Large DNA Fragments Research Articles

Crosstalk between vrille transcripts, proteins, and regulatory elements controlling circadian rhythms and development in Drosophila.

SummaryThe vrille (vri) gene encodes a transcriptional repressor required for Drosophila development as well as circadian behavior in adults. Alternate first exons produce vri transcripts predicted to produce a short VRI isoform during development and long VRI in adults. A vri mutant (vriΔ679) lacking long VRI transcripts is viable, confirming that short VRI is sufficient for developmental functions, yet behavioral rhythms in vriΔ679 flies persist, showing that short VRI is sufficient for clock output. E-box regulatory elements that drive rhythmic long VRI transcript expression are required for developmental expression of short VRI transcripts. Surprisingly, long VRI transcripts primarily produce short VRI in adults, apparently due to a poor Kozak sequence context, demonstrating that short VRI drives circadian behavior. Thus, E-box-driven long VRI transcripts primarily control circadian rhythms via short VRI, whereas the same E-boxes drive short VRI transcripts that control developmental functions using short VRI.

Insights into gene expression changes under conditions that facilitate horizontal gene transfer (mating) of a model archaeon

Horizontal gene transfer is a means by which bacteria, archaea, and eukaryotes are able to trade DNA within and between species. While there are a variety of mechanisms through which this genetic exchange can take place, one means prevalent in the archaeon Haloferax volcanii involves the transient formation of cytoplasmic bridges between cells and is referred to as mating. This process can result in the exchange of very large fragments of DNA between the participating cells. Genes governing the process of mating, including triggers to initiate mating, mechanisms of cell fusion, and DNA exchange, have yet to be characterized. We used a transcriptomic approach to gain a more detailed knowledge of how mating might transpire. By examining the differential expression of genes expressed in cells harvested from mating conditions on a filter over time and comparing them to those expressed in a shaking culture, we were able to identify genes and pathways potentially associated with mating. These analyses provide new insights into both the mechanisms and barriers of mating in Hfx. volcanii.

A CRISPR/Cas9 Cleavage System for Capturing Fungal Secondary Metabolite Gene Clusters.

More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic 'dark matter' to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.

Legal and practical challenges to authorization of gene edited plants in the EU

According to a predominant interpretation of the C-528/16 judgment of the Court of Justice of the European Union, mutants resulting from gene editing, even those featuring only single nucleotide variants, should be subject to the authorization procedures designed for organisms developed through genetic modification (i.e. insertion of large DNA fragments). In this article, we illustrate practical problems with the authorization of products of gene editing in the EU. On the basis of these problems, we analyze the influence of the current interpretation of EU legislation and judgment on the practical ability to authorize and detect such products on the EU market. We show that the predominant interpretation of the judgment leads to legally unacceptable consequences, in particular to the violation of the principle of proportionality with regard to individuals who wish to develop and market products of gene editing. As a result of our considerations, we show that the C-528/16 judgment did not need to be interpreted in the dominant way.

Pulsed-Field Gel Electrophoresis (PFGE) Analysis of Listeria monocytogenes.

PFGE is a valuable tool for assessing L. monocytogenes strain interrelatedness. It is based on the study of total bacterial DNA restriction patterns. Cells are embedded in agarose plugs before being lysed. The released DNA is then digested into large fragments by restriction enzymes. As DNA fragments are too large to be separated by traditional electrophoresis in an agarose gel, changes in the direction of the electrical current are periodically applied in order to allow the proper migration of large DNA fragments. Strains are characterized by the obtained DNA fragment patterns or pulsotypes which vary depending on the number and size of bands.

Mitochondrial DNA Parameters in Blood of Infants Receiving Lopinavir/Ritonavir or Lamivudine Prophylaxis to Prevent Breastfeeding Transmission of HIV-1.

