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Gene Library Synthesis : genomic libraries and cDNA libraries

Gene synthesis has revolutionized genetic research over the past twenty years by allowing the convenience of DNA storage for a wide range of genome sizes. These libraries have allowed researchers to archive the genes of interested and be able to obtain each gene with relative ease at his or her leisure. Another great advantage ofgene synthetic libraries is the convenient location of the genetic foundation of the organism at your disposal. The genetic foundation can either be stored as the entire genome or more specifically the coding regions of the genome. Gene libraries are mainly broken down into two distinct categories: genomic libraries and cDNA libraries. The two have the same principles and similar output, but are created in different ways and have many different aspects.

First, the genomic libraries is representative of the organism’s entire genome, making the library quite large. The process of creating a genomic library is as follows: first the selected DNA is isolated from the cells by a specific restriction endonuclease. The resulting DNA fragments are then inserted into a selected vector using DNA ligase. Once the DNA is located within the vector, the vector is inserted into a bacteriophage to be amplified. After amplification, the cloned DNA is then isolated again and inserted into a genomic library. This process is then repeated until the entirety of the organism’s genome is isolated, amplified, isolated again, and inserted into the genomic library. Since it is the entire organism’s genome is located in these libraries, including both coding and noncoding regions, these libraries can become quite large. Genomic libraries offer many advantages, such as being able to study gene regulation, or off target effects of a particular mutation. The large amounts of data allow researchers to better understand how mutations, located outside of the coding region of a gene, affect the organism. This extra information is essential to better understand certain mutations, but the large amounts of data can sometimes be problematic. In conclusion, genomic libraries offer more information about the organism, but in turn are more laborious to create and are much larger in size when compared to cDNA libaries.

Second, cDNA library is representative of the organism’s exonic regions, meaning that only the coding regions of the genome are recorded and stored in the library. The process of creating a cDNA library is as follows: mRNA is isolated from a cell of interest and collected. Reverse transcriptase is then used to generate the double stranded cDNA corresponding to the mRNA sequence. Once this is complete, the DNA ends are cleaved to become single stranded and is inserted into a selected vector using DNA ligase. The vector is then inserted into a bacteriophage and amplified. After amplification, the cDNA is isolated and collected in the cDNA library. The cDNA library is much smaller than that of genomic libraries as it only represents the exonic portion of the organism’s genome. This small size is not always problematic, cDNA libraries offer a unique approach to study variant mutations present within coding regions of a gene. In addition to studying variant mutations located within a gene, cDNA libraries also offer a more convenient approach to study protein function, interaction and expression.

Whether you are interested in studying an organism with a very small genome or one as complex and large as a human genome, these two types of libraries are one of the best ways to store data. At Synbio Technologies we offer gene synthesis service verify the genes you are interested.A library, either constructed by you or our team of experts, will be generated for a competitive price and in an efficient timeframe. We offer virtually any library type to fit your need, ranging from scanning, to triplet codon, to modular. In addition to this Synbio Technologies also offers error-free gene sequence with a 100 percent guarantee of the generated sequence quality. This also allows us to create a synthetic gene library, generated by genes of interest without the physical copy of the gene being needed. Synbio Technologies offers many quality assessments to make sure that your gene library is exactly how you designed it to be and is as effective as possible. With the high quality library Synbio Technologies offers, and at a competitive price, your library will be synthesized and be ready to analyze with ease and convenience.

Applications of PCR Cloning Technology

PCR cloning technology can amplify trace amounts of DNA, generating millions of copies of a specific DNA sequence. PCR is highly sensitive, extremely specific, and high-yield, while also being easily reproducible, fast, and convenient, making it an amazingly powerful tool in molecular biology. With the rapid development of modern life science, PCR cloning technology has been more and more widely applied to various fields in biological research, medical research, virus detection, and the food industry.

