Individual Bacterial Analysis Report Form Inc: Fill & Download for Free

Scientists have been studying the relationships between pathogens and dementia in general, and between pathogens and Alzheimer's in particular, for decades. Various pathogens are known to cause progressive dementias (some of which may respond to treatment if caught early enough), such as AIDS Dementia Complex, syphilis, neurocysticercosis, herpes encephalitis, and human prion diseases.The possibility that pathogens might somehow cause Alzheimer's itself, and that by preventing or treating a specific infection, we can prevent or treat Alzheimer's, is a fascinating concept, and many studies have been conducted on individual pathogens suspected of having a relationship. The number of potential candidates has been growing by leaps and bounds in recent years. The viruses that have been linked to an increased risk of Alzheimer's include HSV1 / HSV2, Epstein-Barr, cytomegalovirus, hepatitis C, varicella zoster, and HHV-6A and HHV-7 viruses. Other types of infectious pathogens, including Helicobacter pylori, chronic osteomyelitis, Chlamydophila pneumoniae, many spirochetes such as Borrelia burgdorferi, protozoan parasites such as Toxoplasma gondii, various periodontal pathogens, and various yeasts and fungal (e.g., (Alternaria, Botrytis, Candida, Cladosporium, and Malassezia species) are associated with an increased risk of Alzheimer's. Researchers have developed theories about a wide range of possible mechanisms that might lead from infection to Alzheimer's, some specific to a given species, and some generic to broad classes of pathogens.In certain cases, a mountain of evidence has been produced that goes far beyond simple epidemiological studies. Some research has even gotten as far as preliminary clinical trials in humans that appeared to support the contention that treating the suspect pathogen reversed or prevented the dementia. However, efforts to reproduce these results have been unsuccessful, and the reasons are not understood.I assume your question comes from the recent flap over news releases from Cortexyme about their work on Porphyromonas gingivalis, one of the key bacteria in chronic gum disease, and the fact that they have an investigational new drug (IND) in clinical trials. See, e.g.:We may finally know what causes Alzheimer’s – and how to stop itCortexyme, IncAnd speaking of a mountain of evidence, that's exactly what Cortexyme and their collaborators have produced:Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitorsTry plowing through that before you've had your morning coffee!But...It's all supportive evidence, produced in mice (if you know anything about me, you know how I feel about mice), and in vitro solution and cell tissue studies. What happens in model test animals (especially mice) and in vitro experiments may have little or nothing to do with what happens in the human body. Moreover, a lot of the evidence relies on the specificity of certain antibodies (such as the anti-gingipain antibodies) used in the experiments, and while antibodies may appear to be specific when tested against a narrow range of potential analytes in a relatively uncomplicated medium, they could quite conceivably (often do) cross-react with many and diverse epitopes in a complicated milieu such as brain tissue.However, just for the sake of argument, let's suppose that the evidence is all representative of what would happen inside the human body, and the antibodies are highly specific, and so on and so forth. Does that mean they've discovered what causes Alzheimer's and a way to cure, or at least, prevent it?No. Because it doesn't fit in with so many other things that we know about Alzheimer's. In fact, it doesn't fit in with so many other things we know about periodontal disease.Periodontal disease represents a group of oral inflammatory infections initiated by oral pathogens which exist as a complex biofilms on the tooth surface and cause destruction to tooth supporting tissues. The severity of this disease ranges from mild and reversible inflammation of the gingiva (gingivitis) to chronic destruction of connective tissues, the formation of periodontal pocket and ultimately result in loss of teeth. It is now widely accepted that a myriad of bacteria -- not just a single microorganism -- are involved in periodontal diseases. The onset of periodontal tissue inflammation is triggered by the colonization of the subgingival region by periodontal bacteria. On the tooth surfaces, for example, early or primary colonizers are mainly streptococci and actinomyces. Over time, the proportions of these Gram-positive facultatively anaerobic bacteria decrease, and eventually Gram-negative anaerobes become more established, especially at the interface of the teeth and gums. Complex interactions between bacterial flora and the host defense mechanisms (including, possibly, the deficiency of certain immunological factors in the host) significantly influence the balance between bacterial aggression and host protection and determines whether periodontal breakdown occurs. In light of these criteria, a number of experimental findings have indicated that the primary etiological agents of periodontal diseases are generally Gram-negative rods, including Actinobacillus actinomycetemcomitans, Tannerella forsythia, Prevotella, Fusobacterium, and P. gingivalis. None of these microbial species is capable of causing the destructive events involved in the periodontal disease progression on its own; rather, the etiology requires a concerted interaction of these microorganisms to establish their niches in the oral cavity.Among major periodontal pathogens, P. gingivalis appears to be one of the prime etiological agents in the pathogenesis and progression of periodontal disease. However, while it is often found in subgingival plaque samples from patients with chronic periodontitis, it is not always present -- studies have shown that about 40–100% of adult periodontitis patients have been infected with P. gingivalis. Moreover, some strains of P. gingivalis are considered to be non-invasive, based on their inability to form abscesses in a mouse model; and it has been demonstrated that the invasive strains of P. gingivalis possesses more pathogenic activities than the non-invasive strains both in vitro and in vivo. Invasive P. gingivalis strains express many different virulence factors with a wide range of activities, not just gingipains. To date, numerous studies have been done to elucidate the mechanism of virulence compounds secreted by P. gingivalis and the cellular interactions with the host. Improved understanding of the these interactions at the molecular and cellular level, may ultimately have relevance to the overall well-being of the host. In recent years, the use of plant-derived natural compounds has gained more attention to attenuate the action of P. gingivalis. For example, quercetin, resveratrol, and its related compounds, catechin, epicatechin, orcinol, and 4-allylphenol, were found to exhibit an inhibitory effect on the activity of P. gingivalis fimbriae; and polyphenols of Myrothamnus flabellifolia were shown to reduce P. gingivalis adhesion and invasion up to about 50% by interacting with bacterial outer membrane proteins. This anti-adhesive effect is also accompanied by cytoprotective effects which relate to cytokine secretion.Porphyromonas gingivalis: An Overview of Periodontopathic Pathogen below the Gum LineThe