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Prime editor (PE) is a versatile genome editing tool that does not need extra DNA donors or inducing double-strand breaks. However, in vivo implementation of PE remains a challenge because of its oversized composition. In this study, we screened out the smallest truncated Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) with the F155Y mutation to keep gene editing efficiency. We discovered the most efficient gene editing variants of MMLV RT with the smallest size. After optimization of the pegRNAs and incorporation with nick sgRNAs, the mini-PE delivered up to 10% precise editing at target sites in human and mouse cells. It also edited the mouse Hsf1 gene in the mouse retina precisely after delivery with adeno-associated viruses (AAVs), although the editing efficiency was lower than 1%. We will focus on improving the editing efficiency of mini-PE and exploiting its therapeutic potential against human genetic diseases.

Lanthanum ranks first among rare earth elements and accounts for a large proportion of the total rare earth elements. It is rich in resources. La alloys have many applications in hydrogen storage materials, magnetic refrigeration, metal targets, and steel modification. This paper systematically introduces the latest research progress of La-Zn, La-Cu, La-Ni, and La-Al alloys prepared by molten salt electrolysis, compares and summarizes its application fields and the electrochemical behavior of La ions in molten salt. To make suggestions for future basic research and applied research directions.

Cas12a-mediated cytosine (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded target scopes are developed, by combining highly active deaminases with Cas12a variants, enCas12a, RR and RVR, recognizing non-TTTV PAMs.

Plant residue decomposition can significantly affect the water environment of wetland ecosystems. However, little is known about the trajectory and the key drivers of the different initial mass of residue (IMR) that regulate water quality. Here, we conducted a study of 210 days with an in situ experiment to examine the impact of the change in IMR and the key decomposition parameters of Phragmites australis on the water quality in the growing season of 2019 in Baiyangdian wetland in North China. Five IMR of the Phragmites were applied, ranging from 0 to 1000 g with 250 g increments. The chemical oxygen demand (COD_W), total organic carbon (TOC_W), total nitrogen (TN_W) and total phosphorus (TP_W) of the water, and the total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), cellulose, lignin, decomposition rate (k) and the remaining mass of the Phragmites in the bag (RM) were monitored. Our results showed that the IMR of the Phragmites only marginally influenced the decomposition of itself and; the variations in the level of the water indicators were ranked as TN > TP > COD > TOC and; the impacts of IMR were longer on TOC and COD than on TP and TN and; the change in water quality was significantly influenced by the Phragmites decomposition and highly correlated with the change in TOC, cellulose and k of decomposing Phragmites residue. These results indicated that the impact of 250 g of IMR on the water was negligible after 120 days, suggesting that the optimal harvest rate of the Phragmites should be above 75% in the Baiyangdian wetland, despite the strong impact at the beginning and; the impact of change in IMR on water quality can be explained by the change in the decomposition parameters of the decomposing Phragmites residue, where such a decomposition-based explanation might be a promising tool to link wetland plant management and water quality.

Abstract Background Many favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site. Results In this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system. Conclusion The newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.

Base editors can efficiently produce single nucleotide alterations without requiring a double-strand break. Here the authors show base editing at multiple sites simultaneously in pigs.

The estimation of wetland plant biomass is key to understanding the dynamic changes of the structure and functions of wetland ecosystems. Relatively few studies on the spatiotemporal dynamics of wetland plant biomass have been conducted because of the difficulties in obtaining detailed and accurate data. Carex cinerascens meadows are important patches in Poyang Lake in eastern China and provide significant food resources and habitats for wintering waterbirds, particularly geese. We proposed a method to estimate the biomass of C. cinerascens in a subsection of Poyang Lake, i.e. Baisha Lake, employing the digital elevation model and water level gradient of the lake combined with the logistic regression model based on data from field measurements in different elevations. We obtained four C. cinerascens biomass estimation models based on four elevations divided. The maximum biomass decreased with decreasing elevation. Meanwhile, the maximum growth rate initially increased and subsequently decreased. The biomass of C. cinerascens per unit area ranged from 0.10 to 0.58 kg/m2 in the autumn and winter. Significant differences in the growth process of C. cinerascens were observed in different water level gradients. The total biomass of C. cinerascens in Baisha Lake changed following an S-shaped curve and reached 221, 503, and 615 t in the middle of October, November, and December, respectively. The research results can be used to forecast the biomass of C. cinerascens under different hydrological conditions in Poyang Lake, and to provide wetland plant biomass estimation methods for other similar regions.

