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The Combination of DNA Design and Synthesis Empower RNA Synthesis Ability

mRNA vaccines are the third generation of nucleic acid vaccine developed on the basis of the first generation of inactivated vaccines or attenuated live vaccines and the second generation of subunit vaccines. The active component of mRNA vaccines is RNA, which has two forms. One form is non-replicating mRNA, composed of the antigen encoding sequence and two sides of 5’UTR and 3’UTR. The other form is self-replicating mRNA, where the antigen encoding sequence is constructed in the genome of RNA virus and it can be self-amplified with the help of the virus encoding replicase. Ultimately, this improves the expression level of the antigen gene in the cell under the condition of very low injection dose.

Fig.1 Comparison of non-replicating and self-amplifying mRNA vaccines[1]

Why do mRNA vaccines stand out?

mRNA vaccines have many unique advantages, which is the main reason to promote its extensive research and application. Easy preparation is one of the main reasons why mRNA vaccines stand out. Its active RNA component is simple, and it can be obtained by in vitro transcription using linear DNA as a template. In this process, the stability and translation efficiency of mRNA can be improved through codon optimization, nucleoside modification, and an auxiliary delivery system, which ensures high specificity and strong stability of the nucleic acid vaccine. In addition, the safety of the vaccine is also a major factor in promoting its widespread use. The mRNA does not integrate into the host genome, so there is no potential risk of infection or insertion mutation and it can be degraded through normal cellular processes.

Design and Synthesis Technologies Guarantee Every Step of In Vitro Transcription

•    NGTM Codon optimization technology makes codon optimization more convenient.
The secondary structure of mRNA affects the expression and stability of mRNA in cells. Synbio Technologies independently developed our NGTM Codon optimization technology, which can effectively optimize mRNA sequences and improve mRNA translation efficiency by optimizing codon composition, adjusting GC content, simplifying the secondary structure of mRNA, and removing modules that are not conducive to efficient expression.

•    Using a vector containing a poly(A)tail more than 100bp and synthesizing with a 100% accurate DNA template sequence.
The template preparation methods for in vitro transcription include PCR and plasmid linearization. During the linearization of plasmids, flat-terminal enzymes or class II restriction endonuclease enzymes (eg.BspQI) are usually used to produce blunt or 5 ‘end double-stranded DNA templates. Synbio Technologies has created the vector containing the poly(A) tail more than 100bp, providing technical support for template preparation in a fast, efficient method.

Fig.2 The vector map

•    In Vitro Transcription of RNA
Synbio Technologies uses in vitro transcription technology to synthesize various types of RNA such as sgRNA, lncRNA, mRNA, etc. It uses a linear DNA sequence as the template, while using T7, T3, or SP6 RNA polymerase to synthesize RNA sequence from DNA.

Fig.3 The RNA electrophoresis pictures

•    Add Cap, Tail, and Nucleoside Modifications
A mature mRNA is composed of cap structure, 5 ‘UTR, 3′ UTR, an open reading frame (ORF) and a poly(A) tail. The cap structure and poly(A) tail form a “closed loop”, which is closely related to mRNA stability and translation efficiency. Synbio Technologies can add different modifications to RNA sequences according to customers’ needs to further improve the stability and translation efficiency of RNA products.

Fig. 4 mRNA and protein complex

Cap Structure
In eukaryotes, the traditional cap structure has three types: m7GpppXpYp (Cap0), m7GpppXmpYp (Cap1), and m7GpppXmpYmp(Cap2). When the 7th carbon atom of G is methylated to form m7GpppN, the Cap is called “Cap 0”; Based on Cap0, if the 2´-O site of the first nucleotide of the transcript is also methylated, it is called “Cap 1”; If the 2´-O sites of both the first and second nucleotides of the transcript are methylated, it is called “Cap 2”. Usually, the cap is added in vitro by adding vaccinia virus cap enzymes or cap analogizing. Cap1 structure is widely found in mRNA of eukaryotes. Studies have shown that mRNA with Cap1 structure modified by in vitro transcription has higher stability and translation efficiency.

Poly(A) Tail
Most mRNA degradation starts from the Poly(A) tail. The Poly(A) tail of mRNA will be reduced to an oligo A tail structure by deadenylation, so the Poly(A) tail is crucial to resist the degradation of mRNA. According to our experimental results, the stability and translation efficiency of mRNA containing a 120nt Poly(A) tail were higher than those containing a 40nt or 80nt Poly(A) tail length.