virulence factors that Cortexyme is targeting for treating P. gingivalis are gingipains, i.e., cysteine proteases that are essential for P. gingivalis survival and pathogenicity, playing critical roles in host colonization, inactivation of host defenses, iron and nutrient acquisition, and tissue destruction. Gingipains have been shown to mediate the toxicity of P. gingivalis in endothelial cells, fibroblasts, and epithelial cells. Although P. gingivalis has a plethora of virulence factors, much of its pathogenicity is surprisingly related to the overall immunosuppression of the host. While gingipains can contribute to immunosuppression, many other P. gingivalis virulence factors play a wide range of roles in immunosuppression, as well.https://www.tandfonline.com/doi/full/10.3402/jom.v8.33029In addition, P. gingivalis is not alone in being responsible for disease development, neither in periodontal nor in systemic diseases or dementia. Although it can modulate the growth of other bacteria in the subgingival biofilm, periodontitis and its related systemic diseases are not the effect of a single bacterium. Each bacterium in a cluster with quorum sensing-like properties may affect the responses induced by others and vice versa. In fact, a "biofilm concept of Alzheimer's senile plaques" has been proposed as an alternative platform for answering the question on whether microbes are the causative agents of Alzheimer's.Can an Infection Hypothesis Explain the Beta Amyloid Hypothesis of Alzheimer’s Disease?Periodontitis, Microbiomes and their Role in Alzheimer’s DiseaseItzhaki and colleagues claim that HSV1 causes Alzheimer's by direct viral action as well as by virus-induced inflammation, and that the use of anti-viral medications to treat severe active HSV infections can decrease the risk of dementia. However, despite all the evidence they've produced so far, they have failed to persuade me that they have a solid case.Is Alzheimer's or dementia actually caused by a virus like HSV?Similarly, it makes me very squeamish that Cortexyme and colleagues are focusing on a specific pathogen as the cause of Alzheimer's, and a specific drug target that's apparently unique to that bacterial species (i.e., its gingipains) as the cure or prevention.The Alzheimer's brain harbors its own, and apparently diverse, microbiome. The diversity of microbes that have been documented in Alzheimer's brains could be a reflection of the brain donors' other microbiomes (mouth, skin, GI tract), co-morbid states, geographical location, age, diet, oral function (e.g., denture wearing), and lifestyle. Since age is the major risk factor for developing Alzheimer's, evolving microbiomes may provide dynamics of the microbial communities over time. Pathogens may enter the brain via diverse routes. Many viruses can enter the brain via olfactory neural pathways to the basal forebrain, a route also employed by C. pneumoniae, for example. Disruption of the blood-brain barrier (BBB) appears to be an early feature of Alzheimer's, which may facilitate pathogen entry into the brain and/or be caused by certain pathogens or by bacterial cell wall components such as lipopolysaccharide (LPS) or lipoteichoic acid from repeated peripheral bacterial infections (a feature of the elderly population). The BBB can also be damaged by viral infection (vaccinia or HSV-1) or by P. gingivalis. Other pathogens such as B. burgdorferi, C. pneumoniae, or Cryptococcus neoformans (C. neoformans) have found other ways to circumvent this barrier.Periodontal disease has long been established as a major risk factor for Alzheimer's, and many researchers have been actively investigating it. Many of those studies have found a great deal of diversity in the oral microbiome associated with periodontal disease that ends up in the Alzheimer's brain. Some common examples of bacteria in the periodontal microbiome include not only P. gingivalis, but also Tannerella forsythia, Prevotella intermedia, Eikenella corrodens, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, and T. denticola.In fact, some of the best-documented bacterial species associated with periodontal disease were not observed in a 2017 study using 16S ribosomal gene-specific Next generation sequencing (NGS) of extracted brain tissue. Before that study, evidence had relied on methods that require prior knowledge of which bacterial species to look for, and specific methods for detecting the presence of those species. This new study allowed the researchers to determine what actually is in the samples, without going after specific species -- and also enabled the detection of bacteria which cannot be cultured. The researchers noted that these "missing" species could be present at low copy numbers or in discreet areas not sampled cannot be discounted; further sampling and NGS-based experiments exploring more rRNA gene variable regions, different PCR conditions and systematic analysis of 16S DNA in different areas of the brain are required in order to provide a fuller assessment. There are other considerations also: for instance, the cohort assessed in this study was not selected based on periodontal or any other disease; future studies would require specific cohorts selected for the presence and absence of periodontal or other disease. Additionally, any infection which initiates the neuropathology of Alzheimer's may occur 15-20 years pre-mortem; therefore, the bacteria identified in the post-mortem Alzheimer's brain may be due to secondary infection after the blood-brain barrier (BBB) breakdown rather than being the causative agent. In addition, patient cohorts from different geographical regions may differ in their gut, mouth and brain microbiomes. Species vary between global regions and ethnic groups; for example, Spanish periodontitis patients are more likely to harbor oral P. gingivalis than in Netherlands, where A. actinomycetemcomitans is more evident. Furthermore, whereas up to 90% of North American samples of Alzheimer's brains contained C. pneumoniae, in another study from North European patients, C. pneumoniae could not be detected. Similarly, in the 2017 16S ribosomal NGS study, C. pneumoniae was completely absent, as were E. coli K99 and some other periodontal species previously associated with Alzheimer's (as well as some other non-oral bacteria commonly associated with Alzheimer's.)16S rRNA Next Generation Sequencing Analysis Shows Bacteria in Alzheimer’s Post-Mortem BrainSo. Many diverse pathogens have been implicated in Alzheimer's disease in epidemiological studies. Preliminary microbiome studies using unbiased next-generation sequencing have identified many fungi in Alzheimer's brains (Alternaria, Botrytis, Candida, Cladosporium, and Malassezia species) as well as the better studied viruses and bacteria. Many of these viruses, bacteria (or their LPS), fungi, and protozoa increase Aβ production in vitro or in vivo. Several of these pathogens also evoke tau phosphorylation.There are many passionate champions of individual pathogens as the single causative agent for Alzheimer's. Each has some supporting evidence, several have extensive supporting evidence ... but there are flaws or holes in their arguments. The diversity of viruses, bacteria, and fungi contributing to Alzheimer's, Aβ deposition, or dementia suggests a polymicrobial relationship with these conditions. Ergo, rather than a single pathogen being the cause of Alzheimer's, some researchers are beginning to study