PCR cloning Technology Applied to Gene Cloning

Gene cloning and Subcloning via PCR technology plays a significant role in cell biology research. PCR technology can generate millions of copies of a single-copy gene, amplifying a specific DNA fragment that might only be a few picograms. Compared to other gene cloning techniques, PCR omits several tedious processes for cloning of a particular gene fragment from genome DNA, such as enzyme digestion, connection, transformation, DNA library construction, gene screening, gene identification, and Subcloning.

PCR Cloning Technology Applied to DNA Recombination

In molecular biology, PCR cloning technology can be used to construct recombinant DNA molecules by inserting different sources of specific genes or DNA fragments into viruses, plasmids or other vectors in vitro. Recombinant DNA molecules are then imported into reporter cells to amplify and reproduce. After screening, the daughter cells that contain the target gene are further multiply to extract a large amount of DNA. Recombinant DNA technology can be applied to the Human Gene Project, valuable protein expression, gene diagnosis and therapy, genetic modification of animals and plants, and other research fields.

PCR Cloning Technology for Gene Quantification

PCR cloning technology can be applied to quantitatively determine the copy number of a target gene in a sample. The target gene and a single copy reference gene are placed in a tube for PCR cloning. The PCR product is then separated by electrophoretic separation technology and band intensity is observed. Alternatively, the 5’ end of the primer can be marked by a radionucleotide, after which the gene copy number can be determined by radioactivity quantification. PCR cloning technology can also be applied to quantitatively analyze mRNA and tRNA. It can even detect 1TIRNA, which is hard to detect even by Northern blot.

Alteration of endogenous genes and invasion of foreign genes can be threatening to human health. Regardless of whether or not pathogenesis is caused by genetic changes, as long as there is a pathogen, its corresponding existing nucleic acid can be found. With the development of PCR cloning technology and related technologies, PCR can be applied to infectious disease pathogenesis detection and diagnosis, tumor related gene detection, hereditary disease early diagnosis, bone marrow transplant HLA – D locus matches, and evolutionary theory analysis.

Synbio Technologies’s Syno®2.0 platform can clone a target gene to any specific point on provided vectors without depending on restriction enzyme sites, and can quickly and cost-effectively fulfill a wide variety of client requests for myriad applications in synthetic biology.

Gene Synthesis Driving Synthetic Biology Applications

DNA sequencing has been vital to the development of synthetic biology in many ways. Sequencing enables researchers to determine the DNA sequence in just about any gene, and enables the construction of vast databases that can hold entire genomes. Genome databases are an important resource for downstream synthetic biology applications such as protein expression, directed evolution, and metabolic engineering. In addition, the low cost of DNA sequencing enables more efficient quality control of large DNA constructs, a key step in gene synthesis.

Reductions in the production costs of genes and their key raw materials, oligos, are driving demand for synthetic biology products. Gene synthesis is key to many synthetic biology applications, and their availability at low cost increases the number of gene synthesis applications and customers, driving sales up.

The growing proteomics market is driving demand for more efficient protein expression in novel host systems. This in turn is driving the demand for synthetic genes that have been optimized for heterologous gene expression. Such optimized genes allow for expressing the desired protein product more efficiently, since they can be tailored to the intended host cell system. Gene synthesis will penetrate into the genetic engineering market among pharmaceutical and biotech companies developing new products. Gene synthesis provides a high level of flexibility to the customer and, as its cost decreases, its services are rapidly permeating the classical genetic engineering market to become standard tools among end users.

Synthetic biology applications driven by gene synthesis technology:

  • HGP-Write

    • The Human Genome Project – Write, formally announced on 2 June 2016, is a ten-year extension of the Human Genome Project, to synthesize the human genome. The human genome consists of three billion DNA nucleotides, which were described in the Human Genome Project – Read program, completed in 2003. With the advancement of gene synthesis technology, the time and cost of gene synthesis is approaching Moors’ Law. Many researchers expect that the ability to synthesize large portions of the human genome could lead to many scientific and medical advances.

