Engineering of cytosine base editors with DNA damage minimization and editing scope diversification.

Cytosine base editors (CBEs), which enable precise C-to-T substitutions, have been restricted by potential safety risks, including DNA off-target edits, RNA off-target edits and additional genotoxicity such as DNA damages induced by double-strand breaks (DSBs). Though DNA and RNA off-target edits have been ameliorated via various strategies, evaluation and minimization of DSB-associated DNA damage risks for most CBEs remain to be resolved. Here we demonstrate that YE1, an engineered CBE variant with minimized DNA and RNA off-target edits, could induce prominent DSB-associated DNA damage risks, manifested as γH2AX accumulation in human cells. We then perform deaminase engineering for two deaminases lamprey LjCDA1 and human APOBEC3A, and generate divergent CBE variants with eliminated DSB-associated DNA damage risks, in addition to minimized DNA/RNA off-target edits. Furthermore, the editing scopes and sequence preferences of APOBEC3A-derived CBEs could be further diversified by internal fusion strategy. Taken together, this study provides updated evaluation platform for DSB-associated DNA damage risks of CBEs and further generates a series of safer toolkits with diversified editing signatures to expand their applications.

TadA orthologs enable both cytosine and adenine editing of base editors.

Cytidine and adenosine deaminases are required for cytosine and adenine editing of base editors respectively, and no single deaminase could enable concurrent and comparable cytosine and adenine editing. Additionally, distinct properties of cytidine and adenosine deaminases lead to various types of off-target effects, including Cas9-indendepent DNA off-target effects for cytosine base editors (CBEs) and RNA off-target effects particularly severe for adenine base editors (ABEs). Here we demonstrate that 25 TadA orthologs could be engineered to generate functional ABEs, CBEs or ACBEs via single or double mutations, which display minimized Cas9-independent DNA off-target effects and genotoxicity, with orthologs B5ZCW4, Q57LE3, E8WVH3, Q13XZ4 and B3PCY2 as promising candidates for further engineering. Furthermore, RNA off-target effects of TadA ortholog-derived base editors could be further reduced or even eliminated by additional single mutation. Taken together, our work expands the base editing toolkits, and also provides important clues for the potential evolutionary process of deaminases.

TadA reprogramming to generate potent miniature base editors with high precision.

Although miniature CRISPR-Cas12f systems were recently developed, the editing efficacy and targeting range of derived miniature cytosine and adenine base editors (miniCBEs and miniABEs) have not been comprehensively addressed. Moreover, functional miniCBEs have not yet be established. Here we generate various Cas12f-derived miniCBEs and miniABEs with improved editing activities and diversified targeting scopes. We reveal that miniCBEs generated with traditional cytidine deaminases exhibit wide editing windows and high off-targeting effects. To improve the editing signatures of classical CBEs and derived miniCBEs, we engineer TadA deaminase with mutagenesis screening to generate potent miniCBEs with high precision and minimized off-target effects. We show that newly designed miniCBEs and miniABEs are able to correct pathogenic mutations in cell lines and introduce genetic mutations efficiently via adeno-associated virus delivery in the brain in vivo. Together, this study provides alternative strategies for CBE development, expands the toolkits of miniCBEs and miniABEs and offers promising therapeutic tools for clinical applications.

Effects of Forest Type on Nutrient Fluxes in Throughfall, Stemflow, and Litter Leachate within Acid-Polluted Locations in Southwest China.

Although new inputs of acidic anions are decreasing, soil acidification still deserves more academic attention because of the effects of historical stores of SO42- already absorbed into soils. Forest canopy has large, species-specific effects on rainwater chemistry, for which the hydrological mechanism remains unclear. We investigated precipitation, throughfall, stemflow, and litter leachate across three forest types in a severely acid-polluted site located in Southwest China. Precipitation monitored over 4 months, representing summer, fall, winter, and spring, indicated neutral precipitation in Tieshanping with pH ranging from 6.58-7.33. Throughfall and litter leachate in Pinus massoniana Lamb. stands were enriched with greater cation and anion fluxes, as well as more dissolved organic carbon (DOC) flux. Rainwater from pure stands of Cinnamomum camphora (Linn) Presl yielded lower N and DOC inputs to soils with higher base saturation, which would reduce soil acidification and, therefore, improve the sustainability of forest ecosystems.

