CRISPR-Cas12a assisted precise genome editing of Mycolicibacterium neoaurum.

Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum, including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.

Immediate, multiplexed and sequential genome engineering facilitated by CRISPR/Cas9 in Saccharomyces cerevisiae.

A method called Cas-3P allowing for immediate, multiplexed and sequential genome engineering was developed using one plasmid expressing Cas9 and three marked plasmid backbones (P1, P2 and P3) for guide RNA (gRNA) expression. The three marked gRNA plasmid backbones were recurred in a P1-P2-P3 order for sequential gene targeting, without construction of any additional plasmid and elimination of gRNA plasmid by induction in each round. The efficiency of direct gRNA plasmid curing mediated by Cas-3P was more than 40% in sequential gene targeting. Besides, Cas-3P allowed single-, double- and triple-loci gene targeting with an efficiency of 75%, 36.8% and 8.2% within 3-4 days, respectively. Through three sequential rounds of gene targeting within 10 days, S. cerevisiae was optimized for the production of patchoulol by replacing one promoter, overexpressing three genes and disrupting four genes. The work is important for practical application in the cell factory engineering of S. cerevisiae.

Significantly Enhanced Production of Patchoulol in Metabolically Engineered Saccharomyces cerevisiae.

Patchoulol, a natural sesquiterpene compound, is widely used in perfumes and cosmetics. Several strategies were adopted to enhance patchoulol production in Saccharomyces cerevisiae: (i) farnesyl pyrophosphate (FPP) synthase and patchoulol synthase were fused to increase the utilization of FPP precursor; (ii) expression of the limiting genes of the mevalonate pathway was enhanced; (iii) squalene synthase was weakened by a glucose-inducible promoter of HXT1 (promoter for hexose transporter) to reduce metabolic flux from FPP to ergosterol; and (iv) farnesol biosynthesis was inhibited to decrease the consumption of FPP. Glucose was used to balance the trade-off between the competitive squalene and patchoulol pathways. The patchoulol production was 59.2 ± 0.7 mg/L in a shaken flask with a final production of 466.8 ± 12.3 mg/L (20.5 ± 0.5 mg/g dry cell weight) combined with fermentation optimization, which was 7.8-fold higher than the reported maximum production. The work significantly promoted the industrialization process of patchoulol production using biobased microbial platforms.

CRISPR/Cpf1 facilitated large fragment deletion in Saccharomyces cerevisiae.

In this study, we focused on the applicability of CRISPR/Cpf1 in genome simplification of Saccharomyces cerevisiae and established a CRISPR/Cpf1 assisted method for rapid markerless large fragment deletion to facilitate laboratory evolution of geome of S. cerevisiae by rational genetic engineering. This method uses a Cpf1 expression plasmid and a crRNA array expression plasmid. The DNA fragment between two DSBs generated by CRISPR/Cpf1 can be cut off from the chromosome, along with re-ligation of the genomic endpoints of the DSBs. Using this method, the large DNA fragment of ∼38 kb between the two genes of TRM10 and REX4 was successfully and rapidly deleted, which was verified by PCR and Sanger DNA Sequencing. This method is simple and rapid, and can be easily implemented for large fragment deletion in S. cerevisiae.

Cpf1-assisted efficient genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae.

OBJECTIVES: To test the applicability of Cpf1 from Francisella novivida in genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae. RESULTS: An easy-to-use vector toolkit, containing a CEN6/ARS4 plasmid expressing Cpf1 from Francisella novivida (FnCpf1) and a 2 µ plasmid for crRNA or crRNA array expressing, was constructed for Cpf1-assisted genomic integration in S. cerevisiae. Our results showed that FnCpf1 allowed for targeted singleplex, doubleplex, and tripleplex genomic integration of in vivo assembled DNA parts with efficiencies of 95, 52, and 43%, respectively. CONCLUSIONS: CRISPR-Cpf1 system allows for efficient genomic integration of in vivo assembled DNA parts in S. cerevisiae, and thus provides an alternative CRISPR-Cas method for metabolic pathway engineering in addition to CRISPR-Cas9 system previously reported for yeast.

Epigenetic silencing of tumor suppressor long non-coding RNA BM742401 in chronic lymphocytic leukemia.