From November 2016 to April 2017, the decomposition of Carex. cinerascens Kükenth litter along the natural gradients of groundwater level at Poyang Lake wetland beach were simulated using litterbag technique. The depth of the underground water table of the G-L was −25 to −50 cm, while those of G-ML, G-MH and G-H ranged from −15 to −25 cm, −5 to −15 cm, and −5 to 5, respectively. Additionally, the dry weight loss rate increased gradually with increasing days, with that of G-H being highest, followed by G-MH, G-ML and G-L. The decomposition rate of the four gradients increased rapidly to day 30, then decreased slowly until stabilizing. Additionally, the decomposition rate was highest in G-H, followed by G-MH, G-ML and G-L. The Olson negative exponential model could simulate the decomposition good and predicted the time required for 50% decomposition of the G-H, G-MH, G-ML and G-L gradients was about 166, 197, 205 and 247 days, respectively, while required for 95% was about 1.97, 2.23, 2.43 and 2.92 years, respectively. The overall trend in the relative return index of carbon, nitrogen and phosphorus increased with increasing decomposition days, and the order was G-H > G-MH > G-ML > G-L. Differences in soil pH, bulk density, soil total nitrogen, soil organic matter, microbial biomass carbon and soil sand content in the decomposed environment due to groundwater had a significant impact on decomposition and nutrient release. The relative return index of carbon was significantly positively correlated with soil pH, but significantly negatively correlated with soil bulk density, soil nitrogen content, microbial biomass nitrogen and soil organic matter. The above results show that groundwater is the key factor influencing litter decomposition in Poyang Lake wetland during the dry season, with higher groundwater levels promoting the decomposition.

Conventional human pluripotent stem cells (hPSCs) fail to contribute to interspecies chimaeras when injected into mouse blastocysts. Here the authors show that forced expression of BMI1 overcomes apoptosis of hPSCs in blastocysts of mouse, rabbit and pig allowing them to contribute to chimaeras.

Summary: Pig cloning by somatic cell nuclear transfer (SCNT) remains extremely inefficient, and many cloned embryos undergo abnormal development. Here, by profiling transcriptome expression, we observed dysregulated chromosome-wide gene expression in every chromosome and identified a considerable number of genes that are aberrantly expressed in the abnormal cloned embryos. In particular, XIST, a long non-coding RNA gene, showed high ectopic expression in abnormal embryos. We also proved that nullification of the XIST gene in donor cells can normalize aberrant gene expression in cloned embryos and enhance long-term development capacity of the embryos. Furthermore, the increased quality of XIST-deficient embryos was associated with the global H3K9me3 reduction. Injection of H3K9me demethylase Kdm4A into NT embryos could improve the development of pre-implantation stage embryos. However, Kdm4A addition also induced XIST derepression in the active X chromosome and thus was not able to enhance the in vivo long-term developmental capacity of porcine NT embryos. : In this article, Liangxue Lai and colleagues show that nullification of the XIST gene in donor cells can normalize aberrant gene expression in cloned embryos and result in high cloning efficiency in pigs. Keywords: pig somatic cell nuclear transfer, XIST, H3K9me3, H3K9me3 demethylases, H3K9me3 methyltransferases