Children who are human immunodeficiency virus (HIV)-exposed but uninfected (CHEU) accumulate maternal HIV and antiretroviral exposures through pregnancy, postnatal prophylaxis, and breastfeeding. Here, we compared the dynamics of mitochondrial DNA (mtDNA) parameters in African breastfed CHEU receiving lopinavir/ritonavir (LPV/r) or lamivudine (3TC) pre-exposure prophylaxis during the first year of life. The number of mtDNA copies per cell (MCN) and the proportion of deleted mtDNA (MDD) were assessed at day 7 and at week 50 post-delivery (PrEP group). mtDNA depletion was defined as a 50% or more decrease from the initial value, and mtDNA deletions was the detection of mtDNA molecules with large DNA fragment loss. We also performed a sub-analysis with CHEU who did not receive a prophylactic treatment in South Africa (control group). From day seven to week 50, MCN decreased with a median of 41.7% (interquartile range, IQR: 12.1; 64.4) in the PrEP group. The proportion of children with mtDNA depletion was not significantly different between the two prophylactic regimens. Poisson regressions showed that LPV/r and 3TC were associated with mtDNA depletion (reference: control group; LPV/r: PR = 1.75 (CI95%: 1.15–2.68), p < 0.01; 3TC: PR = 1.54 (CI95%: 1.00–2.37), p = 0.05). Moreover, the proportion of children with MDD was unexpectedly high before randomisation in both groups. Long-term health impacts of these mitochondrial DNA parameters should be investigated further for both CHEU and HIV-infected children receiving LPV/r- or 3TC- based regimens.

Redox Properties of Native and Damaged DNA from Mixed Quantum Mechanical/Molecular Mechanics Molecular Dynamics Simulations

The redox properties of two large DNA fragments composed of 39 base pairs, differing only by an 8-oxoguanine (8oxoG) defect replacing a guanine (G), were investigated in physiological conditions using mixed quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations. The quantum region of the native fragment comprised 3 G-C base pairs, while one G was replaced by an 8oxoG in the defect fragment. The calculated values for the redox free energy are 6.55 ± 0.28 eV and 5.62 ± 0.30 eV for the native and the 8oxoG-containing fragment, respectively. The respective estimates for the reorganization free energy are 1.25 ± 0.18 eV and 1.00 ± 0.18 eV. Both reactions follow the Marcus theory for electron transfer. The large difference in redox potential between the two fragments shows that replacement of a G by an 8oxoG renders the DNA more easily oxidizable. This finding is in agreement with the suggestion that DNA fragments containing an 8oxoG defect can act as sinks of oxidative damage that protect the rest of the genome from assault. In addition, the difference in redox potential between the native and the defect DNA fragment indicates that a charge transfer-based mechanism for the recognition of DNA defects might be feasible, in line with recent suggestions based on experimental observations.

Sub3 inhibits Aspergillus flavus growth by disrupting mitochondrial energy metabolism, and has potential biocontrol during peanut storage.

Aspergillus flavus, a saprophytic fungus, is regularly detected in oil-enriched seeds. During colonization, this organism releases aflatoxins that pose a serious risk to food safety and human health. Therefore, an eco-friendly biological approach to inhibit the pathogen is desirable. Experimental results indicated that A. flavus spores could not germinate in potato dextrose broth culture medium, when the concentration of Sub3 exceeded 0.15 g L-1 . Morphological evaluation performed by flow cytometry and scanning electron microscopy indicated that spores were shrunken and pitted following Sub3 exposure. Physiological assessment using propidium iodide, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide, 2,7-dichlorodihydrofluorescein diacetate and 4',6-diamidino-2-phenylindole staining revealed damaged cell membranes, decreased mitochondrial membrane potential, increased intracellular reactive oxygen species levels, and elevated large nuclear condensation and DNA fragmentation. Moreover, mitochondrial dehydrogenase activity was reduced by 29.42% and 45.48% after treatment with 0.1 and 0.15 g L-1 Sub3, respectively. Additionally, colonization capacity in peanut was significantly decreased, and the number of spores on seeds treated with Sub3 was decreased by 26.86% (0.1 g L-1 ) and 77.74% (0.15 g L-1 ) compared with the control group. Sub3 likely inhibits A. flavus by crossing the cell wall and targeting the cell membrane, disrupting mitochondrial energy metabolism, and inducing DNA damage, leading to spore death. Thus, Sub3 may provide a useful biocontrol strategy to control A. flavus growth in peanuts. © 2020 Society of Chemical Industry.

Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum.

Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1-7 nt at the 5' end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the -6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1kb was 79.6%, and the editing efficiencies for 5- and 20kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.

Comparison of Whole Genome (wg-) and Core Genome (cg-) MLST (BioNumericsTM) Versus SNP Variant Calling for Epidemiological Investigation of Pseudomonas aeruginosa.

Whole genome sequencing (WGS) is increasingly used for epidemiological investigations of pathogens. While SNP variant calling is currently considered as the most suitable method, the choice of a representative reference genome and the isolate dependency of results limit standardization and affect resolution in an unknown manner. Whole or core genome Multi Locus Sequence Typing (wg-, cg-MLST) represents an attractive alternative. Here, we assess the accuracy of wg- and cg-MLST by comparing results of four Pseudomonas aeruginosa datasets for which epidemiological and genomic data were previously described. Three datasets included 155 isolates from three different sequence types (ST) of P. aeruginosa collected in our ICUs over a 5-year period. The fourth dataset consisted of 10 isolates from an investigation of P. aeruginosa contaminated hand soap. All isolates were previously analyzed by a core SNP approach. In this study, wg- and cg-MLST were performed in BioNumericsTM using a scheme developed by Applied-Maths. Correlation between SNP calling and wg- or cg-MLST results were evaluated by calculating linear regressions and their coefficient of correlations (R2) between the number of SNPs and the number of allele differences in pairwise comparison of isolates. The number of SNPs and allele difference between isolates with close epidemiological linkage varies between 0–26 and 0–13, respectively. When compared to core-SNP calling, a higher coefficient of correlation was obtained with cgMLST (R2 of 0.92–0.99) than with wgMLST (0.78–0.99). In one dataset, a putative homologous recombination of a large DNA fragment (202 loci) was identified among these isolates, affecting its phylogeny, but with no impact on the epidemiological analysis of outbreak isolates. In conclusion, we showed that the P. aeruginosa wgMLST scheme in BioNumericsTM is as discriminatory as the core-SNP calling approach and apparently useful for outbreak investigations. We also showed that epidemiological linked isolates showed less than 26 SNPs or 13 allele differences. These are important figures for the distinction between outbreak and non-outbreak isolates when interpreting WGS results. However, as P. aeruginosa is highly recombinant, a cgMLST approach is preferable and caution should be addressed to possible recombination of large DNA fragments.

Pseudoscorpion Wolbachia symbionts: diversity and evidence for a new supergroup S

BackgroundWolbachia are the most widely spread endosymbiotic bacteria, present in a wide variety of insects and two families of nematodes. As of now, however, relatively little genomic data has been available. The Wolbachia symbiont can be parasitic, as described for many arthropod systems, an obligate mutualist, as in filarial nematodes or a combination of both in some organisms. They are currently classified into 16 monophyletic lineage groups (“supergroups”). Although the nature of these symbioses remains largely unknown, expanded Wolbachia genomic data will contribute to understanding their diverse symbiotic mechanisms and evolution.ResultsThis report focuses on Wolbachia infections in three pseudoscorpion species infected by two distinct groups of Wolbachia strains, based upon multi-locus phylogenies. Geogarypus minor harbours wGmin and Chthonius ischnocheles harbours wCisc, both closely related to supergroup H, while Atemnus politus harbours wApol, a member of a novel supergroup S along with Wolbachia from the pseudoscorpion Cordylochernes scorpioides (wCsco). Wolbachia supergroup S is most closely related to Wolbachia supergroups C and F. Using target enrichment by hybridization with Wolbachia-specific biotinylated probes to capture large fragments of Wolbachia DNA, we produced two draft genomes of wApol. Annotation of wApol highlights presence of a biotin operon, which is incomplete in many sequenced Wolbachia genomes.ConclusionsThe present study highlights at least two symbiont acquisition events among pseudoscorpion species. Phylogenomic analysis indicates that the Wolbachia from Atemnus politus (wApol), forms a separate supergroup (“S”) with the Wolbachia from Cordylochernes scorpioides (wCsco). Interestingly, the biotin operon, present in wApol, appears to have been horizontally transferred multiple times along Wolbachia evolutionary history.