overall pathogen load as the cause. Some have started by studying viral load, and found that viral load -- rather than specific strains -- was tied to increased expression of components of the amyloid pathway in people with preclinical Alzheimer's. This fits with the related hypothesis that Aβ normally functions as an endogenous antimicrobial defense. Several researchers have reported that the peptide erects a physical barrier against invading bacteria and fungi, cocooning them in amyloid fibrils. Others have reported that Aβ40/42 can prevent HSV-1 entry into cells, and that Aβ can bind and agglutinate HSV-1 and HHV-6 in cells and in mice, preventing acute encephalitis.And this brings us to the "antimicrobial protection hypothesis of Alzheimer's." This is a very intriguing concept that seems to fit so many aspects of Alzheimer's, including the beta amyloid hypothesis, and the possible relationships between many and diverse pathogens and Alzheimer's pathology.Until recently, Aβ had been considered to be a functionless catabolic byproduct; and the pathways leading to Aβ generation were believed to be intrinsically pathological. Now Aβ has been identified as an antimicrobial peptide (AMP.) AMPs are the first line of defense against pathogens and act as potent broad-spectrum antibiotics and immunomodulators that target bacteria, mycobacteria, enveloped viruses, fungi, and protozoans, and in some cases, transformed or cancerous host cells. AMPs are widely expressed and are abundant in brain and other immunoprivileged tissues where actions of the adaptive immune system are constrained. Although AMPs are normally protective, AMP dysregulation can lead to host cell toxicity, chronic inflammation, and degenerative pathologies. Particularly germane to Aβ's role in Alzheimer's, AMPs are deposited as amyloid in several disorders, including senile seminal vesicle amyloid and isolated atrial amyloidosis, two of the most common human amyloidopathies.The protective role for Aβ in innate immunity employs a classic AMP mechanism. When pathogens get into the brain, they activate the production of soluble Aβ proteins which bind to microbial cell wall carbohydrates. Developing protofibrils inhibit pathogen adhesion to host cells. Propagating Aβ fibrils mediate agglutination and final entrapment of microbes. That network of fibers entombs the microbes so they can't infect the cell. It then forms a plaque. This new model posits that in Alzheimer's, normally protective antimicrobial pathways mediated by Aβ oligomerization are overactivated, either by real or incorrectly perceived infection. Ongoing Aβ deposition drives neuroinflammation, leading to neuropathology and widespread neuronal death. Consistent with this model, Salmonella infections of the brains of transgenic 5XFAD mice resulted in rapid seeding and accelerated Aβ deposition, which closely co-localized with the invading bacteria.Overall, the findings raise the intriguing possibility that Aβ may play a protective role in innate immunity. What might drive widespread Aβ deposition in Alzheimer's, however, remains unclear. Among sterile inflammatory diseases, dysregulated innate immune responses rather than infections are emerging as drivers of pathology. Notably, two of the three confirmed AMP amyloidopathies are not linked to obvious infections. However, a large body of data accrued over nearly a century suggests that genuine infection may also play a role in Alzheimer's etiology. The findings to date do not constitute direct evidence of a role for infection in Alzheimer's etiology. However, they do suggest a possible mechanism for pathogen-driven Aβ amyloidosis. The data also suggest the possibility that a range of microbial organisms may be able to induce Aβ deposition, a possible reason for why a single pathogen species has not yet been identified that is overwhelmingly associated with Alzheimer's.This model differs in several important ways from the "pathogen hypothesis" championed by researchers such as Itzhaki and those at Cortexyme. A key insight is that it's not direct killing of brain cells by specific strains of pathogens that causes Alzheimer's; rather, it's the body's innate immune response to the pathogens that leads to brain-damaging neuroinflammation. And while the "pathogen hypothesis" is typically offered as an alternative to the Aβ hypothesis, the "antimicrobial protection hypothesis" is not an alternative, but rather fits within the Aβ-tau-inflammation paradigm. It fills in blanks, offering an explanation for how the process starts and for the true nature of Aβ. Circumstantial evidence for the importance of Aβ is significant; it appears to have developed some 400 million years ago and has not only survived evolutionary pressures to appear in humans today, but is present in 60 percent of vertebrates, including fish, reptiles, and birds.So. How reliable are the new claims linking Alzheimer's disease with chronic gum disease?The concept that chronic gum disease is linked to Alzheimer's is not new, at all, and there may, in fact, be a causal relationship. To claim, however, that Porphyromonas gingivalis, which is one of the key bacteria in periodontic gum disease, is the sole cause of Alzheimer's, and that by targeting small molecule drugs against a tiny handful of its virulence factors, it may be possible to treat or prevent Alzheimer's, is going way out on a limb and, I think, isn't supported by the research literature.

IntroductionThe advancement in contemporary physics has considerably improved such that roughly between 1924 and 1930s, there was an entirely new theoretical approach to dynamics developed to account for sub-atomic behaviour. Quantum mechanics or wave mechanics was popularized with the works of Louis de Broglie who proposed that not only electromagnetic radiation but also that matter could have waves as well as particle aspect. The knowledge of this has great implication in understanding the workings of extra sensory perceptions especially on telepathy and psychokinesis. It is believed that there is really no difference between the radiation, wave or energy experienced by the works of experimental science and what we may call psychic science.Understanding of the Origin of Quantum PhysicsDemocritus of Abdera has been described by Erwin Schrödinger as one of the founders of Quantum mechanics. He was seen as the first quantum physicist not Max Plank as is traditionally known. He holds that all substances consisted of minute particles and that the different qualities of properties of substances were determined by different qualities of the atoms in them [1]. The earth from the position of Democritus and Leucippus emerged from the motion of atoms. Other atoms located outside the earth constitute what we know as the heavenly bodies. Man contained all conceivable types of atom and therefore "a microcosm of the universe" [2]. They went further to posit that properties of physical object like colour, smell, taste, solid, liquid and gaseous composition of the universe was explained by allotting different sizes and shapes to the atoms that constitute them.In trying to explicate the paranormal, metaphysical or things that appear supernatural, he posited that the finest, most perfect, mobile and volatile of all atoms form the souls of animals and man. "It is hardly an exaggeration to say that the theory of Democritus remained in essentials unchanged until the 19th century [3].The philosophy of atomism is