Test case prioritization (TCP) plays an important role in identifying, characterizing, diagnosing, and correcting faults quickly. The TCP has been widely used to order test cases of different types, including model inputs (also called abstract test cases). Model inputs are constructed by modeling the program according to its input parameters, values, and constraints, and has been used in different testing methods, such as combinatorial interaction testing and software product line testing. The Interaction coverage-based TCP (ICTCP) uses interaction coverage information derived from the model input to order inputs. Previous studies have focused generally on the fixed-strength ICTCP, which adopts a fixed strength (i.e., the level of parameter interactions) to support the ICTCP process. It is generally accepted that using more strengths for ICTCP, i.e., mixed-strength ICTCP, may give better ordering than fixed-strength. To confirm whether mixed-strength is better than fixed-strength, in this paper, we report on an extensive empirical study using five real-world programs (written in C), each of which has six versions. The results of the empirical studies show that mixed-strength has better rates of interaction coverage overall than fixed-strength, but they have very similar rates of fault detection. Our results also show that fixed-strength should be used instead of the mixed-strength at the later stage of software testing. Finally, we offer some practical guidelines for testers when using interaction coverage information to prioritize model inputs, under different testing scenarios and resources.

Water table affects litter decomposition in wetlands, but its effects on the degradation of cellulose and lignin are poorly understood. We performed a decomposition experiment in Poyang Lake Wetland, to determine how water table affected the degradation of cellulose and lignin. After 60 days of decomposition at a site with a relatively highly water table, 46.19% of initial cellulose and 41.95% of initial lignin remained. Decay rates of both cellulose and lignin increased as the depth of the water table increased. Principle component analysis showed that the decay rates of cellulose and lignin increased with increasing soil pH, but decreased with increasing contents of clay and fungi: bacteria ratio. The path model accounted for 66% and 79% of the variation in cellulose and lignin decay rates, respectively, and considered the effects of interactions between the water table and related factors. The cellulose decay rate was affected by the water table with a direct coefficient of 0.47, and an indirect coefficient of 0.65, but the lignin decay rate was indirectly affected by water table (coefficient, 1.25) and directly affected by soil property (coefficient, 0.67). Thus, the water table affected the decomposition of cellulose and lignin via different mechanisms.

Abstract Mitochondrial diseases are maternally inherited heterogeneous disorders that are primarily caused by mitochondrial DNA (mtDNA) mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruction of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells (MiPSCs) that harbored a high proportion of m.3243A>G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes (MELAS). We engineered mitochondrial-targeted transcription activator-like effector nucleases (mitoTALENs) and successfully eliminated the m.3243A>G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Furthermore, we successfully achieved reduction in the human m.3243A>G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.

The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed.

Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) is a simple, robust and efficient technique for gene targeting in model organisms such as zebrafish, mice and rats. In this report, we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA (gRNA) into the cytoplasm of pronuclear-stage embryos. We achieved biallelic gene knockout (KO) rabbits by injection of 1 gene (IL2rg) or 2 gene (IL2rg and RAG1) Cas9 mRNA and gRNA with an efficiency of 100%. We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100% for 3 genes (IL2rg, RAG1 and RAG2) and 33.3% for 5 genes (IL2rg, RAG1, RAG2, TIKI1 and ALB). Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.

Rabbits are commonly used as laboratory animal models to investigate human diseases and phylogenetic development. However, pluripotent stem cells that contribute to germline transmission have yet to be established in rabbits. The transcription factor Oct4, also known as Pou5f1, is considered essential for the maintenance of the pluripotency of stem cells. Hence, pluripotent cells can be identified by monitoring Oct4 expression using a well-established Oct4 promoter-based reporter system. This study developed a rabbit Oct4 promoter-based enhanced green fluorescent protein (EGFP) reporter system by transfecting pROP2-EGFP into rabbit fetal fibroblasts (RFFs). The transgenic RFFs were used as donor cells for somatic cell nuclear transfer (SCNT). The EGFP expression was detected in the blastocysts and genital ridges of SCNT fetuses. Fibroblasts and neural stem cells (NSCs) were derived from the SCNT fetuses. EGFP was also reactivated in blastocysts after the second SCNT, and induced pluripotent stem cells (iPSCs) were obtained after reprogramming using Yamanaka's factors. The results above indicated that a rabbit reporter system used to monitor the differentiating status of cells was successfully developed.