  • Antibody Library

    • The traditional humanized antibody library refers to a group of re-expressed antibodies which have been transformed by gene cloning and DNA recombination technology based on mouse monoclonal antibody. Synbio Technologies designed and developed unique antibody humanization strategies based on advanced concepts and technologies in synthetic biology. This, combined with the integration of efficient phage display and cell surface display technology, have allowed Synbio Tech to easily provide fast and efficient humanized antibody services.

  • DNA storage

    • DNA storage (using DNA as a data carrier) technology is a relatively new discovery that may have monumental implications on the future of bioinformatics and data science. Text, images, audio, and video documents could be transformed into “A, T, C, G” format and stored in artificial synthesized DNA. Scientists from Synbio Technologies have mastered next generation gene synthesis technology which greatly reduces the manufacturing cost of DNA synthesis. Combined with our patented DNA StudioTMsoftware, DNA storage is closer than ever to spearheading the next generation of storage technology.

Gene Synthesis Related Services

Synbio Technologies’ Gene Synthesis Process

Synbio Technologies’s proprietary Syno®2.0 gene synthesis process includes computational design of short oligos, which can then be reliably synthesized, assembled, and cloned into the desired vector. An available precursor to this process is optimization of the gene sequence for improved protein expression and other purposes.

gene synthesis process

Fig. 1 Syno®2.0 gene synthesis process
A large number of oligos can be synthesized in parallel on gene chips. The Syno®3.0 next generation DNA synthesis platform offers revolutionary large-scale gene synthesis in an efficient, low cost manner that will open up new avenues for the development and industrialization of synthetic biology applications.

gene synthesis process

Fig. 2 Syno®3.0 gene synthesis process
Gene synthesis process–Codon Optimization:

Codon optimization refers to the use of preferred codons – that is, to avoid the usage of rare codons with low utilization – to simplify secondary structure of mRNA after gene transcription. It also involves the replacement of motifs that hinder efficient expression with those that promote it, as well as the adjusting of GC content and other factors in order to optimize gene expression.

Learn more about Codon Optimization:(click here to learn more:codon optimization)

  • Codon Usage Bias
  • Synbio Technologies’s NGTMCodon Optimization Software
  • Synbio Technologies’s Codon Optimization Strategy

Gene synthesis process–Oligo Synthesis:
Syno®2.0 gene synthesis technology:

  • Higher flexibility
  • Less cost effective
  • Limited throughput

Syno®3.0 gene synthesis technology:

  • Lower price (starting from $0.09/bp)
  • High throughput (synthesis of over 500,000 nucleobases and building DNA strands as long as 30,000bp on a single chip)
  • Limited flexibility

Learn more about Synbio Technologies’ Synotype platform

Gene synthesis process—Gene Assembly:

Comparison of popular DNA assembly technologies:

Gibson AssemblyYeast Assembly
DNA polymerase/Taq ligaseNeedNo need
Yeast has homologous recombination capability
Multiple fragmentOne-time assemblyOne-step assembly
Large DNA fragment/Genome< 20K base pairsUp to 1.08M base pairs

How to order


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+1 732-230-3003
Email
service@synbio-tech.com
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+1 609 228 5911
Online Inquiry
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The Methods of PCR Cloning

PCR Cloning is a technique used to amplify a specific region of DNA strand, and is used in almost every molecular biology lab in the world. PCR can be used on almost any DNA region, provided that suitable primers can be made. Synbio Tech provides one-stop PCR cloning services, including primer design.

PCR Primer Design Procedure

  • Acquisition of DNA sequence: For amplification of known DNA sequences, tried-and-tested primers can be found on the NCBI website. For unknown DNA sequences containing conserved sequence(s) from related species, primers should be designed according to the DNA or RNA of the conserved sequence(s).
  • PCR primer design: Many commercial software products and online tools are used to design primers. Primer Premier 5.0, the most popular primer design software, is both powerful and convenient to use. It can also contrast and comprehensively assess the best choice of primer according to their specificity.
  • Validation of PCR amplification: Once primer synthesis finishes, the accuracy of the primer can be predicted after gel electrophoresis of PCR product.

