The CRISPR-Cas9-based multiplexed gene editing (MGE) provides a powerful method to modify multiple genomic regions simultaneously controlling different agronomic traits in crops. We applied the MGE construct built by combining the tandemly arrayed tRNA–gRNA units to generate heritable mutations in the TaGW2, TaLpx-1, and TaMLO genes of hexaploid wheat. The knockout mutations generated by this construct in all three homoeologous copies of one of the target genes, TaGW2, resulted in a substantial increase in seed size and thousand grain weight. We showed that the non-modified gRNA targets in the early generation plants can be edited by CRISPR-Cas9 in the following generations. Our results demonstrate that transgenerational gene editing activity can serve as the source of novel variation in the progeny of CRISPR-Cas9-expressing plants and suggest that the Cas9-inducible trait transfer for crop improvement can be achieved by crossing the plants expressing the gene editing constructs with the lines of interest.

Key messageCRISPR-Cas9-based genome editing and EMS mutagenesis revealed inter-cultivar differences and additivity in the contribution of TaGW2 homoeologues to grain size and weight in wheat.AbstractThe TaGW2 gene homoeologues have been reported to be negative regulators of grain size (GS) and thousand grain weight (TGW) in wheat. However, the contribution of each homoeologue to trait variation among different wheat cultivars is not well documented. We used the CRISPR-Cas9 system and TILLING to mutagenize each homoeologous gene copy in cultivars Bobwhite and Paragon, respectively. Plants carrying single-copy nonsense mutations in different genomes showed different levels of GS/TGW increase, with TGW increasing by an average of 5.5% (edited lines) and 5.3% (TILLING mutants). In any combination, the double homoeologue mutants showed higher phenotypic effects than the respective single-genome mutants. The double mutants had on average 12.1% (edited) and 10.5% (TILLING) higher TGW with respect to wild-type lines. The highest increase in GS and TGW was shown for triple mutants of both cultivars, with increases in 16.3% (edited) and 20.7% (TILLING) in TGW. The additive effects of the TaGW2 homoeologues were also demonstrated by the negative correlation between the functional gene copy number and GS/TGW in Bobwhite mutants and an F2 population. The highest single-genome increases in GS and TGW in Paragon and Bobwhite were obtained by mutations in the B and D genomes, respectively. These inter-cultivar differences in the phenotypic effects between the TaGW2 gene homoeologues coincide with inter-cultivar differences in the homoeologue expression levels. These results indicate that GS/TGW variation in wheat can be modulated by the dosage of homoeologous genes with inter-cultivar differences in the magnitude of the individual homoeologue effects.Electronic supplementary materialThe online version of this article (10.1007/s00122-018-3166-7) contains supplementary material, which is available to authorized users.

Grain size and weight are important components of a suite of yield-related traits in crops. Here, we showed that the CRISPR-Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1-recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double-copy mutant showing larger effect than the respective single copy mutants. The TaGW7-centered gene co-expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co-localization of TaGW7 with aand b-tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7-associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR-Cas9 system with cross-species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.

Plants have evolved a large number of transcription factors (TF), which are enriched among duplicate genes, highlighting their roles in complex regulatory networks. The APETALA2/EREBP-like genes constitute a large plant TF family and participate in development and stress responses. To probe the conservation and divergence of AP2/EREBP genes, we analyzed the duplication patterns of this family in Brassicaceae and identified interacting proteins of representative Arabidopsis AP2/EREBP proteins. We found that many AP2/EREBP duplicates generated early in Brassicaceae history were quickly lost, but many others were retained in all tested Brassicaceae species, suggesting early functional divergence followed by persistent conservation. In addition, the sequences of the AP2 domain and exon numbers were highly conserved in rosids. Furthermore, we used 16 A. thaliana AP2/EREBP proteins as baits in yeast screens and identified 1,970 potential AP2/EREBP-interacting proteins, with a small subset of interactions verified in planta. Many AP2 genes also exhibit reduced expression in an antherdefective mutant, providing a possible link to developmental regulation. The putative AP2-interacting proteins participate in many functions in development and stress responses, including photomorphogenesis, flower development, pathogenesis, drought and cold responses, abscisic acid and auxin signaling. Our results present the AP2/EREBP evolution patterns in Brassicaceae, and support a proposed interaction network of AP2/EREBP proteins and their putative interacting proteins for further study.

