What Exactly Can CRISPR/Cas9 Do?

CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats, which refers to regularly repeating nucleotide sequences originally noticed in bacterial genomes. It facilitates specific changes in the DNA of humans, animals and plants and modifies DNA in a faster and easier manner compared with earlier techniques.

CRISPR is a critical component of the immune systems of bacteria and microorganism with the immune system being responsible for the protection of the health and well-being of the organism. Cas9 is an RNA-guided DNA endonuclease enzyme associated with the CRISPR adaptive immunity system in Streptococcus pyogenes, among other bacteria. 

The CRISPR/Cas system is an adaptive immune defense mechanism used by Archea and bacteria for the degradation of foreign genetic material. In these organisms, the foreign genetic material from a bacteriophage is acquired and integrated into the CRISPR loci. This new material, also known as a spacer, creates a sequence-specific fragment used for future resistance against a bacteriophage infection. 

The role of the system in providing immunity is a relatively new discovery. Bacterial cell can get invaded by viruses and if a viral infection poses threats to a bacterial cell, the CRISPR immune system is able to combat the attack by destroying the invading virus' genome, preventing continued replication of the virus. The CRISPR immune system therefore provides protection to the bacteria from ongoing viral infection.

Drug developers and researchers from pharmaceutical giants is expecting that gene editing tools such as the CRISPR can lead to the development of new drug or cures for patients with genetic disorders. In a study involving adult mice, researchers were able to demonstrate a cure for a liver disease by replacing the mutant form of the gene with the correct sequence.

CRISPR has potentials in the field of infectious disease by providing a way to develop specific antibiotics that only target the bacterial strains responsible for a disease and sparing the beneficial bacteria. The technology also has become very popular in the lab for making precise changes in the genetic make-up of organisms. What’s more, it also has potential applications in industrial processes that involve bacterial cultures.

Rarely has a new technology generated the level of enthusiasm and interest that is now associated with CRISPR/Cas9, the simple and efficient genomic editing technique with the potential for advancing basic genetic research, gene therapy, and personalized medicine. Scientists across the world, biotech and drug companies, and university licensing officers are capitalizing on the research opportunities presented by this technology and are using this versatile system on a multitude of species.

 

Southern Blotting

What is Southern blotting

Southern blotting is one of the central techniques in molecular biology. This technique was first devised by E. M. Southern in 1975, resulting in transfer of DNA molecules from an electrophoresis gel to a nitrocellulose or nylon sheet referred to as a membrane. By this technique, DNA banding pattern present in the gel is reproduced on the membrane. During transfer or as a result of subsequent treatment,the DNA becomes immobilized on the membrane and can be used as a substrate for hybridization analysis with labelled DNA or RNA probes that specifically target individual restriction fragments in the blotted DNA.

In essence, Southern blotting is a method to detect a specific restriction fragment against a background of many other restriction fragments. The restricted DNA might be a plasmid or bacteriophage clone, and Southern blotting is used to confirm the identity of a cloned fragment or to identify an interesting subfragment from within the cloned DNA. In many cases, Southern blotting is a technique used prior to techniques such as restriction fragment length polymorphism (RFLP) analysis.

The Principle of the Southern Blot

This technique is based on fact that nitrocellulose powder or sheets are able to bind DNA. This ability of nitrocellulose powder or sheets has been known for many years and was utilized in the 1950s and 1960s in various types of nucleic acid hybridization studies. In these early techniques the immobilized DNA was unfractionated, simply consisting of total DNA that was bound to nitrocellulose powder or spotted onto a nitrocellulose sheet. Later in the early 1970s, the introduction of gel electrophoresis methods that enable restriction fragments of DNA to be separated on the basis of their size boosted the development of techniques for the transfer of separated fragments from gel to nitrocellulose support. The procedure involving capillary transfer of DNA from the gel to a nitrocellulose sheet placed on top of it is simple and effective, and has now become routine method used in many molecular biology laboratories.