Notes About PCR Primer Design

  • The length of primer should be around 18-24bp. If the primer is too short, the primer specificity will be too low; if the primer is too long, it may cause base pair mismatches and may reduce the PCR amplification efficiency.
  • The GC% of the primer will affect the denaturation temperature (Tm). Tm should be around 55-80℃ and the annealing temperature difference between the upstream and downstream primers should be within 10℃. Usually, the GC% of primer should be between 40%-60% ,and the GC% difference between the upstream and downstream primers should be within 20% of each other in order to enhance primer specificity.
  • Repetitive structures or high similarity with the template sequence should be avoided as both may lead to possible base pair mismatches.
  • Secondary structure of primers may inhibit the PCR reaction, and should be avoided.

Synbio Technologies provide one-stop PCR cloning services, including primer design. Our Syno®2.0 platform can clone the target gene to any specific point on a provided vector without relying on restriction enzyme sites, to best satisfy the cloning requirements of each client.

PCR Techniques in Biology

Polymerase chain reaction, generally known as PCR and also referred to as in vitro DNA amplification, is one of the most widely used techniques in biology labs across the world.

Traditional DNA amplification method

Traditional DNA amplification involves the construction of a vector containing the target gene, followed by the transfer of the vectors into cells to amplify the target gene and then screening by probe. The process includes enzyme digestion, connection, transformation, culture, and probe hybridization. Although there are no technical difficulties for the traditional method, the complicated operation and long cycle length make it suboptimal as a way to amplify DNA. PCR is faster, more efficient, and cheaper, and has many advantages over traditional DNA amplification.

Advantage of PCR cloning

PCR was first theorized by Kary Mullis in 1983 and was later invented in 1985. PCR can amplify traces of DNA and generate millions of copies of a particular DNA sequence. Because of its high sensitivity, specificity, and yield, along with its reproducibility, speed, and convenience, PCR is now widely used in microbiology, medical science, agriculture, and many other fields. PCR has greatly simplified the process of molecular cloning, enabling researchers to much more easily analyze and identify genes of interest.

Basic principles of PCR cloning

At a temperature of 95℃, DNA denatures and yields single-stranded DNA molecules. The primer then binds to a complementary part of the DNA template. The temperature is then lowered to the optimum activity temperature, which is usually around 72℃. This activates DNA polymerase, which synthesizes a new DNA strand complementary to the DNA template in the 5’ to 3’ direction. The DNA template, primers, and polymerase are then thermocycled through denaturation, annealing, and extension steps, allowing DNA polymerase to replicate a target region of DNA by millions of times or more.

PCR has become an essential part of biology labs around the world, and is widely applied to gene cloning, genetic recombination, DNA sequence analysis, and gene quantification. PCR Cloning is also used in cancer gene detection and early diagnosis of hereditary diseases.

Our Syno®2.0 platform can clone any target gene to any specific point on required vectors
without depending on restriction enzyme sites. We promise both accurate and speedy delivery of any client’s cloning requests.

Codon Usage Bias

The genetic information in DNA is transcribed into mRNA then translated into protein, in which codons played important roles during the process. Total 64 combinations of nucleotide triplet (codon) encode all 22 amino acids. Each amino acid corresponds to at least one codon, e.g. methionine and tryptophan. In other cases, amino acids are encoded by 2 to 6 different codons. The codons encode the same amino acid are referred as synonymous codons. The frequency of synonymous codon usage varies widely among different organisms, and these differences have important implications for the regulation of protein expression. In the process of protein synthesis, a particular species tends to use a set of specific codons, which are called optimal codons. This phenomenon is also known as codon usage bias. So the use of different species in the codon usage bias, optimizing the use of codons can increase the protein expression.

Codon usage bias and protein

At the same time, accurate polypeptide elongation could minimize the energy waste caused by translation errors. Therefore, a codon with optimal translation speed and high fidelity will result high translation efficiency and an increased protein expression. The combination of codons and translation efficiency are not only contributing to the codon usage bias of the whole genome, but also the distribution of codons with different translation efficiency in different regions. The codon usage bias in different regions can regulate genes expression at different stages, such as affecting the mRNA expression during transcription, the speed and accuracy of translation, and the folding of polypeptide.

