Genetic characteristics of yellow seabream Acanthopagrus latus (Houttuyn, 1782) (Teleostei: Sparidae) after stock enhancement in southeastern China coastal waters

Yellowfin seabream is an important economic fish that is widely distributed in the East and South China seas. Many attempts to enhance stocks of yellowfin seabream have occurred in China, but a lack of genetic information for this species after stock release represents an obstacle to its management and conservation. To provide scientific guidance for sustainable germplasm resource development, we sequence the mitochondrial DNA (mtDNA) control region (CR) of 123 yellowfin seabream from 6 sample populations (Xiamen, Dongshan I, Dongshan II, Yangjiang, Fangchenggang, and Beibu Gulf). Populations of both wild and cultured yellowfin seabream have high genetic diversity, which we relate to their breeding habits and growth rate. A neighbor-joining tree of CR haplotypes reveals no specific phylogenetic structure corresponding to location of fish capture. Both neutral test and nucleotide mismatch distribution analyses suggest that yellowfin seabream have experienced population expansion events. Pleistocene glacial periods and recent stock releases have played important roles in the formation of present-day phylogeographical patterns. Our study provides baseline information which will assist future research on genetic structure, genetic diversity, and historical demography of yellowfin seabream after stock release in southeast China coastal waters. The use of exotic seeds should be avoided in stock breeding and release, and relevant follow-up surveys and genetic monitoring should be undertaken to clarify the genetic impact of exotic seed use on wild populations.

Recognition of CRISPR off-target cleavage sites with SeqGAN

Background: The CRISPR system can quickly achieve the editing of different gene loci by changing a small sequence on a single guide RNA. But the off-target event limits the further development of the CRISPR system. How to improve the efficiency and specificity of this technology and minimize the risk of off-target has always been a challenge. For genome-wide CRISPR off-target cleavage sites (OTS) prediction, an important issue is data imbalance, that is, the number of true OTS identified is much less than that of all possible nucleotide mismatch loci. Method: In this work, based on the sequence-generating adversarial network (SeqGAN), positive off-target sequences were generated to amplify the off-target gene locus OTS dataset of Cpf1. Then we trained the data by deep convolutional neural network (CNN) to obtain a predictor with stronger generalization ability and better performance. Results: n 10-fold cross-validation, the AUC value of the CNN classifier after SeqGAN balance was 0.941, which was higher than that of original 0.863 and over-sampling 0.929. In independence testing, AUC value of the CNN classifier after SeqGAN balance was 0.841 which was higher than that of original 0.833 and over-sampling 0.836. the PR value was 0.722 after SeqGAN, which was also about higher 0.16 than original data and higher about 0.03 than over-sampling. Conclusion: The sequence generation antagonistic network SeqGAN was firstly used to deal with data imbalance processing on CRISPR data. All the results showed that the SeqGAN can effectively generate positive data for CRISPR off-target sites.

In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR

A number of RT-qPCR assays for the detection of SARS-CoV-2 have been published and are listed by the WHO as recommended assays. Furthermore, numerous commercial assays with undisclosed primer and probe sequences are on the market. As the SARS-CoV-2 pandemic progresses, the virus accrues mutations, which in some cases - as seen with the B.1.1.7 variant - can outperform and push back other strains of SARS-CoV-2. If mutations occur in primer or probe binding sites, this can impact RT-qPCR results and impede SARS-CoV-2 diagnostics. Here we tested the effect of primer mismatches on RT-qPCR performance in vitro using synthetic mismatch in vitro transcripts. The effects of the mismatches ranged from a shift in ct values from -0.13 to +7.61. Crucially, we found that a mismatch in the forward primer has a more detrimental effect for PCR performance than a mismatch in the reverse primer. Furthermore, we compared the performance of the original Charite RdRP primer set, which has several ambiguities, with a primer version without ambiguities and found that without ambiguities the ct values are ca. 3 ct lower. Finally, we investigated the shift in ct values observed with the Seegene Allplex kit with the B.1.1.7 SARS-CoV-2 variant and found a three-nucleotide mismatch in the forward primer of the N target.

Microsatellite instability (MSI, EMAST) in the pathogenesis of follicular lymphoma