Fig. 5 Case study: The effects of different ploy(A) tail on transcription

Modified Nucleosides
In mammals, common mRNA modifications include N1- and N6-methyladenosine (m1A, m6A, m6Am), 3- and 5-methylcytosine (m3C, m5C), 5-hydroxymethylcytosine (hm5C), pseudouridine (ψ) and 2′ -0-methylation (Nm). Studies founded that mRNAs can activate the cellular innate immune system by stimulating toll-like receptor TLRs (TLR3, TLR7, TLR8). However, most of the TLRs are no longer activated with the synthesized sequence using nucleoside analogues[2], such as pseudouridine (ψ), 5-methylcytidine (m5C), N6-methyladenosine (m6A), 5-methyluridine (m5U), and 2-thiuridine (s2U). Therefore, in the development of mRNA vaccines, modified nucleosides are usually introduced into the sequence to improve the stability of mRNA sequence and reduce its immunogenicity.

Fig. 6 RNA modification[3]

Synbio Technologies, with the leading bio-design and mature experimental technology, constantly improve our own synthesis abilities to meet the market demand and the comprehensive scientific research needs of our customers.

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NG™ Codon Optimization Technology
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Reference
[1] Pardi, N., Hogan, M., Porter, F. et al. mRNA vaccines — a new era in vaccinology. Nat Rev Drug Discov 17, 261–279 (2018).
[2] KORMANN MS, HASENPUSCH G, ANEJA MK, et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice [J]. Nat Biotechnol, 2011, 29(2): 154-157.
[3] Yasmin Granot, Dan Peer,Delivering the right message: Challenges and opportunities in lipid nanoparticles-mediated modified mRNA therapeutics—An innate immune system standpoint, Seminars in Immunology 34, 68-77(2017).

A Simple Trick to Quickly Increase Protein Expression

Codon optimization is one of the most important strategies to improve the expression of heterologous proteins because the use of codons varies greatly among different host cells. This can be summarized as follows: codon preference, codon coordination, and codon sensitivity. Only by understanding the secret of codon usage can we better utilize the appropriate codon to express the target protein efficiently in an experiment.

Codon Preference

Codons are redundant, but host cells do not use every codon equally to encode amino acids. A large number of studies have shown that highly expressed genes usually have a very high frequency of preferred codon use, while low expressed genes tend to random use of codons or contain a large number of rare codons. Also, the mRNA containing the preferred codon was translated faster than the artificially modified mRNA containing the rare codon. There are two hypotheses that can explain codon preference: I: mutation selection equilibrium hypothesis, which states that, under selection pressure, organisms tend to select the optimal codon to encode amino acids; II: The abundance of tRNA, the frequency of use of different codons is positively correlated with the available amount of corresponding tRNA, and the codon bias can reduce the abundance of tRNA, thus reducing the metabolic burden and is more conducive to cell growth [1].

Fig. 1 Codon Preference

Secondly, the use of codons can also affect the secondary structure and stability of mRNA, and affect the efficiency of protein translation. As predicted by the secondary structure of the protein, the unstructured part of the α -helix is more slowly translated. Meanwhile, hairpin structures can significantly affect protein expression, especially at the translation initiation site. Studies on 340 genomes from bacteria, archaea, fungi, plants, insects, birds, and mammals showed that, except birds and mammals, other species can improve the efficiency of translation initiation by reducing the stability of mRNA 5′- end [2].

In addition, the codon preference is also reflected in the GC content of the sequence. Studies have shown that GC content can significantly affect gene expression and regulation [3]. GC content greater than 70% will increase the stability of the RNA’s secondary structure and slow down or suspend translation. However, GC content less than 30% will slow down transcription elongation and is not conducive to protein expression. In eukaryotes, there are also some elements that have important influence on transcription, such as CpG islands, TATA boxes, and other repeat sequences which also have important influence on GC content of sequences.