significant in trying to understand the essence of this paper. Atomism resurfaced in the 17th century when Galileo revived it. He embraced atomism as a physical embodiment from the point of geometry. Newton’s introduced the notion of forces, especially the gravitational force into Mechanics. Attractive forces between the atoms were new ideas and very helpful in understanding how solid and liquid hold together and how new compounds are formed from simpler ones. Robert Boyle promoted the idea that there are different types of atoms known as elements and that matter consists of particles of corpuscles in motion. After John Dalton who posits that atoms can neither be created nor destroyed, J. J. Thomson’s research led to the discovery of the negatively charged electrons and the first idea of the structure of these indivisible atoms. With Ernest Rutherford’s research it showed that some of the atoms consists of a closely packed core of positive charge particles known as the nucleus at the centre and surrounded by negatively charged electrons. He also observed that the energy associated with radioactive atoms is enormous. With prophetic insight Rutherford wrote in 1903 "There is no reason to assume that this enormous store of energy is possessed by radio elements alone. It seems probable that atomic energy in general is of a similar high order of magnitude [4].Albert Einstein in the same model in the early part of the 20th century gave a new conception of the atom in his quantum theory, thus denying the indestructibility of atom. Quantum theory has its major origin in the works of Max Planck, a theoretical physicist. He undertook an experiment in heat radiation and came to the conclusion that atoms never emits energy smoothly and continuously, but only intermittently in definite quantities or pulse. This "Pulse" of energy he calls "Quanta" of energy or photons.Albert Einstein applied the same idea of Planck’s quantum theory in his analysis of the photoelectric effect. It was already known that when light falls on a metal surface it ejects electrons, but this phenomenon cannot be explained by classical philosophers like Democritus. The conclusion is that both light (All electromagnetic radiation) and matter behave either as particles or as waves depending on how we observe them.1.2.What Quantum Physics EntailsIt may be necessary for us to understand what quantum physics entails, bearing in mind that we are not all physicists. A better understanding would give us the opportunity to juxtapose and demystify some acts of extra sensory perceptions within what we have labeled ordinarily as paranormal acts.Quantum physics is the study or theory of how, what and why everything that makes up the universe as well as everything in it both within the confines of the seen and unseen are derived. It studies and tries to determine how everything in the cosmos came into being taking cognizance of its origin from the atomic and sub-atomic sphere. It is the study of the building blocks of the universe, an analysis of various forms of experiences and tracing theme back to molecules and energy. In fact quantum physics describes the universe as nothing more than vibrating strings of energy. This idea invariably posits that everything in the universe is made up of energy and vibration. This includes cells in our bodies and in the elements.1.3.The Metaphysics of Quantum PhysicsThe idea of quantum physics is laden with metaphysical overtone and if we view issues bordering on the paranormal, we also see that they are metaphysical also because there appears to be no clear explanation of the claimed experiences of issues of ESP such as telepathy, psychokinesis, etc. the claims made by the experts in paranormal studies have been seen as uncertain because it cannot be quantified.The uncertainty principle is the main theory in the physical science of quantum mechanics that explains the universe at atomic and sub-atomic levels. This principle emanated in order to explain how to measure the location of an electron around a nucleus. Werner Heisenberg in studying the works of Paul Dirac and Jordan discovered a problem with measurement of basic variables in the equations he was trying to solve. His analysis shows that uncertainties or imprecision always turned up if one tried to measure the position and the momentum of a particle at the same time. He concludes that these uncertainties or imprecision in the measurements are not the faults of the experimenter, but fundamental in nature inherent in quantum mechanics. In the eyes of the neophytes of nature and natural laws these uncertainties are supernatural, paranormal or metaphysical. Reasons for this possible conjecture is predicated on the fact that it is believed that natural sciences and its findings are not only empirically justifiable or quantifiable, but can also be predictable. In Quantum physics, the reverse appears to be the case because you cannot easily predict because the results are uncertain.Courtesy: Joan Solomon.The structure of matterIn juxtaposing religion, metaphysical experiences and paranormal acts as against empirical sciences, certain distinctive features are proposed. These features are that empirical scientific knowledge is accompanied with facts and their results are said to be certain. However, religion, or paranormal predictions or claims are laden with uncertainties and imprecision. The science of quantum physics exhibit uncertainties like that observed in religious circles. Its results are metaphysical because uncertainties or imprecision in measurements of variables are not the faults of the experimenter, they are beyond the scope of the scientist and cannot be predicted by any method or with any theoretical precise measurement. According to Paul levy, an enthusiast of the correlation of quantum physics and spirituality "Although Newtonian physics was and still is ‘partially correct’ from a strictly physical and finite material world view, quantum physics provides a much deeper, profound far more exiting and certainly more empowering understanding as to where all this ‘physical stuff’ (Matter is derived from" [5].The uncertainty principle which is a result of quantum theory shows that at a given time it may be possible to measure the position of an electron with a very high degree of accuracy and it is possible to measure its momentum (and hence velocity) with a very high degree of accuracy; but beyond a certain limit, it is not possible to measure both together with a very high degree of accuracy. It is often thought that this result implies a rejection of determinism; that is the principle that every event is fully determined by antecedent causes [6]. The issue bordering on quantum physics appears to be beyond physics or can be said to be metaphysical.1.4.Extra-Sensory Perception (ESP) and Unknown LawsThe knowledge of ESP may not be new among especially those who are critics or advocate of paranormal phenomena. However, we must confess that there are possibilities of those who are unenlightened on issues of extra-sensory perception. Our study and examples of ESP shall be limited to telepathy and psychokinesis.By telepathy we mean the ability to become aware of another person’s thought or feelings by means beyond the ordinary senses. The term was first used by an English Essayist and poet Fredric W. H. Myers in 1882, as "the communication of impressions of any kind from one mind to another independent of the recognized channels