BM742401 is a tumor suppressor lncRNA downregulated in gastric cancer. As the promoter region and the entire transcript are embedded in a CpG island, we postulated that BM742401 is a tumor suppressor lncRNA inactivated by DNA methylation in chronic lymphocytic leukemia (CLL). The promoter of BM742401 was unmethylated in normal controls including three each of normal bone marrow, peripheral blood buffy coats, and CD19-sorted peripheral B-cells, but methylated in four (57.1%) CLL cell lines. Methylation of BM742401 correlated inversely with expression. In the completely methylated WAC3CD5+ CLL cells, 5-Aza-2'-deoxycytidine treatment led to promoter demethylation and re-expression of BM742401 transcript. Functionally, stable overexpression of BM742401 resulted in inhibition of cellular proliferation and enhanced apoptosis through caspase-9-dependent intrinsic but not caspase-8-dependent extrinsic apoptosis pathway, suggesting a tumor suppressor role of BM742401 in CLL. In primary CLL samples, methylation of BM742401 was detected in 43/98 (43.9%) of patients. Moreover, among CLL patients with standard-risk cytogenetic aberrations, methylation of BM742401 correlated with advanced Rai stage (≥ stage 2)(P = 0.002). Furthermore, BM742401 methylation was associated with miR-129-2 methylation (P = 0.05). BM742401 is a tumor suppressor lncRNA frequently methylated in CLL. The mechanism of BM742401 as a tumor suppressor warrants further studies.

[The new method monitoring crop water content based on NIR-Red spectrum feature space].

Moisture content is an important index of crop water stress condition, timely and effective monitoring of crop water content is of great significance for evaluating crop water deficit balance and guiding agriculture irrigation. The present paper was trying to build a new crop water index for winter wheat vegetation water content based on NIR-Red spectral space. Firstly, canopy spectrums of winter wheat with narrow-band were resampled according to relative spectral response function of HJ-CCD and ZY-3. Then, a new index (PWI) was set up to estimate vegetation water content of winter wheat by improveing PDI (perpendicular drought index) and PVI (perpendicular vegetation index) based on NIR-Red spectral feature space. The results showed that the relationship between PWI and VWC (vegetation water content) was stable based on simulation of wide-band multispectral data HJ-CCD and ZY-3 with R2 being 0.684 and 0.683, respectively. And then VWC was estimated by using PWI with the R2 and RMSE being 0.764 and 0.764, 3.837% and 3.840%, respectively. The results indicated that PWI has certain feasibility to estimate crop water content. At the same time, it provides a new method for monitoring crop water content using remote sensing data HJ-CCD and ZY-3.

Assessment of the AquaCrop model for use in simulation of irrigated winter wheat canopy cover, biomass, and grain yield in the North China Plain.

Improving winter wheat water use efficiency in the North China Plain (NCP), China is essential in light of current irrigation water shortages. In this study, the AquaCrop model was used to calibrate, and validate winter wheat crop performance under various planting dates and irrigation application rates. All experiments were conducted at the Xiaotangshan experimental site in Beijing, China, during seasons of 2008/2009, 2009/2010, 2010/2011 and 2011/2012. This model was first calibrated using data from 2008/2009 and 2009/2010, and subsequently validated using data from 2010/2011 and 2011/2012. The results showed that the simulated canopy cover (CC), biomass yield (BY) and grain yield (GY) were consistent with the measured CC, BY and GY, with corresponding coefficients of determination (R(2)) of 0.93, 0.91 and 0.93, respectively. In addition, relationships between BY, GY and transpiration (T), (R(2) = 0.57 and 0.71, respectively) was observed. These results suggest that frequent irrigation with a small amount of water significantly improved BY and GY. Collectively, these results indicate that the AquaCrop model can be used in the evaluation of various winter wheat irrigation strategies. The AquaCrop model predicted winter wheat CC, BY and GY with acceptable accuracy. Therefore, we concluded that AquaCrop is a useful decision-making tool for use in efforts to optimize wheat winter planting dates, and irrigation strategies.

[Estimating canopy water content in wheat based on new vegetation water index].

Moisture content is an important indicator for crop water stress condition, timely and effective monitoring crop water content is of great significance for evaluate crop water deficit balance and guide agriculture irrigation. In order to improve the saturated problems of different forms of typical NDWI (Normalized Different Water Index), we tried to introduce EVI (Enhanced Vegetation Index) to build new vegetation water indices (NDWI#) to estimate crop water content. Firstly, PROSAIL model was used to study the saturation sensitivity of NDWI, and NDWI# to canopy water content and LAI (Leaf Area Index). Then, the estimated model and verified model were estimated using the spectral data and moisture data in the field. The result showed that the new indices have significant relationships with canopy water content. In particular, by implementing modified standardized for NDWI1450, NDWI1940, NDWI2500. The result indicated that newly developed indices with visible-infrared and shortwave infrared spectral feature may have greater advantage for estimation winter canopy water content.

[Estimation of winter wheat protein content based on new indexes].