In a word, Southern blotting is a technique that enables a specific restriction fragment to be detected against a background of many other restriction fragments. The basic methodology for Southern blotting has not changed since the original technique was described in 1975, but modifications have been introduced many times across years for purpose to speed up the process and achieve a more efficient transfer. Southern blotting has many applications in molecular biology, including the identification of one or more restriction fragments that contain a gene or other DNA sequence of interest, and in the detection of RFLPs used in construction of genomic maps.

Applications of RNA Probes

RNA, which complements a specific mRNA or DNA, is generally used to study virus genes, distribution of specific RNA in tissues and cells, integration of viral DNA into genomes, transcription, etc. Compared with DNA probes which are preferred for detection of the presence of DNA/RNA from specific species or subspecies, RNA probes are commonly for genetic studies. RNA probes can be subdivided by category into plus-sense RNA probes, minus-sense RNA probes, and antisense RNA probes. RNA probes can be used for Northern blotting, RNase protection assays, Southern blotting, downstream of polymerase chain reaction (PCR), and in situ hybridization analysis.

Northern blotting

A Northern blot is a laboratory method used to detect specific RNA molecules. The RNA under study is fractionated by gel electrophoresis. The molecules are then transferred to a membrane that is incubated with the labeled probe(s). Hybridization of complementary sequences allows visualization of target RNA sequences.

RNase protection assays

RPA also depends on the hybridization of probes to target RNAs, but the hybridization takes place in solution. RNA Probes are designed to have non-hybridizing regions. Controlled RNase digestion of the hybrids followed by separation and detection of the remaining probes allows for quantitative analysis of the target RNA in a sample and can provide information on target RNA structure.

In situ hybridization 

ISH experiments are conducted to localize RNA or DNA targets in cells and tissues. This technique involves cultured cells or tissue sections as the substrates for hybridization and detection. Cells or tissues are processed so that their endogenous nucleic acids are fixed in place, but available for hybridization to and detection by labeled probes.

Southern blotting

Southern blotting is a technique for transfer of DNA molecules from an electrophoresis gel to a nitrocellulose or nylon membrane, and is carried out prior to detection of specific molecules by hybridization probing. This technique involves the fractionation and transfer of DNA to membranes. Membranes are then are incubated with the labeled probe. Hybridization of complementary sequences allows visualization of target DNA sequences. 

The RNA Probe Kit 

The RNA Probe Kit allows the rapid and convenient recovery of RNA from any RNA labeling reaction. This kit can also be used for general RNA clean up, where other methods may lead to nuclease contamination or loss of samples. Binding RNA spin-columns and optimized buffers facilitate RNA isolation free of nucleotides, unincorporated labels, enzymes and salts.

Two Main Challenges for Therapeutic RNAi in Neurodegenerative Disease

RNAi has attracted many attentions in therapeutic studies since its discovery, and it is anticipated that RNAi-based therapeutics would rapidly reach the clinic in gene-based medicine research. However, there are still numerous unresolved challenges in nucleic acid-based technologies. And in order to realize the therapeutic potential of RNAi, strategies are being devised to avoid natural barriers to delivery, immune/non-immune toxicities and monitor delivery and therapeutic indices in real-time.

Brain barrier

Delivery of inhibitory RNAs to the CNS is a daunting task due to the blood–brain barrier. The most suitable RNAi delivery modality depends on our understanding of disease pathogenesis and the desired duration of gene silencing. Non-viral-delivered nucleic acids may access the CNS using three major entry routes: through the vasculature, cerebrospinal fluid or by direct intraparenchymal delivery into the brain. Thereby, the limiting factors include stability of the siRNA complexes and their capacity to penetrate target cells without activating immune responses. 