of sense [7]. Psychokinesis on the other hand is the ability to affect matter through a variety of mental force. Other terms that describe it include "remote mental influence, distant mental influences" or ‘mind over matter’. Thus, it can be seen as "… the power of the mind to influence matter directly at a distance without any transfer of physical energy" [8]. Records show that psychokinetic feats have been performed by many individuals, but the force or energy behind it has been unknown.We most times label extra-sensory perceptions paranormal. But the implication of the word paranormal may be misleading, for there is no reason to suppose that there is anything beyond the normal, in other words, outside the laws of nature about it, it is really normal and healthy people who seem to do best in these feats. According to Rosalind Heywood "The faculty is paranormal only in the sense that its cause is not perceptible to human senses or to their extensions, scientific instruments [9].Extra sensory perception (ESP) which in general parlance may be called the sixth sense was used by a parapsychologist J. B. Rhine to describe the reception of knowledge or information not gained through the recognized physical senses but the mind. The mind has been described as a stream of consciousness and simply man at the sub-atomic level. The mind has also been defined as a thinking thing. "As a man thinketh so he is". The mind is seen as the energy within man and is sometimes described as the soul. Democritus described the soul as a smote fine and volatile atom. While Aristotle goes on to affirm that the soul is a form or the "actuality of the body" [10]. The laws governing its workings are unknown that is why it falls within the confines of metaphysical discuss.ESP activities occurs independent of the sense of sight, hearing or other sensory process. Individuals who have extra sensory perception to a high degree are said to be psychic. There is the conception that everybody has ESP; however others think that it is for special individuals with greater abilities. Experiments relating to extra sensory perception as expressed by Rosalind Heywood and from our personal experiences show that "Man is a creature who can make contact with distant events by an unknown process, which does not invoke the use of sign or hearing or touch, taste, smell and which to some extent at least is independent of time. [11].The issue is that the simple understanding that such a faculty exist could lead to a revolution in thought about the nature of living creatures and their relation to their environment and the universe at large. The important point is that the laws which govern these phenomena are not yet known. The question of great philosophical or psychological interest is why is it not acclaimed a great discovery just like the uncertainly principle or the theory of relativity? The answer may be tied to the method of research which arose out of the debate over the issue of demarcating science from non-science or pseudo science.1.5.Dynamics of the Universe and Magnetic MemoriesThe illumination gained through quantum physics exposes us to an understanding of what the extra-sensory perception might look like. This knowledge draws our interest to the conviction that the large scale structure and dynamics of the universe are intimately bound up with life on earth and the structure of atoms.The Copenhagen interpretation of the issue of quantum physics reveals that energy is wave and at other experiment or interpretation of it, other probabilities occur. It shows that the thought and perceptions of the scientist doing the observation or experiment determined which one that manifest, either waves or particles. This implies that the sub-atomic particles (waves) being studied by the researcher respond to as particles (matter) based on the individual thought of the scientist who was studying it. By implication, the mode of thought or belief of the scientist or his perceptions determines the result of the research.The belief by most scientists that life on earth started with a single organism is predicated on the conception that we all have genetic connections of different degrees through this single organism. This may be likened to the monads of Leibnitz. The Greek word Monad means "Unity". Everything is made up of infinite number of monads. He however, denied the reality of matter. One monad always predominates. In the case of human beings the dominant monad is mind. There must be some relation between all the monads which make up the universe [12]. Man has genetic link with one another like a telephone exchange and this is possible because of what we have come to understand as magnetic memory from one person to the other. By this, we opined that all human life share a common branch point, this branch point is a long way in the past.Magnetic Memory is the memory and link due to the storage of data on a magnetized medium. It could be through genetics and wave properties of matter and mind. This medium is conjectured as responsible for the interconnectedness of things in the universe.The interconnectedness of man through magnetic memories may account for the possibility of telepathy, psychokinesis and other psychic phenomena. This is because our thought and being can be transmuted as particles of matter or in form of wave.Quantum physics and the theory of magnetic memories express that the whole of the universe or the entire cosmos is one, which is energy. Our physical body and thought forms are simply energies from different levels.1.6.Magnetic Memories and ESPIn this segment we hope to identify experiences observed in ESP and see its correlation to magnetic memories. Our interest in given examples of some of these extra sensory perceptions are due to the fact that we at various points in our lives have direct experiences of these phenomena. The illumination given by our knowledge of particle physics only helps us to understand why the assumed paranormal should be seen as normal or the perceived supernatural as natural.The science of telepathy and psychokinesis as a case study is perceived to be possible due to the activity of magnetic memory. This magnetic memory is responsible for the interconnectedness of thought waves or particles.There are multiple of cases of interconnectedness between men that makes it possible for us to believe that telepathic and psychokinetic rapour is a fact. For example, twins having some type of pain together, giving birth together or dying together. There was a special issue published in an Australian Magazine – Truth, as recounted by Percy Seymour "… twin sisters Helen and Peg, one night three quarters of an hour before midnight, Peg was killed in a car crash when the steering wheel penetrated her chest. At the same time Helen woke up screaming saying, she has a severe pain in her chest, on her way to the hospital, she died in the ambulance" [13]. This shows that there is interconnectedness between Helen and Peg despite their distance from one another. There is also a mysterious synchronism between twins that justify interconnectedness due to magnetic memory. A particular example recorded by Seymour is between identical twins. It occurred at 4.35 am on a Saturday in July 1948. At this time, Alice Lamba was reading in the palour of her home in Springfield, Illinois, U.S.A. when she suddenly felt a jolt on the left side of her body. This was followed by a feeling of shock and sharp