CRISPR/Cas9 genome editing relies on sgRNA-target DNA base pairing and a short downstream PAM sequence to recognize target DNA. The strict protospacer adjacent motif (PAM) requirement hinders applications of the CRISPR/Cas9 system since it restricts the targetable sites in the genomes. xCas9 and SpCas9-NG are two recently engineered SpCas9 variants that can recognize more relaxed NG PAMs, implying a great potential in addressing the issue of PAM constraint. Here we use stable transgenic lines to evaluate the efficacies of xCas9 and SpCas9-NG in performing gene editing and base editing in rice. We found that xCas9 can efficiently induce mutations at target sites with NG and GAT PAM sequences in rice. However, base editors containing xCas9 failed to edit most of the tested target sites. SpCas9-NG exhibited a robust editing activity at sites with various NG PAMs without showing any preference for the third nucleotide after NG. Moreover, we showed that xCas9 and SpCas9-NG have higher specificity than SpCas9 at the CGG PAM site. We further demonstrated that different forms of cytosine or adenine base editors containing SpCas9-NG worked efficiently in rice with broadened PAM compatibility. Taken together, our work has yielded versatile genome-engineering tools that will significantly expand the target scope in rice and other crops.

No abstract

SummaryPrevious studies revealed that the promoters for driving both Cas9 and sgRNAs are quite important for efficient genome editing by CRISPR/Cas9 in plants. Here, we report our results of targeted genome editing using the maize dmc1 gene promoter combined with the U3 promoter for Cas9 and sgRNA, respectively. Three loci in the maize genome were selected for targeting. The T0 plants regenerated were highly efficiently edited at the target sites with homozygous or bi‐allelic mutants accounting for about 66%. The mutations in T0 plants could be stably transmitted to the T1 generation, and new mutations could be generated in gametes or zygotes. Whole‐genome resequencing indicated that no off‐target mutations could be detected in the predicted loci with sequence similarity to the targeted site. Our results show that the dmc1 promoter‐controlled (DPC) CRISPR/Cas9 system is highly efficient in maize and provide further evidence that the optimization of the promoters used for the CRISPR/Cas9 system is important for enhancing the efficiency of targeted genome editing in plants. The evolutionary conservation of the dmc1 gene suggests its potential for use in other plant species.

No abstract

In this study, we assessed the performance of 34 Coupled Model Intercomparison Project Phase 5 (CMIP5) general climate models (GCMs) for simulating the observed temperature over the Lower Mekong Basin (LMB) in 1961–2004. An improved score-based method was used to rank the performance of the GCMs over the LMB. Two methods of multi-model ensemble (MME), sub-ensemble from the top 25% ranked GCMs and full ensemble from the entire GCMs, were calculated using arithmetic mean (AM) method and downscaled using the Delta method to project future temperature change during two future time periods, the near future (2006–2049) and the far future (2050–2093), under representative concentration pathways (RCP2.6, RCP4.5, and RCP8.5 scenarios) over the LMB. The improved score-based method combining multiple criteria showed a robust assessment of the GCMs performance over the LMB, which can provide good information for projecting future temperature change. The results showed a significant increase in temperature over the LMB under the two ensembles. However, there were differences in the magnitudes of the future temperature increase between the two ensemble methods, with a higher mean annual temperature increase from full ensemble and sub-ensemble at 1.26 °C (1.09 °C), 1.90 °C (1.70 °C), and 2.97 °C (2.78 °C) during 2050–2093 under the RCP2.6, RCP4.5, and RCP8.5 scenarios compared to the values at 0.93 °C (0.87 °C), 0.99 °C (0.95 °C), and 1.09 °C (1.06 °C) during 2006–2049, respectively, relative to the reference time period of 1961–2004. In the future (2006–2093), the temperature is likely to increase at 0.04 °C, 0.16 °C, and 0.37 °C decade-1 under the RCP2.6, RCP4.5, and RCP8.5 scenarios by the sub-ensemble, while a higher temperature increase at 0.05 °C, 0.17 °C, and 0.39 °C was found by the full ensemble over the LMB, relative to the reference time period of 1961–2004. On the whole, the higher warming mainly occurred in the northern and central areas over the LMB, while the lower warming mainly occurred in the southeast and the southwest, especially under the RCP4.5 and RCP8.5 scenarios, with the warming increased with increasing RCP for both ensembles. Moreover, in order to reduce the uncertainty of temperature projection in further studies in the LMB, multiple methods of GCMs ensemble should be considered and compared.

Zebrafish (Danio rerio) is a unique potential model animal for dissecting innate immune signaling. Here we demonstrate that herpes simplex virus 1 (HSV-1) could infect zebrafish at its different developmental stages and trigger the expression of type I interferons (IFNs) as well as interferon-stimulated genes (ISGs) in zebrafish larvae. Silencing of zSTING, but not zMAVS, markedly attenuates the DNA virus-induced antiviral responses. Notably, a conserved serine residue (S373) is essential for the action of zSTING. Unexpectedly, zebrafish cyclic GMP-AMP synthase (cGAS) is dispensable for the STING signaling, whereas zDHX9 and zDDX41 are potential sensors for HSV-1 infection in vivo. Taken together, this proof-of-concept study establishes the zebrafish larva as a feasible model for investigating the cytosolic DNA sensing mechanism, shedding light on the conservation of the STING antiviral signaling pathway. IMPORTANCEThe zebrafish larva provides technical advantages for understanding host-pathogen interactions. In this study, we established the zebrafish larva as a useful model for studying HSV-1 infection. HSV-1 infection triggers strong type I interferon production, which depends on STING expression. In addition, STING-mediated antiviral signaling is conserved in zebrafish. Interestingly, zDHX9 and zDDX41 are indispensable for detecting HSV-1, while cGAS is dispensable. This proof-of-concept study indicates that the zebrafish represents an amenable model for the investigation of cytosolic DNA sensing mechanisms.

No abstract

No abstract