An artificial chromosome ylAC enables efficient assembly of multiple genes in Yarrowia lipolytica for biomanufacturing

The efficient use of the yeast Yarrowia lipolytica as a cell factory is hampered by the lack of powerful genetic engineering tools dedicated for the assembly of large DNA fragments and the robust expression of multiple genes. Here we describe the design and construction of artificial chromosomes (ylAC) that allow easy and efficient assembly of genes and chromosomal elements. We show that metabolic pathways can be rapidly constructed by various assembly of multiple genes in vivo into a complete, independent and linear supplementary chromosome with a yield over 90%. Additionally, our results reveal that ylAC can be genetically maintained over multiple generations either under selective conditions or, without selective pressure, using an essential gene as the selection marker. Overall, the ylACs reported herein are game-changing technology for Y. lipolytica, opening myriad possibilities, including enzyme screening, genome studies and the use of this yeast as a previous unutilized bio-manufacturing platform.

The CRISPR/Cas9 induces large genomic fragment deletions of MSTN and phenotypic changes in sheep

The CRISPR/Cas9 system has been extensively used to engineer genetic loci for the generation of knockouts, insertions, and point mutations in animal models. However, many mutations that have been reported in animals are small insertions or deletions. This study used the CRISPR/Cas9 system to induce large DNA fragment deletions in MSTN via three guide RNAs in sheep. This successfully achieved the precise gene editing of the ovine MSTN gene by injecting both Cas9 mRNA and sgRNAs into embryos at the one-cell stage. Of 10 edited animals, 3 animals (30%) exhibited large genomic fragment deletions (~5 kb). Furthermore, the body weights of these 3 animals were significantly different (P0>0.0001, P15=0.001, P30=0.005, P60=0.027) between lambs with large deletions and wildtype lambs. In addition, the edited lambs were also significantly different (P0>0.0001, P15>0.0001, P30=0.002, P60=0.011) compared with wildtype. These results suggest that the generated MSTN knockout sheep is a reliable and effective animal model for further study. Furthermore, this method is time- and labor-saving, and efficient for the creation of animal models for agriculture, biology, and medicine.

Integration of large heterologous DNA fragments into the genome of Thermococcus kodakarensis.

In this study, a transformation system enabling large-scale gene recombination was developed for the hyperthermophilic archaeon Thermococcus kodakarensis. Using the uracil auxotroph T. kodakarensis KU216 (∆pyrF) as a parent strain, we constructed multiple host strains harboring two 1-kbp DNA regions from the genomes of either the hyperthermophilic archaeon Pyrococcus furiosus or Methanocaldococcus jannaschii. The two regions were selected so that the regions between them on the respective genomes would include pyrF genes, which can potentially be used for selection. Transformation using these host strains and genomic DNA from P. furiosus or M. jannaschii were carried out. Transformants with exogenous pyrF were obtained only using host strains with regions from P. furiosus, and only when the distances between the two regions were relatively short (2-5 kbp) on the P. furiosus genome. To insert longer DNA fragments, we examined the possibilities of using P. furiosus cells to provide intact genomic DNA. A cell pellet of P. furiosus was overlaid with that of T. kodakarensis so that cells were in direct contact. As a result, we were able to isolate T. kodakarensis strains harboring DNA fragments from P. furiosus with lengths of up to 75 kbp in a single transformation step.

Mechanisms of fosfomycin resistance in clinical isolates of carbapenem-resistant Klebsiella pneumoniae