stabbing pain in her side. She ten seems to be knocked off her chair by some unseen force and just before fainting, she said "Something has happened to Dianne" At that very instant, seventy miles away, Dianne her twin was travelling on a train that had just been derailed, and she had been thrown across the carriage. When she woke up in hospital¸ it was to find that she had suffered severe concussion and had fractured two ribs. Alice also complained of pains in her side, and when she was x-rayed it turned out that she had fractures just like her sister at the same two ribs [14].The author most times had telepathic experiences that amazed his friends, this occurs when he reveals the thoughts of these friends during conversations. They sometimes shiver and ask if he was a prophet. What actually took place was transference of thought waves or radiation from their minds to his mind because they were naturally interconnected and his mind was receptive.Many twins have telepathic link just as some individuals that are highly telepathic. There are many examples within our traditional communities that justify the issues of extra-sensory activities. What we wish to stress is that these activities are as natural as the laws in physics.Current research hypothesizes that every part of the body-mental, physical and emotional forms a continuous interconnected bio-energetic communication network. Like the uncertainty principle which we observe has reflected the preconceived idea of the scientists during experiment or research, health and ill health appear to reflect the mind set of individuals. There was a case recounted by J. O. Mume, a woman who gave birth to a male child and became mentally deranged. In addition to her mental problem were malaria fever, cold and headache. During medication on these symptoms, her constant complain was "I have a snake going up and down in my stomach trying to take my life". She wept and asked her doctor, "Doctor, can you remove this snake from my belly?"The doctor being conscious of the inherent powers of the mind in relation to belief and how it affects matter had to exploit his knowledge of psychology. He sent one of his assistants to get a snake dead or alive, this was done. He gave the patient herbal emetic to induce vomit. While she vomited, he smeared pepper into her eyes in order to blindfold her from observing his tricks. At this instance, he brought out the dead snake he concealed in a bottle and placed it among her vomit and asked her to open her eyes, only for her to see a dead snake in front of her. The doctor in addition, instantly shouted in an unusual voice "your trouble is now over, I have killed the snake that is going up and down in your stomach, and I have made you to vomit it [15].With this technique, the woman’s insanity disappeared and she was happy as she pointed at the snake telling her husband "I told you that there was a snake in my body".The lady was mentally ill because she had a belief that a snake was right inside her. Then she was tricked to believe again that the snake had been killed and she became well and sound. This shows that probably there is a clear interconnectedness between the mental and physical and that words have special effects like sound waves with certain vibration that affects the higher dimension of the physical human being.Mume’s experiments may have been successful as a psychological remedy or relief but we do not know if the healing was sustained or mere momentary relief. The question; however is if she gets to know that she was tricked, would she be able to contain the news and remain mentally sound? If not so, then it is the fact that the uncertainty principal and ESP phenomena are controlled by our thoughts.1.7.Human Intensions & PsychokinesisAn interesting area of ESP or psychic science is that of psychokinesis. Psychokinesis is the ability to affect matter through the activity of the mind. It is a term used to describe "ability of the mind to influence matter, time, space or energy without the use of any currently known type of physical means" An example of psychokinesis includes distorting or moving an object [16].Records show that psychokinetic feats have been performed by many individuals, but the force or energy behind it has been unknown. Notable claimants of psychokinetic abilities are Uri Geller, an Israeli, famous for his spoon-bending demonstrations. Nina Kulagina an alleged Soviet psychic of the late 1960s and early 1970s. Another is Felicia Parise, an American Medical Laboratory Technician who allegedly was able to repeatedly demonstrate psychokinetic movement of small objects beginning in the 1970s. In the first reported instance, it was spontaneous and then with practice by intense conscious intention she said, her inspiration for making the attempt was in viewing the black-and-white films of Nina Kulagina performing similar feat". [17] Another individual who developed this ability by viewing the films of Nina was Allan Vinogradovo of the Soviet Union who is said to have been tested successfully under controlled laboratory experiment.Number of researchers has conducted studies to determine whether mere human intention could affect the movement of moving object or affect matter directly. The author has watched with amazement unequal legs of a young lady due to a strange ailment and a man’s left hand shorten by an accident all brought and made equal by an intense conscious intension of a great healer in Warri, Nigeria on the 4th of March, 2016. Within few hours of the last feat, the same healer, with intense conscious intention was able to reverse positively a police officer’s tongue protruded out of his mouth for two years without solution.Extensive research has been conducted by John Hasted at the University of London’s Birkbeck College. He hypothesized that an unknown form of conduction of electrical charge from the subjects bodies through the atmosphere to the sensors of his equipment [18] Other experiments to this are that conscious intention can influence the growth and movement of biological studies. This was conducted by Dr. Carol B. Mash in which bacterial growth was psychokinetically accelerated and retarded according to the intentions of random selected college students.These researches raise many questions regarding the nature of energy or powers involved that appears to transcend experimental scientific explanation. Another penitent question could be, does quantum physics adequately explain ESP?2.Uncertainties in Quantum Physics and ESPThe question we need to ask is does the knowledge of quantum physics explain issues that involve Extra Sensory Perception? We have through the knowledge of particle physics encountered the notion of the uncertainty principle, a situation where imprecision always turned up if one tried to measure the position and the momentum of a particle at the same time. The conclusion made was that uncertainties and imprecision in the measurements are not the faults of the experimenter, but fundamental in nature inherent in quantum mechanics.How does this relate to ESP? The results of Extra-Sensory Perception are uncertain bearing in mind its method. The similarity within the understanding of quantum mechanics, the uncertainty principle with extra-sensory perception cannot be farfetched. The reason is that uncertainties border on each experiment. The researchers may not accurately determine inductively that today’s results would be as accurate as that