In the heterologous protein expression system, selecting the codon combinations that control the speed of translation is essential to avoid formation of inclusion bodies due to high expression or not obtaining any protein at all. Thus a well-tuned system for controlled gene expression is the key component for protein production in industrial and scientific research.

Synbio Tech proprietary NGTMCodon Optimization software can intelligently optimize codons based on different expression systems and effectively improve the protein expression in order to meet the needs of scientific research and industrial production.

Synbio Technologies’ Codon Optimization Strategy

Translation is the last step in protein synthesis that results in stable proteins from DNA replication, mRNA transcription and modification in cells. Codon optimization aims to increase the yield of protein expression in different organisms by rebalancing usage of synonymous codons. High quality protein has a wide range of applications, including study of protein structure and function, biological research, clinical medicine.

Higher protein yield in turn can be a valuable feature in a wide range of applications, including…

  • Codon usage bias

    • There are 64 codons in total, 61 codons encode 20 amino acids and the remaining 3 are stop codons. Redundant codons exist for most amino acids; multiple different codons encode the same amino acid. In general, the expression of heterologous proteins is negatively correlated with the occurrences of rare codons. In simpler terms, when the codon usage of a target protein in its native organism differs significantly from the average codon usage of the expression host, this could cause problems during expression. Therefore, an effective solution is to replace rare codons in the original sequences with major codons that are preferred in the host.

  • The secondary structure of mRNA

    • Translation refers to the conversion of nucleic acid sequences into amino acid sequences, and is greatly affected by complex secondary structures in mRNA. The translation of codons in the non-structural part of the α-helix is the rate-determing step in the translational process. Identifying the hairpin structure in an mRNA can lead to significant optimizations of translational efficiency for that mRNA.

  • Changing regulatory elements and restriction sites

    • The process of optimizing protein expression is affected by many factors: not only basic DNA transcription and translation, but also a series of regulatory elements. By incorporating signal peptides, tags, other regulatory elements, or appropriate restriction sites during gene synthesis, expression of synthetic genes in heterologous host organisms can be significantly improved.

    Synbio Tech NGTMCodon Optimization software can intelligently optimize codon usage to effectively improve protein expression, in order to better meet the needs of scientific research and industrial production.

Synbio Technologies’ NGTMCodon Optimization Software

Synbio Technologies’ proprietary NGTMCodon optimization software provides customers with free codon optimization services bundled with gene synthesis. Our NGTM Codon optimization software optimizes gene sequences based on different expression systems to maximize protein expression.

NGTMCodon optimization software advantages：

  • Elimination of codon usage bias:

    • Codon usage bias refers to the differences in frequency of synonymous codons in coding DNA, and exists in a wide variety of organisms. The most frequently used codon is called the optimal codon, and lower frequency codons are called rare codons. Synbio Technologies’ proprietary codon optimization software, NGTMCodon optimizes the usage of both the optimal codon and the rare codons to achieve superior protein expression.

  • Fine-tuning secondary structure of mRNA

    • The secondary structure of mRNAs play an active role in the translation process. The complexity and stability of mRNA secondary structure both greatly affect the smoothness of the translation process, in particular, secondary structure near the ribosome binding site (RBS) is crucial. NGTMCodon can effectively identify and minimize hairpin structures in mRNA, effectively optimize the original sequence, and generate significant improvements in protein expression.

  • Removal of negative cis-acting elements and restriction sites

    • NGTMCodon was designed to eliminate multiple obstacles during transcription and translation by minimizing negative cis-acting elements that interfere with transcription and translation.