The development of CRISPR-based editors having different protospacer adjacent motif (PAM) recognition specificities, or guide RNA length/structure requirements broadens the range of possible genome editing applications. Here, we evaluated the natural and engineered variants of Cas12a (FnCas12a from Francisella novicida and LbCas12a from Lachnospiraceae bacterium) and Cas9 for wheat genome editing efficiency and ability to induce heritable mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a was able to induce mutations in the wheat genome in the current study, even though with a lower rate than that reported for SpCas9. The eight-fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guide RNAs flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides. A LbCas12a-MGE construct was successfully applied for generating heritable mutations in a gene controlling grain size and weight in wheat. We show that the range of editable loci in the wheat genome could be expanded by using the engineered variants of Cas12a (LbCas12a-RVR) and Cas9 (Cas9-NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9-NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports the set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to Cas9-based modification.

b1 a these authors contributed equally to work b corresponding authors (Cristobal Uauy, email: cristobal.uauy@jic.ac.uk, phone: 0-160-345-0195 and Eduard Akhunov, email: eakhunov@ksu.edu, phone: 1-785-532-1342) (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/327874 doi: bioRxiv preprint first posted online May. 22, 2018; 2 KEY MESSAGE CRISPR-Cas9-based genome editing and EMS mutagenesis revealed inter-cultivar differences and additivity in the contribution of TaGW2 homoeologues to grain size and weight in wheat.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/327874 doi: bioRxiv preprint first posted online May. 22, 2018; 3 AbstractThe TaGW2 gene homoeologues have been reported to be negative regulators of grain size (GS) and thousand grain weight (TGW) in wheat. However, the contribution of each homoeologue to trait variation among different wheat cultivars is not well documented. We used the CRISPR-Cas9 system and TILLING to mutagenize each homoeologous gene copy in cultivars Bobwhite and Paragon, respectively. Plants carrying single-copy nonsense mutations in different genomes showed different levels of GS/TGW increase, with TGW increasing by an average of 5.5% (edited lines) and 5.3% (TILLING mutants). In any combination, the double homoeologue mutants showed higher phenotypic effects than the respective single-genome mutants. The double mutants had on average 12.1% (edited) and 10.5% (TILLING) higher TGW with respect to wild-type lines. The highest increase in GS and TGW was shown for triple mutants of both cultivars, with increases of 16.3% (edited) and 20.7% (TILLING) in TGW. The additive effects of the TaGW2 homoeologues were also demonstrated by the negative correlation between the functional gene copy number and GS/TGW in Bobwhite mutants and an F 2 population. The highest single-genome increases in GS and TGW in Paragon and Bobwhite were obtained by mutations in the B and D genomes, respectively. These inter-cultivar differences in the phenotypic effects between the TaGW2 gene homoeologues coincide with inter-cultivar differences in the homoeologue expression levels. These results indicate that GS/TGW variation in wheat can be modulated by the dosage of homoeologous genes with inter-cultivar differences in the magnitude of the individual homoeologue effects.

Summary The development of CRISPR‐based editors recognizing distinct protospacer‐adjacent motifs (PAMs), or having different spacer length/structure requirements broadens the range of possible genomic applications. We evaluated the natural and engineered variants of Cas12a (FnCas12a and LbCas12a) and Cas9 for their ability to induce mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a‐induced mutations in the wheat genome, even though with a lower rate than that reported for SpCas9. The eight‐fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guides flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides and showed substantial increase after subjecting transgenic plants to high‐temperature treatment. We successfully applied LbCas12a‐MGE for generating heritable mutations in a gene controlling grain size and weight in wheat. We showed that the range of editable loci in the wheat genome could be further expanded by using the engineered variants of Cas12a (LbCas12a‐RVR) and Cas9 (Cas9‐NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9‐NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports a set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to modification using the original SpCas9 nuclease.

Background: Novel coronavirus pneumonia is a novel kind of highly contagious disease without any specific drugs. Considering the successful experience of antiviral therapy combined with glucocorticoids (GCs) in severe acute respiratory syndrome, this study was designed to evaluate the clinical efficacy of GCs in treating patients with coronavirus disease 2019 (COVID-19).Methods: A cohort of 42 patients with COVID-19 admitted to The First Hospital of Jiaxing from January 4, 2020, to February 16, 2020, were included and grouped into a test group (n=20) and control group (n=22) based on their therapeutic regimens. There were no significant differences in baseline characteristics between patients in the two groups. Conventional treatment (antiviral therapy) was given to patients in both groups, while an additional hormone drug (GCs) was used in patients in the test group. Indices including body temperature, blood routine indices [white blood cell (WBC), lymphocyte, monocyte, and C-reactive protein (CRP)], blood biochemical indices [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)], and complications were recorded during the treatment. Time to achieve negative virus nucleic acid (nCoV-RNA) testing, and hospital stays were also observed and compared between the two groups.Results: All included patients completed the trial. After treatment, superior therapeutic efficacy was achieved in patients in the test group, with body temperature dropping more significantly with a much shorter recovery time compared to the control group (P=0.0412). Simultaneously, the percentage of patients with abnormal blood routine indices (WBC), monocyte, and (CRP) in the test group was reduced more sharply, while no noticeable difference was observed in the number of patients who developed abnormal blood biochemical indices during treatment between the two groups. Additionally, a shorter duration of hospital stays was found in the test group relative to the control group (14.84±8.76 vs. 18.25±7.42 days, P>0.05). Patients who received GCs had a shorter recovery time for body temperature and inflammation.Conclusions: Hormonotherapy with GCs can accelerate the recovery time for body temperature as well as inflammation in patients with COVID-19. It deserves promotion and application in the clinical treatment of coronavirus disease as a form of adjuvant medicine. The ongoing focus of research is on long-term adverse events in GCs.