Efforts have been made to address some of these challenges by focusing on incorporating chemical modifications into the sugars, backbone or bases of siRNA duplexes. Certain modifications help to increase stability thus effectively lowering the dose needed to achieve measurable and reproducible gene silencing. And there are still some internal modifications failing to improve CNS entry and uptake after systemic delivery. To date, new efforts have moved towards testing liposomes, nanoparticles and cell-penetrating peptides, among others, to stabilize and navigate siRNAs into and throughout the brain. Future studies will be focused on exploiting the presence of disease-related epitopes as a means to increase further the efficacy, specificity and potency of non-viral siRNA delivery to the brain. Finally, the potential for an adverse immune response to RNAi therapy is an important consideration. In general, innate immune responses to non-viral-delivered siRNAs are mediated by toll-like receptors or by two different dsRNA-sensing proteins, however, the use of chemically modified or nanoparticle-encased siRNA duplexes avoids stimulation of these pathways.

Inability to monitoring

Another challenge with the use RNAi is our limited ability to monitor, in real-time, the delivery, activity and specificity of an RNAi molecule in the brain. Post-treatment sampling is impractical in the brain and thus model systems are required to establish correlations between gene-silencing potency and dose-specific toxicity. Previously, biomarker particular to certain neurodegenerative disease is the best choice to monitor a coincident response to therapeutic RNAi, although for many disorders the validity of a given biomarker to represent a particular disease stage is far from known. But currently advancesin imaging techniques , such as the PCR technique, to track RNA in vivo with quantum dots are showing promise.The monitoring of RNAi will no longer be a problem in near future.

As an experienced bio-tech service supplier, Creative Biogene provides can quality RNAi services for research community. The company has built-up leading scientific, technical and commercial track records, successfully advancing therapeutic programs for numerous major industry and academic partners in a wide range of disease areas. With roots as a pioneer of genome-scale RNAi screening and long-standing contributions towards refining best practices in this field, Creative Biogene provides leading functional genomics technologies including RNA interference (RNAi) tools for gene silencing (siRNA, shRNA, miRNA) and cutting-edge systems for gene editing and gene knockout.

http://www.creative-biogene.com/Services/RNAi-Service.html 

The Best MicroRNAs Products at Creative Biogene

MicroRNAs(miRNAs) are naturally occurring non-coding RNAs of 18-25 nt, which can bind to target RNAs based on sequence complementarity and direct post-transcriptional regulation of gene expression and regulate the stability or translational efficiency of target mRNAs. Numerous studies suggest that miRNA-mediated silencing may play an important role in development and disease, implying that miRNAs can serve as valuable biomarkers for diagnostic approaches.

It was first discovered in 1993 by genetic screens. The following discovery of a second miRNA and its conserved temporal expression patterns reveals that miRNAs is conserved among eukaryotes. Their diversity has been confirmed using bioinformatics and subsequent cloning. The analysis of miRNA knockout mice uncovered the physiological importance of individual miRNAs in mammals.

miRNAs belongs to the large miRNA genes family which has great diversity in genomic organization. Examination of mammalian miRNAs revealed that about one-third are expressed from introns of known protein-coding genes, with the remainder found distant from previously annotated genes. Whereas intronic miRNAs can be transcribed simultaneously with their host genes, intergenic miRNAs can be expressed from independent transcription units. They are frequently found as polycistronic clusters that are coordinately expressed. Although RNA Pol II transcribes the majority of miRNAs, RNA Pol III-transcribed miRNAs have been found.

Creative Biogene is leading in microRNA technologies and can offer full lines of miRNA related products incuding microRNA discovery, expression profiling and functional analysis.

MicroRNA Detection Assays

Quantification of miRNA by real-time PCR is a powerful tool for study of miRNA. Real-time PCR is used to validate the expression of miRNAs discovered during high throughput arrays. It is also a useful tool to discover new miRNAs and to study the expression of individual miRNAs.

MicroRNA Expression Profiling

Creative Biogene provides clones for over-expression of miRNA, miRNA mimics and inhibitors of your choice. All known human, mouse and rat miRNAs in the current miRBase is covered.

