Regulation of Precise DNA Repair by Nuclear Actin Polymerization: A Chance for Improving Gene Therapy?

Although more difficult to detect than in the cytoplasm, it is now clear that actin polymerization occurs in the nucleus and that it plays a role in the specific processes of the nucleus such as transcription, replication, and DNA repair. A number of studies suggest that nuclear actin polymerization is promoting precise DNA repair by homologous recombination, which could potentially be of help for precise genome editing and gene therapy. This review summarizes the findings and describes the challenges and chances in the field.

UAV-based modelling of vegetation recovery under extreme habitat stresses in the water level fluctuation zone of the Three Gorges Reservoir, China.

Impoundment of the Three Gorges Reservoir on the upper Yangtze River has remarkably altered hydrological regime within the dammed reaches, triggering structural and functional changes of the riparian ecosystem. Up to date, how vegetation recovers in response to compound habitat stresses in the water level fluctuation zone remains inexplicitly understood. In this study, plant above-ground biomass (AGB) in a selected water level fluctuation zone was quantified to depict its spatial and temporal pattern using unmanned aerial vehicle (UAV)-derived multispectral images and screened empirical models. The contributions of multiple habitat stressors in governing vegetation recovery dynamics along the environmental gradient were further explored. Screened random forest models indicated relatively higher accuracy in AGB estimation, with R2 being 0.68, 0.79 and 0.62 during the sprouting, growth, and mature periods, respectively. AGB displayed a significant linear increasing trend along the elevational gradient during the sprouting and early growth period, while it showed an inverted U-shaped pattern during late growth and mature period. Flooding duration, magnitude and timing were found to exert greater negative effects on plant sprouting and biomass accumulation and acted as decisive factors in governing the elevation-dependent pattern of AGB. Localized spatial variations in AGB were modulated by other stressors such as sediment burial, soil erosion, soil moisture and nutrient content. Occurrence of episodic summer floods and vegetation distribution were responsible for an inverted U-shaped pattern of AGB during the late growth and mature period. Generally, AGB reached its peak in August, thereafter an obvious decline by an unprecedent dry-hot climatic event. The water level fluctuations with cumulative flooding effects exerted substantial control on AGB temporal dynamics, while climatic condition played a secondary role. Herein, further restorative efforts need to be directed to screening suitable species, maintaining favorable soil condition, and improving vegetation pattern to balance the many trade-offs.

Sedimentary geochemistry mediated by a specific hydrological regime in the water level fluctuation zone of the Three Gorges Reservoir, China.

The water level fluctuation zone (WLFZ) of the Three Gorges Reservoir (TGR) acts as an important sink for inflowing suspended sediment loads over the inundation periods following regular dam operations. This study depicts the sedimentary geochemical dynamics along a sedimentary profile based on the determined chronology and explores its links to the specific hydrological regime created by dam flow regulation and riverine seasonal suspended sediment dynamics. A compact 345-cm-long sediment core was extracted near the base water level (145.3 m) from the WLFZ of the TGR and sectioned at 5-cm intervals. Extracted sediment subsamples were analyzed for grain size composition, organic matter (OM), total nitrogen (TN), and geochemical elements (Na, K, Ca, Mg, Pb, Zn, Ni, Co, Mn, Cr, Fe, and Cu). The sediment core chronology was determined using 137Cs elemental analysis. Sedimentary geochemistry and grain size properties of extracted sediment core exhibited greater variations during initial submergence years till the first complete impoundment of the TGR (2006-2010). Afterward (2011-2013), although upstream inflowing suspended sediments and reservoir water level were comparable, sediment deposition and concentrations of sedimentary geochemical constituents showed considerably fewer variations. Seasonal variations in sediment deposition and geochemical composition were also observed during the rainy (October-April) and dry (May-September) seasons, in addition to annual variations. Grain size, OM, and other sediment geochemical constituents all had significant correlations with each other and with sediment core depth. The concentrations of geochemical elements in various sediment stratigraphic layers exhibited staggering associations with each other and were dependent on each other in several ways. The arrangement of geochemical elements in various stratigraphic layers of the extracted core illustrated amalgamation with inputs from upstream seasonal suspended sediment dynamics and reservoir water levels. During shortened submergence periods and higher input sediment loads, geochemical elements demonstrated impulsive distributions. Alternatively, during longer submergence periods, elemental distributions were relatively uniform attributed to higher settling time to deposit according to grain size and geochemical affinities. Higher suspended sediment loads in association with seasonal floods also resulted in rough sediment deposition patterns, imparting variations in the distributions of geochemical elements. Interim mediations in geochemical element concentrations are associated with seasonal distal flash floods and local terrace bank collapses, which generate significant amounts of distal sediment loads that are quickly deposited and are not sorted hydrodynamically. Overall, although a specific mechanism was devised to circumvent the siltation process, a considerable amount of sediment is trapped at pre-dam sites. In addition, siltation caused nutrients and geochemical elements' enrichment.