Codon Coordination

The codon preference strategy can be applied to the expression of most heterologous proteins. When the amino acid sequence is preserved unchanged, the host cell preference codon is used for replacement, and the abundant tRNA in the host is used for translation, which can accelerate the translation efficiency and promote the protein expression. However, in the actual process of protein synthesis, not all codons are high-frequency codons. If the introduced codon frequency is inappropriate, the natural structure and function of the protein may be changed. Therefore, the selection of codons with appropriate frequency is a key factor for the successful expression of active protein [4]. At present, it has been reported that codon coordination optimization strategies have been successfully applied to the development and preparation of enzymes and vaccines.

Fig. 2 Codon Coordination

Codon Sensitivity

High codon sensitivity refers to the fact that, in the absence of amino acids in cells, the binding ability of tRNA to amino acids decreases significantly with the decrease of amino acid concentration. This results in suspension or even cessation of protein expression [5]. Although the optimization strategy of codon sensitivity has not been promoted, it has important guiding significance for protein expression under extreme conditions or strict requirements on the culture medium.

Fig. 3 Codon Sensitivity

Synbio Technologies NG™ Codon Optimization

Based on Codon preference, GC content, tandem short repeat sequences, hairpin structures, and other factors affecting the expression of heterologous proteins, Synbio Technologies independently developed our NG™ Codon optimization software. Our proprietary NG™ Codon optimization software can significantly improve the success rate of prokaryotic proteins expression and protein solubility.

Case Study 1

Protein R0103 has a theoretical molecular weight of 53.48kDa. The WT and optimized sequences were constructed on pET-28a(+) vector respectively, then heterologous proteins were expressed at 37 ℃ and 16 ℃. The results are shown in Fig. 1 and Fig. 2:

Results: The target protein was not expressed with the WT sequence at 37°C and 16°C. While the sequence optimized by NG™ Codon optimization software of Synbio Technologies showed significant protein expression at 37°C and 16°C.

Case Study 2

Protein R0118 has a theoretical molecular weight of 35.77kDa. The WT and optimized sequences were constructed on pET-28a (+) vector respectively, then heterologous protein expression were expressed at 37°C and 16°C. The results were shown in Fig. 3 and Fig. 4:

Results: The target protein was not expressed with the WT sequence at 37°C and 16°C. While the sequence optimized by NG™ Codon optimization software of Synbio Technologies showed significant protein expression and high ratio soluble protein expression at 37°C and 16°C.

References

[1] Gustafsson C, Govindarajan S, Minshull J. Codon bias and heterologous protein expression [J], Trends Biotechnol, 2004, 22(7):346-353.
[2] Gu W, Zhou T, Wilke CO. A universal trend of reduced mRNA stability near the translation-initiation site in Prokaryotes and Eukaryotes [J], PLoS Comput Biol, 2010, 6(2):e1000664.
[3] Newman ZR, Young JM, Ingolia NT, et al. Differences in codon bias and GC content contribute
to the balanced expression of TLR7 and TLR9. Proc Natl Acad Sci USA, 2016, 113(10): E1362–E1371.
[4] Angov E, Legler PM, Mease RM. Adjustment of codon usage frequencies by codon harmonization improves protein expression and folding//Evans TC Jr, Xu MQ, Eds. Heterologous Gene Expression in E. coli: Methods and Protocols. Totowa, NJ: Humana Press, 2011: 1–13.
[5] Dittmar KA, Sørensen MA, Elf J, et al. Selective charging of tRNA isoacceptors induced by amino-acid starvation. EMBO Rep, 2005, 6(2): 151–157.

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RNAi therapy: Human miRNA miR-675 Inhibits DUX4 Expression and Potential Treatment for Facioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a potentially devastating myopathy caused by de-repression of the DUX4 gene in skeletal muscles. Effective therapies will likely involve DUX4 inhibition. miRNAs are single-stranded, small, non-coding RNA of 22 nucleotides that play important roles as endogenous gene regulators by mediating translation repression or promoting degradation of target mRNA. While the normal expression and function of miRNAs are vital for physiological processes, aberrant expression of miRNAs has been proven to be closely related to the occurrence of various cancers. Perturbation of endogeneous miRNAs help to study the function of specific miRNAs and hold the potential for therapeutics.