Integration of Multimodal Data for Deciphering Brain Disorders.

The accumulation of vast amounts of multimodal data for the human brain, in both normal and disease conditions, has provided unprecedented opportunities for understanding why and how brain disorders arise. Compared with traditional analyses of single datasets, the integration of multimodal datasets covering different types of data (i.e., genomics, transcriptomics, imaging, etc.) has shed light on the mechanisms underlying brain disorders in greater detail across both the microscopic and macroscopic levels. In this review, we first briefly introduce the popular large datasets for the brain. Then, we discuss in detail how integration of multimodal human brain datasets can reveal the genetic predispositions and the abnormal molecular pathways of brain disorders. Finally, we present an outlook on how future data integration efforts may advance the diagnosis and treatment of brain disorders.

Restoration Efficacy of Picea likiangensis var. rubescens Rehder & E. H. Wilson Plantations on the Soil Microbial Community Structure and Function in a Subalpine Area.

The knowledge concerning the relationship between vegetation restoration and soil microorganisms is limited, especially at high altitudes. In order to evaluate the restoration efficacy of the reforestation on the soil microbial community, we have examined vegetation composition, edaphic properties and structure and function of different soil microbial groups in two different aged (25- and 40-year-old) Picea likiangensis var. rubescens Rehder & E. H. Wilson (P. rubescens) plantations and the primeval coniferous forest (PCF) dominated by Abies squamata Masters by plot-level inventories and sampling in western Sichuan Province, China. Our results suggested that only the fungal samples in 25-year-old P. rubescens plantation could be distinguished from those in the PCF in both structure and function. The structure and function of the fungal community recovered relatively slowly compared with bacterial and archaeal communities. In addition to the soil chemical properties and tree species composition, the shrub composition was also a key factor influencing the soil microbial community. The P. rubescens plantations were conducive to restoring the soil microbial community in both structure and function. However, there were uncertainties in the variations of the bacterial and archaeal communities with increasing the P. rubescens plantation age.

Nitrogen Fertilization Modified the Responses of Schima superba Seedlings to Elevated CO2 in Subtropical China.

There are very few studies about the effects of relatively higher CO2 concentration (e.g., 1000 µmol·mol-1) or plus N fertilization on woody plants. In this study, Schima superba seedings were exposed to ambient or eCO2 (550, 750, and 1000 µmol·mol-1) and N fertilization (0 and 10 g·m-2·yr-1, hereafter: low N, high N, respectively) for one growth season to explore the potential responses in a subtropical site with low soil N availability. N fertilization strongly increased leaf mass-based N by 118.38%, 116.68%, 106.78%, and 138.95%, respectively, in different CO2 treatments and decreased starch, with a half reduction in leaf C:N ratio. Leaf N was significantly decreased by eCO2 in both low N and high N treatments, and N fertilization stimulated the decrease of leaf N and mitigated the increase of leaf C:N by eCO2. In low N treatments, photosynthetic rate (Pn) was maximized at 733 µmol·mol-1 CO2 in August and September, while, in high N treatments, Pn was continuously increased with elevation of CO2. N fertilization significantly increased plant biomass especially at highly elevated CO2, although no response of biomass to eCO2 alone. These findings indicated that N fertilization would modify the response of S. superba to eCO2.

Effects of natural and artificial restoration on plant community characteristics of alpine cutting blank in Western Sichuan, China.

To investigate the plant community characteristics of alpine cutting blanks under different restoration approaches, we conducted a field survey on cutting blanks experienced either natural restoration (40 years) or artificial restoration (30, 40 and 50 years) in western Sichuan, with natural forests as the reference. Our results showed that after 40 years natural succession, cutting blank was replaced by the secondary shrub of Spiraea alpina, while artificial restoration plantation was dominated by Picea likiangensis var. rubescens. The similarity indices between these communities and natural forests were low (0.19) and medium (0.28-0.49), respectively. Cutting blank through natural and artificial restoration had lower species diversity in the shrub layer but higher diversity in the herb layer than that of natural forests. With the increases of recovery time, total cross-sectional area at breast height, wood volume, index of species diameter class distribution, diversity indices, and similarity indices between plantations and natural forests gradually increased, while stand density gradually decreased. Compared with natural forests, plantations were facing with problems including high stand density, unreasonable structure, pure stands of cohorts and poor regeneration.

Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination.

Base editing tools with diversified editing scopes and minimized RNA off-target activities are required for broad applications. Nevertheless, current Streptococcus pyogenes Cas9 (SpCas9)-based adenine base editors (ABEs) with minimized RNA off-target activities display constrained editing scopes with efficient editing activities at positions 4-8. Here, functional ABE variants with diversified editing scopes and reduced RNA off-target activities are identified using domain insertion profiling inside SpCas9 and with different combinations of TadA variants. Engineered ABE variants in this study display narrowed, expanded or shifted editing scopes with efficient editing activities across protospacer positions 2-16. And when combined with deaminase engineering, the RNA off-target activities of engineered ABE variants are further minimized. Thus, domain insertion profiling provides a framework to improve and expand ABE toolkits, and its combination with other strategies for ABE engineering deserves comprehensive explorations in the future.

Elevated O3 alters soil bacterial and fungal communities and the dynamics of carbon and nitrogen.

Although many studies have reported the negative effects of elevated O3 on plant physiological characteristics, the influence of elevated O3 on below-ground processes and soil microbial functioning is less studied. In this study, we examined the effects of elevated O3 on soil properties, soil microbial biomass, as well as microbial community composition using high-throughput sequencing. Throughout one growing season, one-year old seedlings of two important endemic trees in subtropical China: Taxus chinensis (Pilger) Rehd. var. chinensis, and Machilus ichangensis Rehd. Et Wils, were exposed to charcoal-filtered air (CF as control), 100 nl l-1 (E100) or 150 nl l-1 (E150) O3-enriched air, in open top chambers (OTCs). We found that only higher O3 exposure (E150) significantly decreased soil microbial biomass carbon and nitrogen in M. ichangensis, and the contents of organic matter were significantly decreased by E150 in both tree species. Although both levels of O3 exposure decreased NO3-N in T. chinensis, only E150 increased NO3-N in M. ichangensis, and there were no effects of O3 on NH4-N. Moreover, elevated O3 elicited changes in soil microbial community structure and decreased fungal diversity in both M. ichangensis and T. chinensis. However, even though O3 exposure reduced bacterial diversity in M. ichangensis, no effect of O3 exposure on bacterial diversity was detected in soil grown with T. chinensis. Our results showed that elevated O3 altered the abundance of bacteria and fungi in general, and in particular reduced nitrifiers and increased the relative abundance of some fungal taxa capable of denitrification, which may stimulate N2O emissions. Overall, our findings indicate that elevated O3 not only impacts the soil microbial community structure, but may also exert an influence on the functioning of microbial communities.

Effects of Elevated Ozone Levels on Photosynthesis, Biomass and Non-structural Carbohydrates of Phoebe bournei and Phoebe zhennan in Subtropical China.

To assess the impacts of ozone (O3) on carbon metabolism of subtropical broadleaved tree species, seedlings of Phoebe bournei and Phoebe zhennan were exposed to elevated O3 levels in open-top chambers (OTCs) from June to November 2014. Three treatments were conducted in nine total OTCs, including charcoal-filter air (CF) as a control treatment, low O3 treatment 'O3-1' (∼100 nl l-1), and high O3 treatment 'O3-2' (∼150 nl l-1). Our findings demonstrated that elevated O3 levels significantly decreased the net photosynthesis rates (Pn ) and leaf, root, and total biomass of both species, while it did not significantly affect the root/shoot ratio in P. bournei and P. zhennan. O3-1 treatments significantly increased water soluble carbohydrates (WSC) in leaves of both tree species, while only increased the total non-structural carbohydrates (TNC) and starch in leaves of P. bournei; effects on P. zhennan were equivalent in comparison to the control treatment (CF). Likewise, there was no effect of treatment on the polysaccharide content of both tree species. The contents of polysaccharide, starch contents in fine roots of both species, and TNC in fine roots of P. bournei increased significantly in O3-1 compared to CF. O3-2 treatment significantly decreased starch and TNC in the fine roots of P. bournei, and significantly decreased polysaccharide, starch, WSC, and TNC in the fine roots of P. zhennan. Elevated O3 had no effects on leaf polysaccharide in both species, but O3-1 significantly increased polysaccharide in the fine roots of both species, and O3-1 significantly increased WSC in the leaves while decreased that in the fine roots of both species. These results suggested that elevated O3 levels have significant impacts on the carbon metabolism of both tree species in our study, with differential responses between tree species and among leaves and roots.