Sangerbox: A comprehensive, interaction-friendly clinical bioinformatics analysis platform.

In recent decades, with the continuous development of high-throughput sequencing technology, data volume in medical research has increased, at the same time, almost all clinical researchers have their own independent omics data, which provided a better condition for data mining and a deeper understanding of gene functions. However, for these large amounts of data, many common and cutting-edge effective bioinformatics research methods still cannot be widely used. This has encouraged the establishment of many analytical platforms, a portion of databases or platforms were designed to solve the special analysis needs of users, for instance, MG RAST, IMG/M, Qiita, BIGSdb, and TRAPR were developed for specific omics research, and some databases or servers provide solutions for special problems solutions. Metascape was designed to only provide functional annotations of genes as well as function enrichment analysis; BioNumerics and RidomSeqSphere+ perform multilocus sequence typing; CARD provides only antimicrobial resistance annotations. Additionally, some web services are outdated, and inefficient interaction often fails to meet the needs of researchers, such as our previous versions of the platform. Therefore, the demand to complete massive data processing tasks urgently requires a comprehensive bioinformatics analysis platform. Hence, we have developed a website platform, Sangerbox 3.0 (http://vip.sangerbox.com/), a web-based tool platform. On a user-friendly interface that also supports differential analysis, the platform provides interactive customizable analysis tools, including various kinds of correlation analyses, pathway enrichment analysis, weighted correlation network analysis, and other common tools and functions, users only need to upload their own corresponding data into Sangerbox 3.0, select required parameters, submit, and wait for the results after the task has been completed. We have also established a new interactive plotting system that allows users to adjust the parameters in the image; moreover, optimized plotting performance enables users to adjust large-capacity vector maps on the web site. At the same time, we have integrated GEO, TCGA, ICGC, and other databases and processed data in batches, greatly reducing the difficulty to obtain data and improving the efficiency of bioimformatics study for users. Finally, we also provide users with rich sources of bioinformatics analysis courses, offering a platform for researchers to share and exchange knowledge.

Engineered FnCas12a with enhanced activity through directional evolution in human cells.

Clustered regularly interspaced short palindromic repeat-Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity.

Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNAGln-processing system in human cells.

CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNAGln-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifically, acting on previous observations that small guide RNAs (sgRNAs) harboring an extra A or G (A/G) in the first 5' nucleotide greatly affect the activity of high-fidelity Cas9 variants and that tRNA-sgRNA fusions improve Cas9 activity, we investigated whether a GN20 sgRNA fused to different tRNAs (G-tRNA-N20) could restore the activity of SpCas9 variants in human cells. Using flow cytometry, a T7E1 assay, deep sequencing-based DNA cleavage activity assays, and HEK-293 cells, we observed that a tRNAGln-sgRNA fusion system enhanced the activity of Cas9 variants, which could be harnessed for efficient correction of a pathogenic mutation in the retinoschisin 1 (RS1) gene, resulting in 6- to 8-fold improved Cas9 activity. We propose that the tRNA-processing system developed here specifically for human cells could facilitate high-fidelity Cas9-mediated human genome-editing applications.

Efficient Human Genome Editing Using SaCas9 Ribonucleoprotein Complexes.

Genome editing using RNA-guided nucleases in their ribonucleoprotein (RNP) form represents a promising strategy for gene modification and therapy because they are free of exogenous DNA integration and have reduced toxicity in vivo and ex vivo. However, genome editing by Cas9 nuclease from Staphylococcus aureus (SaCas9) has not been reported in its RNP form, which recognizes a longer protospacer adjacent motif (PAM), 5'-NNGRRT-3', compared with Streptococcus pyogenes Cas9 (SpCas9) of 5'-NGG-3' PAM. Here, SaCas9-RNP-mediated genome editing is reported in human cells. The SaCas9-RNP displayed efficient genome editing activities of enhanced green fluorescent protein (EGFP) coding gene as well as three endogenous genes (OPA1, RS1, and VEGFA). Further, SaCas9-RNP is successfully implemented to correct a pathogenic RS1 mutation for X-linked juvenile retinoschisis. It is also shown that off-target effects triggered by SaCas9-RNP are undetectable by targeted deep sequencing. Collectively, this study demonstrates the potential of SaCas9-RNP-mediated genome editing in human cells, which could facilitate genome-editing-based therapy.

