe dwarfism, strongly reduced panicle size, and delayed flowering, suggesting that OsmiR156 and OsSPL target genes are involved in various developmental processes, especially the flower development of rice. Different patterns of transcript changes (decreased or unchanged) of different target genes in same tissue and of same target gene in different tissues detected in the OsmiR156-overexpressing plants suggested diverse interactions between OsmiR156 and OsSPL target genes in a tissue-specific manner.