RNA interference (RNAi) is one powerful approach to inhibit DUX4, and previously described a RNAi gene therapy to achieve DUX4 silencing in FSHD cells and mice using engineered microRNAs. This paper reported a strategy to direct RNAi against DUX4 using the natural microRNA miR-675, which is derived from the lncRNA H19. Human miR-675 inhibits DUX4 expression and associated outcomes in FSHD cell models. In addition, miR-675 delivery using gene therapy protects muscles from DUX4-associated death in mice. Finally, This research shows that three known miR-675-upregulating small molecules inhibit DUX4 and DUX4-activated FSHD biomarkers in FSHD patient-derived myotubes.

RNA Synthesis and RNA Modifications | Synbio Technologies

Synbio Technologies offers a wide range of high quality miRNA synthesis products, including miRNA mimics/inhibitors, miRNA agomirs/antagomirs and miRNA negative controls, etc. to support your miRNA functional research.

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References:
Saad Nizar Y,Al-Kharsan Mustafa,Garwick-Coppens Sara E et al. Human miRNA miR-675 inhibits DUX4 expression and may be exploited as a potential treatment for Facioscapulohumeral muscular dystrophy.[J] .Nat Commun, 2021, 12: 7128.

DNA Assembly for Nanopore Data Storage Readout

The digital revolution has led to the explosive growth of electronic data, and a large amount of data needs to be stored. With the development of life science, synthetic DNA sequences have been listed as storage media with research prospects and application value. The technology of high-throughput synthesis of DNA and sequencing of DNA sequences has also developed rapidly in recent years. Commercial nanopore sequencing is more portable, inexpensive, and automated than Illumina’s sequencing by synthesis (SBS), but nanopore sequencing itself also has some problems, such as higher error rate and lower sequencing throughput than SBS.

This paper reports a method that combines random access, DNA assembly, and nanopore sequencing to decode the information stored in DNA. Multiple digital files were mapped into 150 nucleotide DNA sequences and synthesized. Gibson assembly and Overlap-Extension PCR (OE-PCR) successfully decoded 1.67MB of digital information stored in DNA. Nanopore sequencing can estimate the decoding coverage of digital files stored in DNA in real time. Four different DNA coding files were amplified, assembled, and sequenced using the ONT MinION platform. An assembly of 6, 10, or 24 fragments were built for each file. By storing 1.67 megabytes of digital information in 111,499 oligonucleotides, the ability of sequencing and decoding has been improved by two orders of magnitude, and a faster and more flexible DNA storage scheme has been developed.

Fig.1 Overview of the DNA data storage workflow

Gene Synthesis and DNA Data Storage | Synbio Technologies
Synbio Technology has a complete synthesis platform, providing services such as DNA fragment synthesis, gene synthesis, pathway synthesis and assembly, and genome synthesis and assembly. We have rich experience in complex and long gene synthesis. We guarantee the delivery of 100% accurate gene sequences, so that you can obtain high-quality products with minimal project funds.

We have also developed DNA StudioTM to achieve bi-directional transcoding between “A, T, C, G ” and digital information. Then, according to the code, DNA sequences are synthesized accurately on chips, rapidly as well as massively. We invite you to join in developing DNA digital storage systems.

Reference
Randolph Lopez, Yuan-Jyue Chen, et al. DNA assembly for nanopore data storage readout. Nat Commun. 2019; 10: 2933.

Antisense Oligonucleotide Drugs: Targeting LINC02273 with ASOs Showed Potential in Mitigating Breast Cancer Metastasis

Although the overall survival rate and prognosis of breast cancer patients has improved in recent years, metastasis is still the leading cause of mortality in breast cancer patients. Emerging evidence has shown that ncRNAs, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in particular, play crucial roles in various types of cancer through regulating coding gene expression and epigenetic signatures. Increased LINC02273 promoted breast cancer metastasis in vitro and in vivo. This study revealed the molecular mechanism of lncRNA driving breast cancer metastasis through epigenetic increase of AGR2 transcription. Targeting LINC02273 with ASOs showed potential in mitigating breast cancer metastasis, which has important significance for the development of ASO drugs for the treatment of metastatic breast cancer.

Recently, antisense oligonucleotide (ASO) drugs have gained increasing attention with their ability to target diverse RNAs, which has been validated both in vitro and in vivo. To explore the possibility of whether LINC02273 could be interfered by ASOs, three antisense oligonucleotides (ASOs) specifically targeting LINC02273 and one negative control targeting no known sequence in the human genome were designed for the study (Fig. 1a). LINC02273 mRNA expression was significantly inhibited by all three ASOs in MDA-MB-231, LM2, and BT549 cells (Fig. 1b). Accordingly, AGR2 expression was dramatically decreased when LINC02273 was suppressed (Fig. 1c).