MicroRNA 3'UTR Target Cloning

Creative Biogene offers genome-wide human, mouse and rat miRNA 3'UTR target clones in mammalian expression vectors. The 3'UTR plasmids provide a convenient solution for quantitative assessment of the inhibitory effect between miRNAs and their potential target genes. Creative Biogene also provides 3'UTR Virus Particles for delivery of 3' UTR into a wide range of cell types including difficult-to-transfect cells.

shRNA Used for Application in Gene Therapy

shRNA (full name is small hairpin RNA or short hairpin RNA) is a sequence of RNA with a tight hairpin turn that can be used to silence target gene expression by inducing RNA interference(RNAi ). shRNA is created in the cell from a DNA construct encoding a sequence of single stranded RNA and its complement, separated by a stuffer fragment, allowing the RNA molecule to fold back on itself, creating a dsRNA molecule with a hairpin loop.

shRNA has the ability to provide specific, long-lasting, gene silencing. Scientists ,therefore, have been considering about using it for gene therapy applications. Some shRNA-based therapies have already been put into trail now.

shRNA has been considered an advantageous mediator of RNAi because it has a relatively low rate of degradation and turnover. The disadvantage of using it lies in that its expression in cells needs the assistance of plasmids, viral or bacterial vectors, which may cause some safety concerns.

There are still many challenges in using shRNA expression. In the past, viral based gene therapy approaches have proved dangerous in clinical trials. Besides, If the shRNA is expressed at levels that are too high the cell might not be able to correctly process the endogenous RNA, which could cause significant problems. It is possible that the patient will generate an immune response against the therapy. And it is also probable that the shRNA could silence other unintended genes.

About RNA interference

RNAi is the best way to effectively knock down gene expression and study protein function in a wide range of cell types and it is a powerful genetic tool for conducting functional studies. This technique is usually mediated by the use of siRNAs or shRNAs. Previous studies have showed that vector-based shRNA expression strategy is capable of inducing RNAi in viable cells. And results have showed that using vectors to perform RNAi experiments can expand experimental possibilities beyond the traditional siRNA approach.

About Creative Biogene
Creative Biogene is a US based biotech product and service supplier for academic and governmental research institutes, pharmaceutical and biotechnology industry. The company provides a series of high quality shRNAs, which could be used for silencing target gene expression via RNA interference.


http://www.creative-biogene.com/Product/shRNA 

General Introduction of AAV, rAAV and rAAV Vector

Adeno-associated virus (AAV) is inherently replication-deficient virus that belongs to the family Parvoviridae. It is single-stranded DNA virus with very simple structure. Adeno-associated viral which has been artificially recombined called recombinant adeno-associated viral (rAAV). And recombinant adeno-associated viral used in scientific research is unusually called rAAV vector.

The rAAV vectors consist of a simple capsid with a single-stranded DNA genome and no viral coding sequences. Its limited ability to transduce dendritic cells results in its limited immune responses.They are nonimmunogenic and can transduce both dividing and nondividing cells. Different rAAV serotypes may transduce diverse cell types. All those features make rAAV vectors excellent tools to study the function of neuropeptides in local brain areas. And they can also be used to locally or systemically enhance or silence gene expression.

In recent years rAAV vectors have become increasingly valuable for in vivo studies in animals and are also currently being tested in human clinical trials. rAAV vector has proven to be very useful vector for efficient and long-term gene transfer in a variety of tissues including lung, muscle, brain, spinal cord, retina and liver, thus the use of rAAV vectors holds great promise for human gene therapy. Its advantages observed in numerous disease paradigms, such as, the broad host range, low level of immune response, and longevity of gene expression has enabled the initiation of a number of clinical trials.

In the past, rAAV was most often generated through the co-transfection of rAAV vector plasmid and wild-type AAV helper plasmid into Ad-infected cells. Now Ad-infected cells is no longer necessary due to the improvements in AAV helper design as well as construction of non-infectious mini-Ad plasmid helper, which has improved the yield of rAAV per transfected cell in a crude lysate. Scalable methods of rAAV production have been developed too, which means that rAAV production will no longer rely on DNA transfection. More scale-up production of rAAV has become possible in some laboratories.