Wheat protein content is an important indicator often employed in wheat sale price. Spectral indexes and concurrent winter wheat protein content (WWPC) samples were obtained across three years. Data from 2008/2009 and 2009/2010 were utilized to build the new ratio indexes and product indexes, and then selected grey relational method and partial least squares method were used to improve the estimation accuracy of WWPC, data from 2011/2012 was utilized to validate model. The results showed that the correlation coefficients between ratio indexes and WWPC were better than that between single indexesand WWPC. The r of single indexes and ratio indexes were 0.726 and 0.751, respectively, and the product indexes were used to improve the parts of single indexes. The estimation accuracy of WWPC was improved by using GRA-PLS, the determination coefficients (R2) of single indexes, ratio indexes and product indexes were 0.537, 0.631 and 0.521, respectively, and corresponding root mean square errors (RMSE) were 0.665%, 0.564% and 0.574%, respectively. The results indicated that it was feasible to estimate WWPC by building new ratio indexes and product indexes, and then applying the GRA-PLS.

Flow-injection determination of hydrogen peroxide based on fluorescence quenching of chromotropic acid catalyzed with Fe(II).

Flow-injection analysis system (FIA system), which was based on Fe(II)-catalyzed oxidation of chromotropic acid with hydrogen peroxide, was developed for the determination of hydrogen peroxide. The chromotropic acid has a fluorescence measured at lambda(em)=440 nm (emission wavelength) with lambda(ex)=235 nm (excitation wavelength), and the fluorescence intensity at lambda(em)=440 nm quietly decreased in the presence of hydrogen peroxide and Fe(II), which was caused by Fe(II)-catalyzed oxidation of chromotropic acid with hydrogen peroxide. By measuring the difference of fluorescence intensity, hydrogen peroxide (1.0 x 10(-8)-1.0 x 10(-3) mol L(-1)) could be determined by the proposed FIA system, whose analytical throughput was 40 samples h(-1). The relative standard deviation (RSD) was 1.03% (n=10) for 4.0 x 10(-8) mol L(-1) hydrogen peroxide. The proposed FIA technique could be applied to the determination of hydrogen peroxide in rain water samples.

[Analysis of part of M gene and genotyping for M segment of hantavirus detected from HFRS patients' sera in Qingdao region during 2000 - 2003].

OBJECTIVE: To study the molecular epidemiological characteristics of hantavirus seen during 2000-2003 in Qingdao region of Shandong province. METHODS: Sera were collected from 64 patients with hemorrhagic fever with renal syndrome (HFRS) and viral RNA was extracted from the sera. HTN and SEO universal primers were designed as outer primers and HTN and SEO specific primers as inner primers. G1 gene region of M segment from hantavirus was amplified by using RT-nest-PCR for sequencing. The data of nucleotide sequences were analyzed by DNA star software. RESULTS: Six cases were positive by HTN specific primer of total cases (9%); 25 of 64 cases by SEO specific primer (39%); total positive rate was 48%. In general, SEO type was a prevalent type of hantavirus in Qingdao region. The variation of the nucleotide sequences among SEO viruses (nucleotide sequence divergence ranged from 0.3% approximately 8.9%) was lower than that among HTN type (nucleotide sequence divergence ranged from 2.6% approximately 11.2% ). CONCLUSION: Majority of hantavirus found in Qingdao region belonged to SEO type and still a few strains belonged to HTN type. Most of the HTN viruses were detected in Jiaonan county.

Trace and ultratrace analysis methods for the determination of phosphorus by flow-injection techniques.

Trace (< or =1 mg/l or 30 microM) and ultratrace (< or =1 microg/l or 30 nM) analysis methods for phosphorus determination by flow-injection analysis are reviewed. Most of the methods cited in this review are fundamentally based on the reaction of orthophosphate with molybdate to form heteropoly acids, such as molybdenum yellow and molybdenum blue, and some of the methods are based on the formation of such secondary reactions as ion associates and their aggregates with bulky cations, such as cationic dyes and quaternary ammonium ions. The heteropoly acids themselves can be measured by spectrophotometry, and the ion associate formed with a cationic dye, Malachite Green (MG), can be measured based on the coloration of MG. Light scattering detection methods can be used for measuring the aggregates of ion associates formed with bulky cations. Highly sensitive detection of phosphorus can be accomplished by fluorophotometry; Rhodamine B (RB) and its analogues react with molybdophosphate to form ion associates, which shows fluorescence quenching of RB: LOD is about 5 nM. The detection method based on the chemiluminescence of luminal oxidized with molybdophosphoric acids is probably the most sensitive of all the detection methods reported so far: LOD of the method is as low as 1nM. The LOD of the molybdenum blue method can be improved by using a liquid core waveguide: LOD is 0.5 nM.