    Case study：Targeting protein was reproducibly expressed in E. coli after codon optimization by NGTMCodon.

    ng-codon

    NGTMCodon improves codon usage efficiency

Four Steps of Subcloning Technology Protocol

Subcloning refers to the technique of re-cloning a DNA fragment from one vector to another, so that we can more easily perform analysis, transformation, and recombination of the target gene(s). Subcloning is an important tool in any molecular biologist’s toolkit, helping to elucidate the function of a target gene and to easily analyze its phenotype. There are four steps in the subcloning process: obtain the target fragment, connect enzyme vector and target fragment, transform in host cell, identify and screen.

  1. Subcloning Technology Protocol- obtain the target fragment
    • Find the target fragment in a gene library
    • The cDNA sequence was reverse transcribed with mRNA as a template through PCR, so we can obtain the target fragment from the cDNA library
    • Cut the DNA into many fragments with restriction enzymes and introduce into cells; we can screen for cells containing the target gene(s)
    • Synthesize the gene sequences in vitro
  2. Subcloning Technology Protocol- connect enzyme vector and target fragment

    3. Choose an appropriate restriction enzyme to cleave the target fragment from the vector. Cleavage in this way usually generates symmetrical cohesive ends, non-symmetric cohesive ends, or blunt ends. For subcloning, non-symmetric cohesive ends are preferred.

  3. Subcloning Technology Protocol- transform into host cell

    4. The two most common methods of transforming the target fragment into cells are transformation / transfection and transduction. In the first method, a recombinant plasmid or phage is simply transformed into a treated host cell; in the second, a host cell is transducted with a virus harboring exogenous DNA. In general, transduction is more efficient than transformation.

  4. Subcloning Technology Protocol-identify & screen

    5. Vectors with recognizable genetic markers can be used to help distinguish and separate cells transformed with recombinant DNA. For example, color can be used as such a marker, as the color of a colony may change in some vectors with exogenous genes. Other effective and widely used screening methods also exist, such as screening with drugs or with selective media.

With our Syno®2.0 gene synthesis platform and experienced technical team, Synbio Technologies provides one-stop subcloning services including target gene synthesis, vector construction and transformation, identification, and screening.

Synbio Technologies’ PCR cloning and Subcloning Technology

PCR cloning and subcloning technology, first developed in the 1970s, is now a staple in every molecular biology lab in the world. Cloning allows researchers to much more easily understand gene function at a deeper level, and greatly facilitates gene editing. PCR cloning and subcloning technology is not only revolutionary for the field of biology, but has profound implications on fields like agriculture, industry, and medicine as well.

PCR cloning technology

PCR cloning technology is similar to natural DNA replication, and contains three basic reaction steps: modification-annealing-extension. We can obtain a desired target gene sequence with appropriate primer design. PCR can then be used to amplify this gene sequence, preparing it for use in cloning.

Subcloning technology

In molecular cloning, target DNA is assembled into a vector plasmid through restriction enzymes and screening. In subcloning, a gene of interest is transferred from one vector to another. Both processes consist of several key steps, such as screening of the target fragment, cloning vector preparation, transformation/transduction of the product into cells, and screening for cells containing recombinant plasmids.

Both PCR cloning and subcloning technology can insert a target gene into a plasmid of choice in vitro through recombinant technology. The main forms of target gene transfer into a plasmid are transformation and transduction. This allows researchers to have an enormous amount of customization available to them when trying to study a gene of interest, making cloning and sub-cloning two extremely powerful tools in a molecular biologist’s arsenal.

With our proprietary Syno®2.0 gene synthesis platform, Synbio Technologies can provide one-step services for gene synthesis, vector construction, PCR cloning, and subcloning. Customers only need to offer the sequence information, and we can help design amplification primers and clone the PCR products to the specific sites of the new plasmid. We also provide sequencing services in order to confirm that the correct product was accurately synthesized.

Genome Editing Technology vs Transgenic Technology

Genome editing technology

Genome editing technology enables genetic engineering where DNA is replaced, deleted or inserted in the genome of a living organism, and the emergence of CRISPR-Cas9 system has further facilitated the realization of precise genetic modifications. Gene mapping and precise genetic modifications by inducing targeted DNA double-strand breaks opened up new avenues for the application of genome editing technology in drug development, gene therapy, agricultural breeding, environmental protection and endangered animal rescue.