This study further developed LINC02273-targeting ASO-1/ASO-2 and control ASO-NC with modifications optimized for in vivo study. Free uptake assays showed dose-dependent inhibition of LINC02273 in LM2 cells by ASO-1 and ASO-2 compared to NC (Fig. 1d). An orthotopic xenograft tumor model was used to determine the therapeutic efficacy of LINC02273 ASO treatment. Wild type LM2 cells were inoculated into mammary fat pads of NOD/SCID mice (Fig. 1e). After 2 weeks, mice were randomly assigned into three groups (ASO-1, ASO-2, and ASO-NC) and given respective ASO treatment by tail vein injection twice a week. Compared to ASO-NC, tumor growth was significantly decreased in the ASO-1 and ASO-2 groups (Fig. 1f). More prominently, significant reduction of lung metastasis was observed by bioluminescent imaging, which was further confirmed by H&E staining (Fig. 1g). Moreover, This study confirmed that the expression of LINC02273 and AGR2 mRNA levels were decreased in tumors tissues treated with ASO-1 and ASO-2, compared to the ASO-NC group (Fig. 1h). Collectively, this data demonstrated that LINC02273 played critical role in promoting breast cancer metastasis and targeting LINC02273 with ASOs might serve as an effective therapeutic approach to mitigate breast metastasis.

Fig.1 Potential therapeutic role of LINC02273 in breast cancer

Synbio Technologies has established production workshops that meet the quality management requirements of ISO 9001 and ISO 13485, with a standard production process and excellent synthesis & purification technologies. Our synthetic antisense oligonucleotides strictly comply with QC testing standards. HPLC purity detection is used to ensure the high quality output of all our antisense oligonucleotide products.

Service Specifications:
ServiceYieldPurificationModificationsDeliverables
ASO SynthesisR&D Level: μg-mgHPLCPTO, 2’-OME, 2’-MOE, LNA, Cholesterol, GalNAc, etc.• Dry powder delivery, according to the customer’s packing needs.
• COA files.
Manufacturing Level: gHPLCPTO, 2’-OME, 2’-MOE, LNA, Cholesterol, GalNAc, etc.• Dry powder delivery, according to the customer’s packing needs.
• COA files
If you need other special modifications or pricing, please contact us for a quote at quote@synbio-tech.com.

Learn more: https://www.synbio-tech.com/oligo-synthesis/antisense-oligonucleotides/

References: https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-019-1115-y

Immunogenicity Research of SARS-CoV-2 mRNA Vaccine

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes significant morbidity and mortality, particularly among vulnerable immunosuppressed cancer patients. From observational studies, messenger RNA (mRNA) SARS-CoV-2 vaccines, BNT162b2, and mRNA-1273 show reduced antibody response in cancer patients compared to healthy individuals. Patients with selected hematological malignancies and those receiving specific anticancer treatments may have low or no antibody response following vaccination. While it is believed that vaccine-induced responses are robust up to six months, and clinically significant breakthrough infections appear to be rare in healthy individuals, the kinetics of immune response and incidence of breakthrough infections are unclear in patients receiving concurrent therapy for malignancy. There is an urgent need to understand long-term immunogenicity of SARS-CoV-2 vaccines among cancer patients to inform evidence-based guidance for subsequent timing and frequency of booster vaccinations.