of yesterday because imprecision always turned up. Research by John Hasted on ESP at the University of London’s Birbeck College hypothesized that an unknown form of conduction of electrical charge was observed through the atmosphere from the bodies of the subject. The statement "unknown form of conduction" indicates similarity with uncertainty in procedures and results.Albert Einstein’s E=Mc2 proved that all things broken down to their most basic form consist of the same stuff. What is this stuff-Pure energy? Energy makes up all things. Without a basic understanding of quantum physics, we may think that there are different energies such as energy that powers our homes, which make us move about, fuels our cars.The cosmos from the foregoing is just a form of universal interconnected energy which obeys the science of physics based on the principles of everything is energy. And energy follows thought as expressed in the uncertainty principle and extra sensory perception and that whatever man focuses on, he gets more of it. Our thoughts and feelings whether in the process of research or not send out magnetic field, and universal energy matches our alteration by giving us more of the same resonant energy. Just as the sub atomic particles took whatever form the scientist studying them, expected them to take based on their thoughts, events, conditions and circumstances that make up our reality take shape as we expect and believe they will. Intense conscious intention observed in faith healing and psychokinesisis is an expressions of energy in motion from one form to the other.Quantum Physics tells us that it is the act of observing an object (events, conditions and circumstances) that causes it to be there and the outcome is based only on how we observe it.Quantum physics appears to reconcile science and religion. It tends to say "As a man thinketh, so is he". If our thoughts can be telepathically transferred and psychokinetically move an object by intention, it implies that if we think negatively or have negative belief system about ourselves, we will manifest illness in our bodies. Thus, thought is energy. As we "believe so shall we receive. Within the religious parlance, we can say "Faith (positive thought energy) is the substance of things hoped for, the evidence of things not seen". Whatsoever ye desire when ye pray (thought in motion) believed that ye receive and it shall be given to you". If we can conceive and believe in the materialization of our hopes¸ dreams and desires, they already exist as a wave in the quantum field (which is infinite in nature) as a probability of existence only awaiting you to make them real.3.ConclusionThis research concludes, bearing in mind that our knowledge of Quantum Physics and uncertainty principle exposes that Extra-Sensory Perception (ESP) are due to natural laws expressed in better terms as the universe vibrating in strings of energy. That an understanding of this, will position us to know that our thoughts are not only energy, but exist as a wave in the quantum field as a probability of existence that can be controlled or channeled to accomplish a desire in the universe. It posits that the cosmos is a universal interconnected energy which obeys the law of physics and made earlier by inherent magnetic memories in the universe.This paper does not classify ESP phenomena as religious experiences with the conception that our belief might be replicated as it is backed by faith, but that the content of religious faith and belief expresses the theory of quantum physics and uncertainty principle. Our study here sees ESP as the melting point of science and religion.ReferencesV. Smilga, In the Search for Beauty. Moscow: Mir Publishers. 1970. Pp. 19-20.Stephen F. Mason A history of Science. New York: Collier Books. 1970. Pp. 33-34.W. K. C. Guthrie The Greek Philosophers: from Thales to Aristotle. London: Methuen &Company Ltd. 1976. P. 57.Joan Solomon. The Structure of Matter. England: David & Charles. 1973. P. 97.Paul Levy, Quantum Physics. /quantum-physics-the. Retrieved Now 2015Thomas Meitner (ed) The Penguin Dictionary of Philosophy. London: Penguin Books. 2000. P. 578.W. A. Redmond Telepathy. Microsoft Student. DVD Microsoft Co-operation, 2008.Rosalind Heywood. Beyond the Preach of Sense: An Inquiry into Extra Sensory Perception. New York: E. P. Dutton and Company, Inc. 1974. P. 162.Ibid, P. 15.Aristotle, On the Soul, Book 2. 412. a 15-28.Rosalind Heywood. Opcit P. 10Samuel Enoch Stumpf. Philosophy: History &Problems. New York: Mc Graw-Hill Inc. 1989. P. 256.Percy Seymour. The Paranormal: Beyond Sensory Science. London: Arkana Penguin Books. 1992. P. 132.Ibid. p. 130J. O. Mume "How I Acquired the knowledge of traditional medicine" in Traditional Healing: New Science or New Colonialism (Essay in critique of medical Anthropology). Philip Singer (ed) London: Couch Magazine Ltd. 1977. Pp. 153-156.Farlex Partner, Medical Dictionary: Psychakinesis. http//medical-dictionary. The free dictionary. copsychokinesis. Retrieved July 16, 2014.Mind over Matter (Volume of Mysteries of the Unknown Encyclopedia Series). New York: Time-Life Book. 1988. P. 27.The Roots of Consciousness: science, Psychokinesis.Armstrong, D. M. A Materialistic Theory of the Mind. London: Rutledge and Kegan Paul. 1968.Aristotle, On the Soul. Book 2. 412.Baker, R. Human Navigation and the Sixth Sense. Machester University Press. 1988.Barley, Cyril, The Greek Atomists and Epicurus. New York: Russel and Russel Inc. 1964.Bell, John S. Speakable and Unspeakable in Quantum Mechanics. Cambridge University Press. 1988.Broughton, Richard S. Parapsychology: The Controversial Science London: Ballantine Books. 1991.Berger, Arthur, S.; Berger, Joyce. The Encyclopedia of Parapsychological & Psychical Research. New York: Paragon House. 1991.Capra F., The Tao of Physics, New York: Fontara Press. 983Davies, P. W. C. Superstrings: A Theory for Everything. Cambridge University Press. 1988.Frazer, J. G. The Golden Bough: A Study in Magic and Religion. Abridged Edition, London: Macmillan & company. 1963.Guiley, Rosemary Ellen, Encyclopedia of the Strange, Mystical and Unexplained. New York: Grammercy Book. 1991.Guthrie, W. K. The Greek Philosophers: From Thales to Aristotle. London: Methuen & Company Ltd. 1976.Heywood, Rosalind, Beyond the Reach of Sense. New York EP, Dutton and Co. Inc. 1974.Inglis, Brain. The Paranormal: An Encyclopedia of Psychic Phenomena. London: Paladin. 1986.Mume J. O. "How I Acquired the Knowledge of traditional Medicine" in Traditional Healing New Science or New Colonialism (Essays in Critique of Medical Anthropology) Philip Singer (ed). London: Conch Magazine Ltd. 1977.Mason, F. Stephen A History of the Sciences; New York: Collier Books, 1979.Milton, Richard Forbidden Science: Suppressed Research that Could Change our Lives; London: Fourth Estate. 1994.Solomon, Joan The Structure of Matter: The Growth of Man’s Idea on the Nature of Matter. Newton Abbot Devon David & Charles. 1973.Seymour, Percy The Paranormal: Beyond Sensory Science, London: Arkana Penguin Books 1992.Seymour, Percy Cosmic Magnetism London & Adam Hilger, 1986.Smith, C. & Best S. Electromagnetic Man. Dento 1989.Talbot, M. Beyond the Quantum; New York, Bantam Books, 1988.