Basic and Clinical Application of Adeno-Associated Virus-Mediated Genome Editing.

Traditional gene therapy (gene replacement) has made a breakthrough in treating inherited diseases. Adeno-associated virus (AAV) has emerged as a highly promising vector with innate ability, boosting the development of gene replacement and gene targeting. With the recent advance of engineered nucleases that work efficiently in human cells, AAV mediated-genome editing with nucleases has raised hopes for in situ gene therapy of inherited and non-inherited diseases. Here, the applications of AAV-mediated genome editing are highlighted, and the prospect of AAV and nucleases that will render extension of such success in clinical gene therapy is discussed.

Engineering the Direct Repeat Sequence of crRNA for Optimization of FnCpf1-Mediated Genome Editing in Human Cells.

FnCpf1-mediated genome-editing technologies have enabled a broad range of research and medical applications. Recently, we reported that FnCpf1 possesses activity in human cells and recognizes a more compatible PAM (protospacer adjacent motif, 5'-KYTV-3'), compared with the other two commonly used Cpf1 enzymes (AsCpf1 and LbCpf1), which requires a 5'-TTTN-3' PAM. However, due to the efficiency and fidelity, FnCpf1-based clinical and basic applications remain a challenge. The direct repeat (DR) sequence is one of the key elements for FnCpf1-mediated genome editing. In principle, its engineering should influence the corresponding genome-editing activity and fidelity. Here we showed that the DR mutants [G(-9)A and U(-7)A] could modulate FnCpf1 performance in human cells, enabling enhancement of both genome-editing efficiency and fidelity. These newly identified features will facilitate the design and optimization of CRISPR-Cpf1-based genome-editing strategies.

Particle size differentiation explains flow regulation controls on sediment sorting in the water-level fluctuation zone of the Three Gorges Reservoir, China.

The Three Gorges Dam has significantly interrupted fluvial continuity and modified the mass transfer regime along river continuums. Flow regulation following regular dam operations drives dramatic hydrological regime shifts, which facilitates sediment dispersal in the water-level fluctuation zone over episodic inundation periods. How flow regulation modulates sediment redistribution, however, remains unclear. In this study, we depict absolute particle size composition of suspended sediment and sink sediment in the water-level fluctuation zone, and these are interpreted in the context of flow regulation controls on sediment sorting. Multiple sampling strategies were applied at different spatial and temporal scales, to overcome limitations of labour and cost input in a large-scale field study and to collect representative samples. The results revealed a longitudinal fining trend and seasonal variability in particle size composition for suspended sediment. Sink sediment collected from the water-level fluctuation zone during a single summer flood event displayed a similar longitudinal fining trend, reflecting preferential settling of coarser fractions in the backwater reaches where flow velocity declines sharply. Surface sediment demonstrated a laterally coarsening trend with increasing elevations along a slope profile. Flooding duration, frequency and timing represent key factors in determining the elevation-dependent variations in the magnitude of sedimentation and its source inputs. Relatively longer flooding duration and frequent intermediate summer floods with high suspended sediment flux are responsible for high sedimentation rates in the lower portions with distal upstream source inputs, while low sedimentation rates in the upper portions are principally associated with water impoundment and sediment produced from local bank erosion. Vertical particle size variability was observed along a sedimentary core profile, which most likely reflects seasonal differences in source supply with contrasting particle size characteristics. We conclude that absolute particle size differentiation explains flow regulation controls on sediment sorting in the water-level fluctuation zone of the Three Gorges Reservoir.

Sedimentary chronology reinterpreted from Changshou Lake of the Three Gorges Reservoir Area reveals natural and anthropogenic controls on sediment production.

