Summary The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9‐induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9‐induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large‐scale off‐targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off‐target sites of a large number of T0 plants, low‐frequency mutations were found in only one off‐target site where the sequence had 1‐bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering.

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Improvements in plant agricultural productivity are urgently needed to reduce the dependency on limited natural resources and produce enough food for a growing world population. Human intervention over thousands of years has improved the yield of important crops; however, it is increasingly difficult to find new targets for genetic improvement. MicroRNAs (miRNAs) are promising targets for crop improvement, but their inactivation is technically challenging and has hampered functional analyses. We have produced a large collection of transgenic short tandem target mimic (STTM) lines silencing 35 miRNA families in rice as a resource for functional studies and crop improvement. Visual assessment of field-grown miRNA-silenced lines uncovered alterations in many valuable agronomic traits, including plant height, tiller number, and grain number, that remained stable for up to five generations. We show that manipulation of miR398 can increase panicle length, grain number, and grain size in rice. In addition, we discovered additional agronomic functions for several known miRNAs, including miR172 and miR156. Our collection of STTM lines thus represents a valuable resource for functional analysis of rice miRNAs, as well as for agronomic improvement that can be readily transferred to other important food crops.small RNA | microRNA | silencing | yield | crop

SummaryCRISPR/Cas9 and TALEN are currently the two systems of choice for genome editing. We have studied the efficiency of the TALEN system in rice as well as the nature and inheritability of TALEN-induced mutations and found important features of this technology. The N287C230 TALEN backbone resulted in low mutation rates (0-6.6%), but truncations in its C-terminal domain dramatically increased efficiency to 25%. In most transgenic T0 plants, TALEN produced a single prevalent mutation accompanied by a variety of low-frequency mutations. For each independent T0 plant, the prevalent mutation was present in most tissues within a single tiller as well as in all tillers examined, suggesting that TALEN-induced mutations occurred very early in the development of the shoot apical meristem. Multigenerational analysis showed that TALENinduced mutations were stably transmitted to the T1 and T2 populations in a normal Mendelian fashion. In our study, the vast majority of TALEN-induced mutations (~81%) affected multiple bases and~70% of them were deletions. Our results contrast with published reports for the CRISPR/Cas9 system in rice, in which the predominant mutations affected single bases and deletions accounted for only 3.3% of the overall mutations.