Effects of elevated O3 on physiological and biochemical responses in three kinds of trees native to subtropical forest in China during non-growing period.

Numerous studies have documented the negative effects of ozone (O3) on tree species in growing season, however, little is done in non-growing season. Three evergreen tree species, Phoebe bournei (Hemsl.) Yang (P. bournei), Machilus pauhoi Kanehira (M. pauhoi) and Taxus chinensis (Pilger) Rehd (T. chinensis), were exposed to non-filtered air, 100 nmol mol-1 O3 air (E1) and 150 nmol mol-1 O3 air (E2) in open-top chambers in subtropical China. In the entire period of experiment, O3 fumigation decreased net photosynthesis rate (Pn) through stomatal limitation during the transition period from growing to non-growing season (TGN), and through non-stomatal limitation during the period of non-growing season (NGS) in all species tested. Meanwhile, O3 fumigation reduced and delayed the resilience of Pn in all species tested during the transition period from non-growing to growing season (TNG). O3 fumigation significantly decreased chlorophyll contents during NGS, whereas no obvious injury symptoms were observed till the end of experiment. O3 fumigation induced increases in levels of malondialdehyde, superoxide dismutase, total phenolics and reduced ascorbic acid, and changes in four plant endogenous hormones as well in all species tested during NGS. During NGS, E1 and E2 reduced Pn by an average of 80.11% in P. bournei, 94.56% in M. pauhoi and 12.57% in T. chinensis, indicating that the O3 sensitivity was in an order of M. pauhoi > P. bournei > T. chinensis. Overall, O3 fumigation inhibited carbon fixation in all species tested during NGS. Furthermore, O3-induced physiological activities also consumed the dry matter. All these suggested that elevated O3, which is likely to come true during NGS in the future, will adversely affect the accumulation of dry matter and the resilience of Pn during TNG in evergreen tree species, and further inhibit their growth and development in the upcoming growing season.

How important is woody tissue photosynthesis in EuCahetus dunnii Maiden and Osmanthus fragrans (Thunb.) Lour. under O3 stress?

Numerous studies have demonstrated the negative effects of elevated O3 on leaf photosynthesis. Within trees, a portion of respired CO2 is assimilated by woody tissue photosynthesis, but its response to elevated O3 remains unclear. Saplings of two evergreen tree species, EuCahetus dunnii Maiden (E. dunnii) and Osmanthus fragrans (Thunb.) Lour. (O. fragrans), were exposed to non-filtered air (NF), 100 nmol mol-1 O3 air (E1) and 150 nmol mol-1 O3 air (E2) in open-top chambers from May 5 to September 5, 2016 (8 h a day; 7 days a week) in subtropical China. In this study, O3 fumigation significantly reduced leaf net photosynthesis rate in both two tree species on most measurements. However, compared with leaf net photosynthesis rate, woody tissue gross photosynthesis rate showed less negative response to O3 fumigation and was even stimulated to increase. Refixation rate reflects the utilization efficiency of the respired CO2 by woody tissue photosynthesis. During the experiment period, E1 and E2 both increased refixation rate in O. fragrans compared with NF. Whereas for E. dunnii, E1 increased refixation rate until 81 days after starting of fumigation and then decreased it, and E2 decreased it all the time. Refixation rate had a significant positive correlation with woody tissue chlorophyll contents, indicating that the response of refixation rate to elevated O3 may relate to chlorophyll contents. All these suggested that under O3 fumigation, when atmospheric CO2 uptake and fixation by leaf is limited, woody tissue photosynthesis can contribute more to the total carbon assimilation in trees. The findings help to understand the significance of woody tissue photosynthesis under elevated O3 conditions.

Physiological and biochemical responses of Machilus ichangensis Rehd. et Wils and Taxus chinensis (Pilger) Rehd. to elevated O3 in subtropical China.