Transgenic technology

Transgenics describes the process of introducing foreign DNA into a host organism’s genome. The foreign DNA, or “transgene,” that is transferred to the recipient can be from other individuals of the same species, from different species or even from artificially synthesized DNA. The foreign DNA was incorporated into the host genome by either homologous recombination or non-homologous recombination, and the following trait selection on a population allows cultivar development within a species to create offspring with desirable traits.

The difference between genome editing technology and transgenic technology

Both Genome editing technology and transgenic techniques can alter the genome of an organism so that the desirable trait can be inherited, but there is a big difference between the two. Genome editing is the manipulation of the genome of the organism itself by knocking out or replacing targeted gene which resulting in individuals with intentionally selected and desired traits, while transgenic technology can only introduce biologically nonexisting foreign genes to the original organisms in order to tailor the species with new traits. Therefore, the use of gene editing technology, can be fast, accurate and without the introduction of exogenous DNA fragments in the case of the organism genome transformation. The US Department of Agriculture (USDA) has shown lenient to genetically modified crops comparing to its controversial transgenic sibling. The decision means that the genetically modified crops can be cultivated and sold without passing through the agency’s regulatory process. The green light from USDA allows the flourish of valuable and disease-resistant crops while bypassing the use of controversial transgenic technologies.

The Comparison Between Three Generation Genome Editing Technologies

Genome editing refers to a type of genetic engineering in which DNA is replaced, deleted or inserted in the genome of a living organism using engineered nucleases. These engineered nucleases enable efficient and precise genetic modifications by inducing targeted DNA double-strand breaks (DSBs) that stimulate the cellular DNA repair mechanisms, There are currently three families of engineered nucleases being used, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the CRISPR-Cas system.

The mechanism of ZFN and TALEN system

ZFN and TALENs undergo similar molecular mechanisms for executing genome editing. ZFN and TALENs are both engineered nucleases composed of a DNA binding domain that recognize a specific nucleotide triplet based on the residues in their helix and a FokI nuclease motif that have a strong catalytic cleavage capability for specific nucleotides. The FokI domains must dimerize for activity, thus increasing target specificity by ensuring that two proximal DNA-binding events must occur to achieve a double-strand break (DSBs). These chimeric nucleases enable efficient and precise genetic modifications by inducing targeted DNA DSBs that stimulate the cellular DNA repair mechanisms, including error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR).

The mechanism of CRISPR system

CRISPR is a ubiquitous family of clustered repetitive DNA elements present in 90% of Archaea and 40% of sequenced Bacteria. The CRISPR system was first identified as an adaptive defensive mechanism that confers resistance to foreign genetic elements. Later on, CRISPR-Cas system was engineered into a versatile gene-editing tool enabling manipulation of protospacer adjacent motif (PAM) downstream DNA. CRISPR-Cas9 genome editing system consists of two components: a “guide” RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9). The Cas9 protein is an endonuclease that uses guide RNA molecule (gRNA) to form base pairs with DNA target sequences, enabling Cas9 to introduce a site-specific DSB in the DNA. The CRISPR-Cas9 system offers unprecedented advantages over the ZFN and TALEN strategies. Due to its simplicity and efficiency, CRISPR-Cas9 system has quickly become the go-to genome engineering tool for animal model construction, drug development, gene therapy, agricultural breeding and many other applications.

Based on our knowledge and years of experience in DNA technology, Synbio Technologies has developed CRISPR-Cas9 gene/genome editing platform. We offer a one-stop solution for CRISPR-Cas9 projects to achieve high genome editing efficiency, including CRISPR-Cas9 sgRNA design, CRISPR-Cas9 sgRNA library design and genome editing.