Sedimentary archives preserved in geomorphic sinks provide records of historical sediment dynamics and its related natural and anthropogenic controls. This study reinterpreted sedimentary processes in Changshou Lake of the Three Gorges Reservoir Area in China by combining a rainfall erosivity index with multiple tracing proxies, and the impacts of natural and anthropogenic drivers on sediment production were also explored. Erosive rainfalls with low frequency and large magnitude in the rainy season contribute to a substantial proportion of annual total rainfall, which thus can be used to infer erosion and sediment yield events. The sedimentary chronology was determined by comparing rainfall erosivity index with depth distribution of 137Cs and absolute particle size, which revealed annual sedimentation rates ranging from 1.1 to 2.3 cm a-1. The multi-proxy dating index and variation of sedimentation rate divided the sediment profile into three major periods. The reference period (1956-1982) displays low variability of TOC, TN, trace metal concentrations, and mean sedimentation rate. In the stressed period (1982-1998), industrial and sewerage discharge led to input and deposition of TOC, TN, and trace metals (e.g., Cd, Co, Cu, Cr, and Ni). The highest annual sediment accumulation rate of 2.3 cm a-1 may be ascribed to the 1982 big flood event. In the present period (1998-2013), increased TOC, TN and decreased trace metals in the top layers of the sediment core indicated changes in lake ecology. Fish farming promoted algal growth and primary productivity which caused eutrophication until 2004-2005. The reduced mean sedimentation rate of 1.7 cm a-1 between 1998 and 2004, and thereafter, may be attributed to soil and water conservation and reforestation policies implemented in the Longxi catchment. Human activities such as deforestation, cultural and industrial revolution, and lake eutrophication associated with fish farming since 1989, therefore led to appreciable limnological variations. Overall, the dated sedimentary profile from Changshou Lake displays high consistency with archived historical events and reflects the impact of both natural and anthropogenic controls on sediment production.

SaCas9 Requires 5'-NNGRRT-3' PAM for Sufficient Cleavage and Possesses Higher Cleavage Activity than SpCas9 or FnCpf1 in Human Cells.

CRISPR/Cas9-mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9-mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells. To address this issue, the authors develop a dual fluorescence reporter system that could be harnessed for identifying functional PAMs for genome editing endonuclease, including Cas9. With this system, the authors investigate the effects of different PAM sequences for SaCas9, which is small and has the advantage of allowing in vivo genome editing, and found only 5'-NNGRRT-3' PAM could induced sufficient target cleavage with multi-sites. The authors also found SaCas9 possesses higher activity than SpCas9 or FnCpf1 via plasmids (episomal) and chromosomes with integrated eGFP-based comparison. Taken together, the authors show that a dual fluorescence reporter system is a means to identifying a functional PAM and quantitatively comparing the efficiency of different genome editing endonucleases with the similar or identical target sequence in human cells.

[Genome-editing: focus on the off-target effects].

Breakthroughs of genome-editing in recent years have paved the way to develop new therapeutic strategies. These genome-editing tools mainly include Zinc-finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases. However, off-target effects are still the major issue in genome editing, and limit the application in gene therapy. Here, we summarized the cause and compared different detection methods of off-targets.

A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells.

Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5'-TTTN-3' protospacer adjacent motif (PAM) at the 5' end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5'-TTN-3' as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications.

Sediment transfer at different spatial and temporal scales in the Sichuan Hilly Basin, China: Synthesizing data from multiple approaches and preliminary interpretation in the context of climatic and anthropogenic drivers.

Quantifying sediment production and transfer at different spatial and temporal scales in a changing environment is critical in understanding the potential effects of climatic and anthropogenic drivers. Accordingly, estimates of soil erosion and sediment production at hillslope field, first-order small catchment (<0.25km2) and river basin scales in the Sichuan Hilly Basin of Southwestern China, generated using a variety of techniques, including fallout radionuclide tracing, runoff plot observations, core chronology dating and conventional sediment flux monitoring, were synthesized and interpreted in the context of potential climatic and human controls. Mean annual soil erosion rates ranged from 800Mg·km-2·yr-1 to 4500Mg·km-2·yr-1 on the basis of fallout radionuclide tracing and from 600Mg·km-2·yr-1 to 3300Mg·km-2·yr-1 using runoff plot monitoring on selected cultivated hillslopes. A high slope-channel sediment delivery ratio was observed, meaning that a substantial proportion of eroded sediment was delivered into downstream drainage channels. An obvious temporal trend of decreasing sediment transfer to the river channels in the first-order catchments was identified, which may be driven by change in regional precipitation regime and the implementation of multiple soil conservation and reforestation practices over recent decades.

Linking sedimentary total organic carbon to 210Pbex chronology from Changshou Lake in the Three Gorges Reservoir Region, China.

