Understanding CRISPR-Cas9’s capacity to produce native overexpression (OX) alleles would accelerate agronomic gains achievable by gene editing. To generate OX alleles with increased RNA and protein abundance, we leveraged multiplexed CRISPR-Cas9 mutagenesis of noncoding sequences upstream of the rice PSBS1 gene. We isolated 120 gene-edited alleles with varying non-photochemical quenching (NPQ) capacity in vivo—from knockout to overexpression—using a high-throughput screening pipeline. Overexpression increased OsPsbS1 protein abundance two- to threefold, matching fold changes obtained by transgenesis. Increased PsbS protein abundance enhanced NPQ capacity and water-use efficiency. Across our resolved genetic variation, we identify the role of 5′UTR indels and inversions in driving knockout/knockdown and overexpression phenotypes, respectively. Complex structural variants, such as the 252-kb duplication/inversion generated here, evidence the potential of CRISPR-Cas9 to facilitate significant genomic changes with negligible off-target transcriptomic perturbations. Our results may inform future gene-editing strategies for hypermorphic alleles and have advanced the pursuit of gene-edited, non-transgenic rice plants with accelerated relaxation of photoprotection.

Cis-regulatory element editing can generate quantitative trait variation that mitigates extreme phenotypes and harmful pleiotropy associated with coding sequence mutations. Here, we applied a multiplexed CRISPR/Cas9 approach, informed by bioinformatic datasets, to generate genotypic variation in the promoter of OsSTOMAGEN, a positive regulator of rice stomatal density. Engineered genotypic variation corresponded to broad and continuous variation in stomatal density, ranging from 70% to 120% of wild-type stomatal density. This panel of stomatal variants was leveraged in physiological assays to establish discrete relationships between stomatal morphological variation and stomatal conductance, carbon assimilation and intrinsic water use efficiency in steady-state and fluctuating light conditions. Additionally, promoter alleles were subjected to vegetative drought regimes to assay the effects of the edited alleles on developmental response to drought. Notably, the capacity for drought-responsive stomatal density reprogramming in stomagen and two cis-regulatory edited alleles was reduced. Collectively our data demonstrate that cis-regulatory element editing can generate near-isogenic trait variation that can be leveraged for establishing relationships between anatomy and physiology, providing a basis for optimizing traits across diverse environments.

In flowering plants, rapid activation of the zygotic genome occurs after fertilization1-3, but there is limited knowledge of the molecular pathways underlying embryo initiation4. In rice, a key role is played by the transcription factor BABY BOOM 1 (OsBBM1), initially expressed from the paternal genome1. Ectopic OsBBM1 expression in the egg cell can override the fertilization requirement, giving rise to parthenogenetic progeny5. Here we show that the WOX-family transcription factor DWARF TILLER1 (OsDWT1)/WUSCHEL-LIKE HOMEODOMAIN 9 (OsWOX9A)6, another gene paternally expressed in zygotes, is a strong enhancer of embryo initiation by OsBBM1. Co-expression of OsWOX9A and OsBBM1 in egg cells results in 86-91% parthenogenesis, representing 4- to 15-fold increases over OsBBM1 alone. These results suggest that embryo initiation is promoted by the synergistic action of paternal-genome-expressed transcription factors in the fertilized egg cell. These findings can be utilized for the efficient production of haploids, as well as clonal hybrid seeds in crop plants7,8. The efficient induction of embryos without fertilization, which is important for crop breeding and hybrid seed production, can be achieved by combined expression in the egg of BBM1 and WOX9A, two rice transcription factors normally expressed from the male genome.

Cis-regulatory element editing can generate quantitative trait variation while mitigating against extreme phenotypes and harmful pleiotropy associated with coding sequence mutations. Here, we applied a multiplexed guide RNA design approach, informed by bioinformatic datasets, to generate genotypic variation in the promoter of OsSTOMAGEN, a positive regulator of stomatal density in rice. Engineered genotypic variation corresponded to broad and continuous variation in stomatal density, ranging from 70% to 120% of wild-type stomatal density. This near-isogenic panel of stomatal variants was leveraged in physiological assays to establish discrete relationships between stomatal morphological variation and stomatal conductance, carbon assimilation, and intrinsic water use efficiency in steady-state and fluctuating light conditions. Additionally, promoter alleles were subjected to vegetative drought regimes to assay the effects of the edited alleles on developmental response to drought. Notably, the capacity for drought-responsive stomatal density reprogramming in stomagen and two cis-regulatory edited alleles was reduced. Collectively our data demonstrate that cis-regulatory element editing can generate near-isogenic trait variation that can be leveraged for establishing relationships between anatomy, physiology, and crop improvement along diverse environmental clines.### Competing Interest StatementThe authors have declared no competing interest.