Considerable researches have documented the negative effects of ozone on woody species in North America and Europe; however, little is known about how woody tree species respond to elevated O3 in subtropical China, and most of the previous studies were conducted using pot experiment. In the present study, Machilus ichangensis Rehd. et Wils (M. ichangensis) and Taxus chinensis (Pilger) Rehd. (T. chinensis), evergreen tree species in subtropical China, were exposed to non-filtered air (NF), 100 nmol mol-1 O3 (E1) and 150 nmol mol-1 O3 (E2), in open-top chambers under field conditions from 21st March to 2nd November 2015. In this study, O3 fumigation significantly reduced net photosynthesis rate (Pn) in M. ichangensis in the three measurements and in T. chinensis in the last measurement. Also, non-stomatal factors should be primarily responsible for the decreased Pn. O3 fumigation-induced increase in malondialdehyde, superoxide dismutase, and reduced ascorbic acid levels indicated that antioxidant defense mechanism had been stimulated to prevent O3 stress and repair the oxidative damage. Yet, the increase of antioxidant ability was not enough to counteract the harm of O3 fumigation. Because of the decrease in CO2 assimilation, the growth of the two tree species was restrained ultimately. The sensitivity of the two tree species to O3 can be determined: M. ichangensis > T. chinensis. It suggests a close link between the rising O3 concentrations and the health risk of some tree species in subtropics in the near future.

Effects of grazing intensity on soil labile organic carbon fractions in a desert steppe area in Inner Mongolia.

Grazing can cause changes in soil carbon (C) level. This study aimed to elucidate the response of soil labile organic carbon (SLOC) under four different grazing intensities: non grazing (NG), 0 sheep·ha(-1); light grazing (LG), 0.91 sheep·ha(-1); moderate grazing (MG), 1.82 sheep·ha(-1), and heavy grazing (HG), 2.73 sheep·ha(-1). Results showed that there was no significant difference in total soil organic carbon (TOC) and soil inorganic carbon (SIC) content from three soil depths (0-15 cm, 15-30 cm, and 30-45 cm) under different grazing intensities. However, the SLOC including particulate organic carbon (POC), light fraction organic carbon (LFOC), and readily oxidizable carbon (ROC) content at a depth of 0-15 cm decreased with the increasing grazing intensity among LG, MG and HG. The SLOC content at depths of 15-30 cm under the NG and LG were significantly higher than that under the MG and the HG. The TOC and SLOC content decreased with increasing depths of soil horizons, but SIC content increased. The variation trend of the density of different soil carbon fractions and the ratio of individual SLOC fractions to TOC were similar to that of the soil carbon content of corresponding fractions. These results indicated that MG and HG treatments caused C loss at 0-30 cm; and SLOC was more sensitive than TOC in response to different grazing intensities.

Response of soil respiration to soil temperature and moisture in a 50-year-old oriental arborvitae plantation in China.

China possesses large areas of plantation forests which take up great quantities of carbon. However, studies on soil respiration in these plantation forests are rather scarce and their soil carbon flux remains an uncertainty. In this study, we used an automatic chamber system to measure soil surface flux of a 50-year-old mature plantation of Platycladus orientalis at Jiufeng Mountain, Beijing, China. Mean daily soil respiration rates (R(s)) ranged from 0.09 to 4.87 µmol CO(2) m(-2) s(-1), with the highest values observed in August and the lowest in the winter months. A logistic model gave the best fit to the relationship between hourly R(s) and soil temperature (T(s)), explaining 82% of the variation in R(s) over the annual cycle. The annual total of soil respiration estimated from the logistic model was 645±5 g C m(-2) year(-1). The performance of the logistic model was poorest during periods of high soil temperature or low soil volumetric water content (VWC), which limits the model's ability to predict the seasonal dynamics of R(s). The logistic model will potentially overestimate R(s) at high T(s) and low VWC. Seasonally, R(s) increased significantly and linearly with increasing VWC in May and July, in which VWC was low. In the months from August to November, inclusive, in which VWC was not limiting, R(s) showed a positively exponential relationship with T(s). The seasonal sensitivity of soil respiration to T(s) (Q(10)) ranged from 0.76 in May to 4.38 in October. It was suggested that soil temperature was the main determinant of soil respiration when soil water was not limiting.