The influences of total organic carbon (TOC) and total nitrogen (TN) on Lead-210 (210Pb) dating have recently been of increasing concern in lacustrine research. Sediment core from Changshou Lake in the Longxi catchment was investigated for influence of TOC on 210Pb dating. Lead-210 excess (210Pbex), Cesium-137 (137Cs) activities, TOC, TN, and particle size were measured. We proposed a dating index based on 137Cs chronology and particle size distribution of the lake sediment profile and rainfall erosivities calculated from Longxi catchment metrological records. Increasing trends in TOC and TN were specifically caused by commercial cage fish farming after 1989. The statistically significant correlation between 210Pbex activity, TOC (0.61, p = 0.04) and TN (0.51, p = 0.04), respectively explained post-1989 210Pb scavenging. The 210Pbex activity was closely related with coupled peaks of TOC and TN from mass depth 5-10 g cm-2. Higher TOC/TN ratio (8.33) indicated submerged macrophytes and native aquatic algal growth as main source of carbon from enhanced primary productivity because of massive fertilizer use and coherent climate warming. The study supported key hypothesis on vital role of fertilizer usage and algal derived TOC in controlling sedimentary 210Pbex activity at Changshou Lake sediment. 137Cs profile and erosive events as time markers provided reliable and consistent sedimentation rate of (1.6 cm y-1). 210Pbex activity decayed exponentially after peak at mass depth 5.68 g cm-2. Therefore, violation of 210Pb dating primary assumptions made it inappropriate for sediment dating at Changshou Lake. TOC content must be considered while using 210Pb as dating tool for lake sediment profiles.

Recapitulating X-Linked Juvenile Retinoschisis in Mouse Model by Knock-In Patient-Specific Novel Mutation.

X-linked juvenile retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding retinoschisin (RS1), which leads to a significant proportion of visual impairment and blindness. To develop personalized genome editing based gene therapy, knock-in animal disease models that have the exact mutation identified in the patients is extremely crucial, and that the way which genome editing in knock-in animals could be easily transferred to the patients. Here we recruited a family diagnosed with XLRS and identified the causative mutation (RS1, p.Y65X), then a knock-in mouse model harboring this disease-causative mutation was generated via TALEN (transcription activator-like effector nucleases). We found that the b-wave amplitude of the ERG of the RS1-KI mice was significantly decreased. Moreover, we observed that the structure of retina in RS1-KI mice has become disordered, including the disarray of inner nuclear layer and outer nuclear layer, chaos of outer plexiform layer, decreased inner segments of photoreceptor and the loss of outer segments. The novel knock-in mice (RS1-KI) harboring patient-specific mutation will be valuable for development of treatment via genome editing mediated gene correction.

Flow regulation manipulates contemporary seasonal sedimentary dynamics in the reservoir fluctuation zone of the Three Gorges Reservoir, China.

Since the launch of the Three Gorges Dam on the Yangtze River, a distinctive reservoir fluctuation zone has been created and significantly modified by regular dam operations. Sediment redistribution within this artificial landscape differs substantially from that in natural fluvial riparian zones, due to a specific hydrological regime comprising steps of water impoundment with increasing magnitudes and seasonal water level fluctuation holding a range of sediment fluxes. This study reinterpreted post-dam sedimentary dynamics in the reservoir fluctuation zone by stratigraphy determination of a 345-cm long sediment core, and related it to impact of the hydrological regime. Seasonality in absolute grain-size composition of suspended sediment was applied as a methodological basis for stratigraphic differentiation. Sedimentary laminations with relatively higher proportions of sandy fractions were ascribed to sedimentation during the dry season when proximal subsurface bank erosion dominates source contributions, while stratigraphy with a lower proportion of sandy fractions is possibly contributed by sedimentation during the wet season when distal upstream surface erosion prevails. Chronology determination revealed non-linear and high annual sedimentation rates ranging from 21.7 to 152.1cm/yr. Although channel geomorphology may primarily determine the spatial extent of sedimentation, seasonal sedimentary dynamics was predominantly governed by the frequency, magnitude, and duration of flooding. Summer inundation by natural floods with enhanced sediment loads produced from upstream basins induced higher sedimentation rates than water impoundment during the dry season when distal sediment supply was limited. We thus conclude that flow regulation manipulates contemporary seasonal sedimentary dynamics in the reservoir fluctuation zone, though little impact on total sediment retention rate was detected. Ongoing reductions in flow and sediment supply under human disturbance may have profound implications in affecting sedimentary equilibrium in the reservoir fluctuation zone. The results herein provide insights of how big dams have disrupted the sediment conveyance processes of large scale fluvial systems.