ObjectivesOlder antimicrobials such as fosfomycin are being considered as alternative agents in the treatment of drug-resistant organisms. However, there are limited data on the usefulness of fosfomycin against carbapenem-resistant Klebsiella pneumoniae (CRKP). This study aimed to investigate the prevalence of fosfomycin resistance and associated mechanisms in CRKP. MethodsA total of 99 clinical CRKP isolates were collected in the Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) between January 2017 and June 2018. Fosfomycin susceptibility testing was performed by the agar dilution method. Carbapenemase and fosfomycin resistance genes were detected by PCR. Analysis of the murA, glpT, uhpT, uhpA, ptsI and cyaA genes was performed by PCR and sequencing of four fosfomycin-resistant fosA3-negative CRKP strains. Conjugation experiments were employed to determine the mobility of the fosA3 gene. ResultsOf the 99 CRKP isolates, fosfomycin-non-susceptibility was detected in 48 (48.5%) isolates, among which the fosA3 gene was detected in 44. Among the four fosfomycin-resistant fosA3-negative CRKP isolates, one isolate possessed a single nucleotide insertion and deletion mutations as well as 219 nucleotide substitution mutations in murA, two isolates possessed deletion or mutation of large DNA fragments in glpT, and one isolate possessed a fragment insertion sequence in glpT. Transfection into Escherichia coli J53 via plasmid conjugation was successful for 19 (43.2%) of the 44 fosA3-positive CRKP isolates. ConclusionThe fosA3 gene is the primary mechanism of fosfomycin resistance in CRKP and can be transmitted widely by plasmid in hospitals. Mutations in murA and glpT were found in fosfomycin-resistant fosA3-negative CRKP.

Correlated Evolution of Large DNA Fragments in the 3D Genome of Arabidopsis thaliana

In eukaryotes, the three-dimensional (3D) conformation of the genome is far from random, and this nonrandom chromatin organization is strongly correlated with gene expression and protein function, which are two critical determinants of the selective constraints and evolutionary rates of genes. However, whether genes and other elements that are located close to each other in the 3D genome evolve in a coordinated way has not been investigated in any organism. To address this question, we constructed chromatin interaction networks (CINs) in Arabidopsis thaliana based on high-throughput chromosome conformation capture data and demonstrated that adjacent large DNA fragments in the CIN indeed exhibit more similar levels of polymorphism and evolutionary rates than random fragment pairs. Using simulations that account for the linear distance between fragments, we proved that the 3D chromosomal organization plays a role in the observed correlated evolution. Spatially interacting fragments also exhibit more similar mutation rates and functional constraints in both coding and noncoding regions than the random expectations, indicating that the correlated evolution between 3D neighbors is a result of combined evolutionary forces. A collection of 39 genomic and epigenomic features can explain much of the variance in genetic diversity and evolutionary rates across the genome. Moreover, features that have a greater effect on the evolution of regional sequences tend to show higher similarity between neighboring fragments in the CIN, suggesting a pivotal role of epigenetic modifications and chromatin organization in determining the correlated evolution of large DNA fragments in the 3D genome.

Effect of DNA sizes and reactive oxygen species on degradation of sulphonamide resistance sul1 genes by combined UV/free chlorine processes

Nowadays, antibiotic resistance genes (ARGs) have been characterized as an emerging environmental contaminant, as the spread of ARGs may increase the difficulty of bacterial infection treatments. This study evaluates the combination of ultraviolet (UV) irradiation and chlorination, the two most commonly applied disinfection methods, on the degradation of sulphonamide resistance sul1 genes. The results revealed that although both of individual UV and chlorination processes were relatively less effective, two of the four combined processes, namely UV followed by chlorination (UV-Cl2) and simultaneous combination of UV and chlorination (UV/Cl2), delivered a better removal rate (up to 1.5 logs) with an observation of synergetic effects up to 0.609 log. The mechanisms analysis found that the difference of DNA size affected sul1 genes degradation by UV and chlorination; targeted genes on larger DNA fragments could be more effectively degraded by UV (1.09 logs for large fragments and 0.12 log for small fragments when UV dose reached 432 mJ/cm2), while to degrade ARGs on smaller DNA fragments required less free chlorine dosage (10 mg/L for small fragments and 40 mg/L for large fragments). The sequential combination of UV and chlorination (UV-Cl2) used the corresponding reactivity of both processes, which could be the reason for the synergetic effect. For UV/Cl2 process, the formation of reactive oxygen species (ROS) contributed to the synergetic effect. Scavenger analysis showed that the contribution of ROS to the sul1 gene reduction was 0.004 to 0.273 log (up to 45.5 % of the total synergy values), and among the two major reactive species in UV/Cl2 system, HO was the more important radical, while the contribution of Cl was negligible. Besides, UV/Cl2 process also used the corresponding reactivity of both processes to generate the remaining synergy values when excluding the contribution by reactive radicals. These findings provide a thorough understanding of the effects of UV and free chlorine on the degradation of ARGs and indicate the potential to utilize the combined processes of UV and free chlorine in water or wastewater treatment practice to control the dissemination of antibiotic resistance.