To optimize clinical experiments, longitudinal studies of SARS-CoV-2 vaccine-induced immune responses in cancer patients are needed. In a prospective cohort study of 366 (291 vaccinated) patients, antibody levels [anti-spike (IgG-(S-RBD)) and anti-nucleocapsid immunoglobulin were measured at three timepoints. (1.enrollment (prior to dose 1 vaccination for vaccinated patients, n=112); 2.peak response (2–12 weeks post two-dose for vaccinated patients, n=147) and 3.sustained response (16–28 weeks post two-dose for vaccinated patients, n=124). IgG-(S-RBD) at peak response was higher and remained higher after 4-6 months in patients receiving mRNA-1273 compared to BNT162b2. Patients with solid tumors attained higher peak levels and sustained levels after 4-6 months (p<0.001) compared to those with hematologic malignancies. Solid tumor patients receiving immune checkpoint inhibitors before vaccination had lower sustained antibody levels than those who received treatment after vaccination (p=0.043). Two (0.69%) vaccinated and one (1.9%) unvaccinated patient had severe COVID-19 illness during follow-up. Differences in antibody responses were partly explained by differences in treatment regimens, timing between vaccination and treatment, mRNA vaccine type, age, and ethnicity. All cases of severe illness/death occurred in hematologic malignancy patients; additional approaches may be needed to protect these individuals from COVID-19. This study shows variation in sustained antibody responses across cancer populations receiving various therapeutic modalities with important implications for vaccine booster timing and patient selection.

With the combination of our proprietary Syno® platforms and high-throughput antibody preparation, Synbio Technologies not only provides RNA synthesis, mRNA in vitro transcription service along with add cap structure, poly(A) tails, and modified nucleosides, but we also provide a one-stop solution to antibody research, such as accurately obtaining antibody sequences from hybridoma, antibody gene synthesis, and recombinant antibody preparation. We are committed to support your scientific research in both a timely and cost-effective manner.

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References DOI : 10.1158/0008-5472.CAN-21-3554

A Brief Introduction of CRISPR-Cas Technologies

In nature, the CRISPR-Cas system is a prokaryotic (bacteria and archaea) adaptive immune mechanism used to cut invading nucleic acids. The CRISPR-Cas9 system has the advantages of simplicity, convenience, and quickness, ultimately leading to its widespread and rapid use while promoting the change of research technology. In the past decade, CRISPR-Cas9 has changed genomic engineering by relying on base pairing of nucleic acids rather than traditional protein-DNA recognition.

Common gene editing tools include CRISPR-Cas9, CRISPR-Cas12a, Cascade, and Cas3. The CRISPR-Cas9 system is the most widely used genome editing tool. Streptococcus pyogenes Cas9 (SpCas9) is the first one used outside prokaryotic cells and reprogrammed for mammalian cell genome editing. It is the most commonly used Cas9. After DNA target recognition, SpCas9 usually produces a blunt double strand break (DSB). Cas12a generates a staggered cut with a 5’ overhang at DNA target sites without a tracrRNA. Before recruiting Cas3 targeted DNA sequences, Cascade first binds to DNA through PAM and spacer recognition. Due to its promiscuous recognition of PAM sequences, Cascade provides greater target site flexibility.

Fig 1 Overview of the main CRISPR–Cas gene editing tools.

Gene Regulation by CRISPR-Cas
In addition to gene editing by forming DNA breaks, the catalytic activity of Cas9 can be destroyed by mutating RuvC (D10A) and HNH (H840A) sites of Cas9, while maintaining its RNA-guided DNA targeting ability to form dCas9. dCas9 is combined with a variety of effectors, such as transcription inhibitors or activators, epigenetic modifiers, and fluorophores, so as to expand the application range of CRISPR-Cas system.

Fig 2 Targeted gene regulation and other applications

Future
The application of the CRISPR-Cas system to eukaryotic cells has completely changed the field of genome engineering. The progress of CRISPR gRNA libraries and next-generation sequencing makes genome-wide genetic and epigenetic screening easy, which is helpful to find new therapeutic targets.

As CRISPR-Cas-based therapies enter clinical trials, although there are some application limitations, such as small adeno-associated virus vector, off-target effect, immunogenicity of Cas9 protein, etc., the use of CRISPR-Cas shows great potential in correcting genetic diseases and enhancing cell therapy.

Synbio Technologies’s CRISPR-Cas9 Gene Editing Services
Synbio Technologies provides one-stop CRISPR-Cas9 gene editing services, including sgRNA design, chip synthesis, sgRNA library construction, NGS verification, virus packaging, and bioinformatics analysis. At present, Synbio Technologies has delivered hundreds of high-quality CRISPR-Cas9 sgRNA libraries covering more than 20 species including animals, plants, and microorganisms. Our offered libraries include various genomic knockout libraries, interference libraries, and activation libraries, which can meet the customized needs of many different customers. We always strive to provide our customers around the globe with accurate, fast, and convenient gene editing services.