Background. Genetic instability, an important phenomenon involved in oncogenic transformation and tumor progression, is associated with the insufficiency of the multicomponent DNA repair complex, in particular, the nucleotide mismatch repair (MMR) system. The MMR defect manifests itself as abnormalities in DNA microsatellite repeats, or microsatellite instability (MSI). In the studies of colorectal cancer, the role of MSI in prognostication of the disease, and defining the choice of specific therapy with immune checkpoint inhibitors has been proven.However, in lymphatic system tumors, the significance of this phenomenon is poorly understood. Determination of genetic instability in the onset of follicular lymphoma, a disease characterized by a heterogeneous course, may have prognostic value.Objective: to determine the genetic instability at the onset of follicular lymphoma.Materials and methods. Here we report an analysis of 24 microsatellite repeats and amelogenin loci in tumor cells of 46 follicular lymphoma patients.Results. In the studied cohort, lesions in microsatellite repeats were presented by MSI in 9 cases (19.6 %) and the loss of heterozygosity (LOH) in 19 cases (41.3 %). Most frequent lesions were found for the SE33 marker located at the q14 locus of chromosome 6. A significant association was shown between MSI and the double-hit follicular lymphoma group with rearrangements of the MYC and BCL2/BCL6 genes.Conclusion. Thus, our data indicate that the MSI phenomenon might be involved in the pathogenesis of the lymphatic tumors and particularly follicular lymphoma. However further studies on the expanded cohorts of patients are required to define the possible prognostic value of MSI in lymphatic tumors.

Low-Cost Graphene-Based Digital Microfluidic System

In this work, the laser-scribing technique was used as a low-cost, rapid and facile method for fabricating digital microfluidic (DMF) systems. Laser-scribed graphene (LSG) electrodes are directly synthesized on flexible substrates to pattern the DMF electrode arrays. This facilitates the DMF electrodes’ fabrication process by eliminating many microfabrication steps. An electrowetting test was performed to investigate the effectiveness of the LSG DMF electrodes in changing the contact angles of droplets. Different DMF operations were successfully performed using the proposed LSG DMF chips in both open and closed DMF systems. The quality and output resolution were examined to assess the performance of such patterned electrodes in the DMF systems. To verify the efficacy of the LSG DMF chips, a one-step direct assay for the detection of Legionellapneumophila deoxyribonucleic acid (DNA) was performed on the chip without the need for any washing step. The high specificity in distinguishing a single-nucleotide mismatch was achieved by detecting target DNA concentrations as low as 1 nM. Our findings suggest that the proposed rapid and easy fabrication method for LSG DMF electrodes offers a great platform for low-cost and easily accessible point-of-care diagnostic devices.

Plasmonic Metasensors Based on 2D Hybrid Atomically Thin Perovskite Nanomaterials

In this work, we have designed highly sensitive plasmonic metasensors based on atomically thin perovskite nanomaterials with a detection limit up to 10−10 refractive index units (RIU) for the target sample solutions. More importantly, we have improved phase singularity detection with the Goos–Hänchen (GH) effect. The GH shift is known to be closely related to optical phase signal changes; it is much more sensitive and sharp than the phase signal in the plasmonic condition, while the experimental measurement setup is much more compact than that of the commonly used interferometer scheme to exact the phase signals. Here, we have demonstrated that plasmonic sensitivity can reach a record-high value of 1.2862 × 109 µm/RIU with the optimum configurations for the plasmonic metasensors. The phase singularity-induced GH shift is more than three orders of magnitude larger than those achievable in other metamaterial schemes, including Ag/TiO2 hyperbolic multilayer metamaterials (HMMs), metal–insulator–metal (MIM) multilayer waveguides with plasmon-induced transparency (PIT), and metasurface devices with a large phase gradient. GH sensitivity has been improved by more than 106 times with the atomically thin perovskite metasurfaces (1.2862 × 109 µm/RIU) than those without (918.9167 µm/RIU). The atomically thin perovskite nanomaterials with high absorption rates enable precise tuning of the depth of the plasmonic resonance dip. As such, one can optimize the structure to reach near zero-reflection at the resonance angle and the associated sharp phase singularity, which leads to a strongly enhanced GH lateral shift at the sensor interface. By integrating the 2D perovskite nanolayer into a metasurface structure, a strong localized electric field enhancement can be realized and GH sensitivity was further improved to 1.5458 × 109 µm/RIU. We believe that this enhanced electric field together with the significantly improved GH shift would enable single molecular or even submolecular detection for hard-to-identify chemical and biological markers, including single nucleotide mismatch in the DNA sequence, toxic heavy metal ions, and tumor necrosis factor-α (TNFα).

A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells

CRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3′ end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.

Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene

SUMMARYThe mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. On this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeast’s URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutLE36K). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G< C:C< G:A< C:A, A:A, G:G, T:T, T:G, A:C, C:T< G:T, T:C as the one prevalent in Pseudomonas putida. This information was vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.Originality-Significance StatementSingle-stranded DNA (ssDNA) recombineering has emerged in recent years as one of the most powerful technologies of genome editing in E. coli and other Enterobacteria. However, the efforts to expand the concept and the methods towards environmental microorganisms such as Pseudomonas putida have been limited thus far by several gaps in our fundamental knowledge of how nucleotide mismatch repair (MMR) operates in such non-model species. One critical bottleneck is the hierarchy of recognition of different types of base mispairings as well as the need of setting up strategies for counteracting MMR and thus enabling tolerance to all types of changes. The work presented here tackles both issues and makes P. putida amenable to sophisticated genetic manipulations that were impossible before.