Multigene human artificial chromosome vector delivery with herpes simplex virus 1 amplicons

Gene expression studies and gene therapy require efficient gene delivery into cells. Different technologies by viral and non-viral mechanisms have been used for gene delivery into cells. Small gene vectors transfer across the cell membrane with a relatively high efficiency, but not large genes or entire loci spanning several kilobases, which do not remain intact following introduction. Previously, we developed an efficient delivery system based on herpes virus simplex type 1 (HSV-1) amplicons to transfer large fragments of DNA incorporated in human artificial chromosome (HAC) vectors into the nucleus of human cells. The HSV-1 amplicon lacks the signals for cleavage and replication of its own genome, yet each amplicon has the capacity to incorporate up to 150 kb of exogenous DNA. In this study, we investigated whether the capacity of gene delivery could be increased by simultaneously introducing multiple HSV-1 modified amplicons carrying a gene expressing HAC vector into cells with the aim of generating a single artificial chromosome containing the desired genes. Following co-transduction of two HSV-1 HAC amplicons, artificial chromosomes were successfully generated containing the introduced genes, which were appropriately expressed in different human cell types.

Efficient Generation of Large-Fragment Knock-In Mouse Models Using 2-Cell (2C)-Homologous Recombination (HR)-CRISPR.

Generating large-fragment knock-ins, such as reporters, conditional alleles, or humanized alleles, directly in mouse embryos is still a challenging feat. We have developed 2C-HR-CRISPR, a technology that allows highly efficient (10-50%) and rapid (generating founders in 2 months) targeting of large DNA fragments. Key to this strategy is the delivery of CRISPR reagents into 2-cell-stage mouse embryos, taking advantage of the high homologous recombination activity during the long G2 cell cycle phase at this stage. Furthermore, by exploiting a Cas9-monomeric streptavidin (Cas-mSA) and biotinylated PCR template (BioPCR) system to localize the repair template to specific double strand breaks, the efficiency can be further improved to up to 95%. Here we provide a procedure to generate large-fragment knock-in mouse models using 2C-HR-CRISPR. We first describe the principles for designing single guide RNAs and repair templates but refer to published manuscripts and protocols for molecular cloning methods or commercial sources for these reagents. We then describe two unique aspects of 2C-HR-CRISPR that are critical for success: (1) production of the CRISPR reagents for 2C-HR-CRISPR, particularly for applying the Cas9-mSA/BioPCR method, and (2) microinjection of mouse embryos at the 2-cell stage. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Single guide RNA and repair template design Basic Protocol 2: Preparing reagents for 2C-HR-CRISPR Basic Protocol 3: Microinjecting 2-cell-stage mouse embryos.

The characteristic of agarose extraction from dimethyl sulfide and isopropanol

Introduction. This study aimed to obtain agarose product with market standard quality which could be applied for electrophoresis. Methods. The material used in agarose extraction process was dried agar – agar bar extracted using dimethyl sulfide and soaked in isopropanol solution with 85%, 90%, and 95% of isopropanol concentration. The parameter which observed in this study was consisted of yield, water content, sulfate content, ash content, gel strength and electrophoresis gel. Results. The average score of agarose yield using different concentrations of isopropanol (IPA) was around 46.76% - 57.53% with the lowest yield of 46.76% and the highest yield of 57.53%. Meanwhile the average water content ranged from 18.32% to 18.64%. The average ash content was around 0.15% - 0.16% and the average sulfate content was 0.14% - 0.15%. The average strength of a 1% gel prepared in isopropanol was 1170 – 1183.33 gr/cm2. Best IPA concentration to soak agarose extract was on 95% concentration, with market standard characteristic as 57.54% of yield, 18.35% of water content, 0.14% of sulfate content, 0.15% of ash content and 1183.33 of gel strength. Conclusions.The application of electrophoresis of agarose gel showed that the bands of large molecular DNA fragment could not separate. Partially hydrolyzed protein samples had higher protein content, lighter color, and lower degree of denaturation and better functional properties compared to the traditional protein isolates.