Rice (Oryza sativa) is of paramount importance for global nutrition, supplying at least 20% of global calories. However, water scarcity and increased drought severity are anticipated to reduce rice yields globally. We explored stomatal developmental genetics as a mechanism for improving drought resilience in rice while maintaining yield under climate stress. CRISPR/Cas9-mediated knockouts of the positive regulator of stomatal development STOMAGEN and its paralog EPIDERMAL PATTERNING FACTOR-LIKE10 (EPFL10) yielded lines with ∼25% and 80% of wild-type stomatal density, respectively. epfl10 lines with moderate reductions in stomatal density were able to conserve water to similar extents as stomagen lines but did not suffer from the concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation observed in stomagen knockouts. Moderate reductions in stomatal density achieved by editing EPFL10 present a climate-adaptive approach for safeguarding yield in rice. Editing the paralog of STOMAGEN in other species may provide a means for tuning stomatal density in agriculturally important crops beyond rice.

rice 3 4 Nicholas G. Karavolias, Dhruv Patel, Kyungyong Seong, Michelle Tjahjadi, Gloria5 Alexandra Gueorguieva, Jaclyn Tanaka, Douglas Dahlbeck, Myeong-Je Cho, Krishna K. 6 Niyogi, Brian J. Staskawicz 7 8 9 Summary 10 11 Rice production is of paramount importance for global nutrition and potential yields will 12 be detrimentally affected by climate change. Rice stomatal developmental genetics were 13 explored as a mechanism to improve water use efficiency while maintaining yield under 14 climate stress. 15 16 Gene-editing of STOMAGEN and its paralog, EPFL10, using CRISPR/Cas9 in rice cv. 17 Nipponbare yielded lines with altered stomatal densities that were functionally 18 characterized. CRISPR/Cas9 mediated knockouts of EPFL10 and STOMAGEN yielded 19 lines with c. 80% and 25% of wild-type stomata, respectively. 20 21 epfl10 lines with small reductions in stomatal densities are able to conserve water to similar 22 extents as stomagen lines with large stomatal density reductions but do not suffer from any 23 concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation. 24 25 The duplicate of STOMAGEN, EPFL10, is a weak positive regulator of stomatal 26 development in rice. epfl10 lines maintained wild-type physiological characteristics while 27 conserving more water. Modest reductions in stomatal densities may be a climate-adaptive 28 approach in rice that can safeguard yield. 29 30 3

SOG1 governs the programmed breakdown and reconstruction of the root stem cell niche after acute DNA damage. In Arabidopsis, DNA damage-induced programmed cell death is limited to the meristematic stem cell niche and its early descendants. The significance of this cell-type-specific programmed cell death is unclear. Here, we demonstrate in roots that it is the programmed destruction of the mitotically compromised stem cell niche that triggers its regeneration, enabling growth recovery. In contrast to wild-type plants, sog1 plants, which are defective in damage-induced programmed cell death, maintain the cell identities and stereotypical structure of the stem cell niche after irradiation, but these cells fail to undergo cell division, terminating root growth. We propose DNA damage-induced programmed cell death is employed by plants as a developmental response, contrasting with its role as an anticarcinogenic response in animals. This role in plants may have evolved to restore the growth of embryos after the accumulation of DNA damage in seeds.

In Arabidopsis, DNA damage-induced programmed cell death is limited to the meristematic stem cell niche and its early descendants. The significance of this cell-type-specific programmed cell death is unclear. Here, we demonstrate in roots that it is the programmed destruction of the mitotically compromised stem cell niche that triggers its regeneration, enabling growth recovery. In contrast to wild-type plants, sog1 plants, which are defective in damage-induced programmed cell death, maintain the cell identities and stereotypical structure of the stem cell niche after irradiation, but these cells fail to undergo cell division, terminating root growth. We propose DNA damage-induced programmed cell death is employed by plants as a developmental response, contrasting with its role as an anticarcinogenic response in animals. This role in plants may have evolved to restore the growth of embryos after the accumulation of DNA damage in seeds.