Reference
[1] Adrian Pickar-Oliver and Charles A.Gersbach. The next generation of CRISPR Cas technologies and applications. NatRevMolCellBiol. 2019Aug; 20(8):490–507. doi:10.1038/s41580-019-0131-5

Customized Immunologic Adjuvant—CpG ODNs

CpG-oligodeoxynucleotides (CpG-ODN) are potent immune stimuli being developed for use as adjuvants in different species. In this study, using a cell-based activation assay, researchers developed a type of CpG-ODN containing a GACGTT or AACGTT motif in 12 phosphorothioate-modified deoxynucleotides with potent stimulatory activity for rabbit TLR9. The results of this study suggest that both the choice of CpG-motif and its length are important factors for CpG-ODN to effectively activate rabbit TLR9 mediated immune responses.

Different types of CpG ODNs have different structural characteristics and immune effects. They are generally divided into three types: Class A, Class B, and Class C. Synbio Technologies can provide these three types of CpG ODNs products, as well as synthesize the CpG sequences that you provide.

References: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108808

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RNA Sequencing Describes the Expression Profile of Iron Regulatory Genes in Human Islets

Eukaryotic and prokaryotic RNA sequencing is used to discover expressed genes in cells, tissues, or individuals under different physiological or pathological conditions. A transcriptome bonds a genome’s genetic information and biological functions. Nowadays, RNA sequencing is widely applied to a wide variety of biological research as well as clinical diagnosis and drug development.

Epidemiological and clinical studies have shown that cellular damage resulting from iron overload has been linked with type 2 diabetes (T2D). Iron overload promotes the generation of reactive oxygen species (ROS). Pancreatic β-cells can counter oxidative stress through multiple anti-oxidant responses. However, the core molecular signature of the protective anti-oxidant response to iron overload is not clear. Therefore, RNA sequencing is utilized to describe the expression profile of iron regulatory genes in human islets with or without diabetes. Functional experiments including siRNA silencing, qPCR, western blotting, cell viability, ELISA, and RNA sequencing were performed as a means of identifying the genetic signature of the protective response following iron overload-induced stress in human islets and INS-1.

With Synbio Technologies’s high-throughput sequencing technology, almost all RNA information of a tissue or organ can be sequenced comprehensively. We have rich experience in library construction for RNA sequencing to reach optimal rRNA removal efficiency. It also has high coverage, strand-specific RNA-seq libraries, comprehensive analysis, integrative analysis of multiomics services, etc.

Applications

Medical Research: Disease markers, disease diagnosis and classification, disease recurrence diagnosis, disease mechanism, clinical efficacy evaluation, drug toxicology evaluation, and personalized therapy.

Life Science Research: Abiotic environmental relationships, plants and microorganisms, phenotypic identification, metabolic pathway and functional genomic studies, and medicinal plants.

Data Analysis

Reference: DOI : 10.1016/j.mce.2021.111462

Breakthrough in Artificial Starch Synthesis Technology

Starch is a stored form of carbohydrate that is the main source of calories in the human diet as well as a crucial raw material in many bio-industries. At present, starch is produced primarily through photosynthesis in plants. However, a research team from China recently published their research achievements with synthetic starch on “Science”.

Through rational design, modular assembly, and three pivotal enzyme optimizations, the team constructed 11 reactions of unnatural carbon sequestration and starch synthesis pathways. This is the first time that scientists have synthesized starch molecules from carbon dioxide in the lab. NMR and other tests showed that the structure of the synthetic starch was consistent with that of natural starch. Preliminary laboratory tests showed that synthetic starch is about 8.5 times more efficient than starch produced by conventional agriculture.

Fig 1. Design and modular assembly of synthetic starch metabolic pathways

Synthetic biology makes synthetic starch possible. Using synthetic starch instead of natural starch can help save more than 90% of natural resources, avoid the negative effects of pesticides, reduce chemical fertilizer impact on the environment, improve worldwide levels of food security, promote economic development of carbon neutral creatures, and increase the sustainable development of biology-based societies.

Synbio Technologies, as a DNA synthesis company, commits to providing efficient DNA sequence design and synthesis services around the global. Through modular assembly, we can provide you with any well-designed DNA sequence.

References: https://www.science.org/doi/10.1126/science.abh4049

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