The Applications of Genome Editing Technology

Genome editing technology enables manipulations at genome level where DNA is replaced, deleted or inserted in a living organism. Classic genome editing approaches depend on homology-directed repair and the totipotency of stem cells to facilitate the modification of the individual gene. The Nobel Prize in Physiology or Medicine 2007 was awarded to the discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells. Because the traditional HDR approach has disadvantages of low efficiency, high technical requirements and high cost, which seriously restricted its applications in large-scale genomic manipulation research. In 2013 the discovery of the type II CRISPR-Cas9 system has promoted the development of precise genetic modifications. This new RNA-mediated DNA editing approach opens up new avenues for the application of genome editing technology in Animal model construction, genetic disease treatment and agricultural breeding.

Genome editing technology——Generation of Animal models

CRISPR-Cas9 system performs precise targeting and editing a specific DNA sequence of interest via a programmable mechanism, and provides a versatile approach to establish transgenic animal models. While mouse models have been widely used, the CRISPR-Cas9 gene-editing approach has been established in many other animal models, including worm, rat, rabbit, pig and monkey. New mouse models can be generated with CRISPR-Cas9 by injecting Cas9 mRNA and guide RNAs (sgRNA) directly into mouse embryos to generate precise genomic edits into specific loci with an efficiency of 100%. CRISPR-Cas9 mediated targeting and editing has facilitated the generation of knockin and knockout mouse models, dramatically decreases the time and resource consumption comparing to traditional methods.

Genome editing technology——Curing genetic diseases

Although CRISPR-Cas9 has already has been widely used as a research tool, a particularly exciting future direction is the development of CRISPR-Cas9 as a therapeutic technology for treating genetic disorders. Researchers at Chinese Academy of Sciences reported successful correction of disease-causing mutations in cataract mouse models via the CRISPR-Cas9 system. Upon injection of CRISPR-Cas9 into zygotes, 1/3 genetic defect in the cataract mouse model could be corrected at the organism level and more importantly the corrected trait was successfully transmitted to the next generation through the germline.

Genome editing technology——Agricultural breeding

The CRISPR-Cas9 technology opens up exciting possibilities for creating crop varieties with desirable traits without introducing foreign DNA. Precision breeding crops with desirable traits, such as disease resistance and drought tolerance not only help reduce pesticide, fertilizer and water usage, but also improve food quality and safety. Researchers at Penn State University created mushrooms with reduced production of a specific enzyme that causes mushrooms to blemishes caused by handling or mechanical harvesting. It becomes the first CRISPR-edited organism to receive a green light from the US government, means that the mushroom can be cultivated and sold without passing through the agency’s regulatory process.

CRISPR-Cas9 is an emerging technology that enables precise genome modification without introducing foreign genes. This transformative tool holds great promises to revolutionize biological research and expand our ability correcting the genetic causes behind many diseases.

Synbio Technologies establish the CRISPR-Cas9 gene/genome editing platform which can offer a one-stop solution for CRISPR-Cas9 projects. We can offer the services including: CRISPR-Cas9 sgRNA design, CRISPR-Cas9 sgRNA library design and genome editing.

A Brief Introduction of Molecular Biology Techniques

Molecular biology studies the morphology, structural features, biological relevance and regulatory mechanism of biological macromolecules including nucleic acid, protein and others. Molecular biology truly uncovered the mysteries of the biological world from the molecular level; it signaled the transition from passive adaption from nature to actively restructuring and reengineering the nature.

The main research contents of molecular biology span a broad spectrum: applications of DNA recombinant technology (also known as genetic engineering) in genetic engineered drugs; transgenic plants and somatic cell nuclear transfer; gene expression and regulation; structure-function relationship of biological macromolecules; functional genomics and bioinformatics studies.

Molecular biology is the cornerstone of current life sciences and medicine-related disciplines. Molecular biology techniques are the most commonly used tools in life science, however the viability and reliability of experiment result are dependence of many factors, including experience and expertise.

Empowered by Syno®1.0 and Syno®2.0 platforms, Synbio Technologies provides comprehensive and accurate molecular biology service for worldwide customers, ranging from DNA cloning, site-directed mutagenesis, vector construction, protein expression, gene/genome sequencing, gene synthesis technology and many other services.