Hi there...Just go through this NEB article. It has beautiful explanation about the methods and uses of this technology.CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular BiologyThe development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects (6). Other recent approaches to targeted genome modification – zinc-finger nucleases [ZFNs, (7)] and transcription-activator like effector nucleases [TALENs (8)]– enable researchers to generate permanent mutations by introducing doublestranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.The Biology of Cas9The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coli (9), but their function wasn’t confirmed until 2007 by Barrangou and colleagues, who demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus (10).Three types of CRISPR mechanisms have been identified, of which type II is the most studied. In this case, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity (Figure 1) (11).Figure 1. Cas9 in vivo: Bacterial Adaptive ImmunityIn the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence. (Figure not drawn to scale.)One Cas protein, Cas9 (also known as Csn1), has been shown, through knockdown and rescue experiments to be a key player in certain CRISPR mechanisms (specifically type II CRISPR systems). The type II CRISPR mechanism is unique compared to other CRISPR systems, as only one Cas protein (Cas9) is required for gene silencing (12). In type II systems, Cas9 participates in the processing of crRNAs (12), and is responsible for the destruction of the target DNA (11). Cas9’s function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein (13).To achieve site-specific DNA recognition and cleavage, Cas9 must be complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA (11). The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9 (12).During the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double-stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript (11, 14). The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence, (2–5 nts) known as protospacer-associated motif (PAM), follows immediately 3´- of the crRNA complementary sequence (15). In fact, even fully complementary sequences are ignored by Cas9-RNA in the absence of a PAM sequence (16).Cas9 and CRISPR as a New Tool in Molecular BiologyThe simplicity of the type II CRISPR nuclease, with only three required components (Cas9 along with the crRNA and trRNA) makes this system amenable to adaptation for genome editing. This potential was realized in 2012 by the Doudna and Charpentier labs (11). Based on the type II CRISPR system described previously, the authors developed a simplified two-component system by combining trRNA and crRNA into a single synthetic single guide RNA (sgRNA). sgRNAprogrammed Cas9 was shown to be as effective as Cas9 programmed with separate trRNA and crRNA in guiding targeted gene alterations (Figure 2A).To date, three different variants of the Cas9 nuclease have been adopted in genome-editing protocols. The first is wild-type Cas9, which can site-specifically cleave double-stranded DNA, resulting in the activation of the doublestrand break (DSB) repair machinery. DSBs can be repaired by the cellular Non-Homologous End Joining (NHEJ) pathway (17), resulting in insertions and/or deletions (indels) which disrupt the targeted locus. Alternatively, if a donor template with homology to the targeted locus is supplied, the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise replacement mutations to be made (Figure 2A) (17, 18).Cong and colleagues (1) took the Cas9 system a step further towards increased precision by developing a mutant form, known as Cas9D10A, with only nickase activity. This means it cleaves only one DNA strand, and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only, resulting in reduced indel mutations (1, 11, 19). Cas9D10A is even more appealing in terms of target specificity when loci are targeted by paired Cas9 complexes designed to generate adjacent DNA nicks (20) (see further details about “paired nickases” in Figure 2B).The third variant is a nuclease-deficient Cas9 (dCas9, Figure 2C) (21). Mutations H840A in the HNH domain and D10A in the RuvC domain inactivate cleavage activity, but do not prevent DNA binding (11, 22). Therefore, this variant can be used to sequence-specifically target any region of the genome without cleavage. Instead, by fusing with various effector domains, dCas9 can be used either as a gene silencing or activation tool (21, 23–26). Furthermore, it can be used as a visualization tool. For instance, Chen and colleagues used dCas9 fused to Enhanced Green Fluorescent Protein (EGFP) to visualize repetitive DNA sequences with a single sgRNA or nonrepetitive loci using multiple sgRNAs (27).Figure 2. CRISPR/Cas9 System ApplicationsA. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery. In the absence of a homologous repair template non-homologous end joining can result in indels disrupting the target sequence. Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway.B. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered double-stranded break which can then undergo homology directed repair.C. Nuclease-deficient Cas9 can be fused with various effector domains allowing specific localization. For example, transcriptional activators, repressors, and fluorescent proteins.Targeting Efficiency and Off-target MutationsTargeting efficiency, or the percentage of desired mutation achieved, is one of the most important parameters by which to assess a genome-editing tool. The targeting efficiency of Cas9 compares favorably with more established methods, such as TALENs or ZFNs (8). For example, in human cells, custom-designed ZFNs and TALENs could only achieve efficiencies ranging from 1% to 50% (29–31). In contrast, the Cas9 system has been reported to have efficiencies up to >70% in zebrafish (32) and plants (33), and ranging from 2–5% in induced pluripotent stem cells (34). In addition, Zhou and colleagues were able to improve genome targeting up to 78% in one-cell mouse embryos, and achieved effective germline transmission through the use of dual sgRNAs to simultaneously target an individual gene (35).A widely used method to identify mutations is the T7 Endonuclease I mutation detection assay (36, 37) (Figure 3). This assay detects heteroduplex DNA that results from the annealing of a DNA strand, including desired mutations, with a wildtype DNA strand (37).Figure 3. T7 Endonuclease I Targeting Efficiency AssayGenomic DNA is amplified with primers bracketing the modified locus. PCR products are then denatured and re-annealed yielding 3 possible structures. Duplexes containing a mismatch are digested by T7 Endonuclease I. The DNA is then electrophoretically separated and fragment analysis is used to calculate targeting efficiency.Another important parameter is the incidence of off-target mutations. Such mutations are likely to appear in sites that have differences of only a few nucleotides compared to the original sequence, as long as they are adjacent to a PAM sequence. This occurs as Cas9 can tolerate up to 5 base mismatches within the protospacer region (36) or a single base difference in the PAM sequence (38). Off-target mutations are generally more difficult to detect, requiring whole-genome sequencing to rule them out completely.Recent improvements to the CRISPR system for reducing off-target mutations have been made through the use of truncated gRNA (truncated within the crRNA-derived sequence) or by adding two extra guanine (G) nucleotides to the 5´ end (28, 37). Another way researchers have attempted to minimize off-target effects is with the use of “paired nickases” (20). This strategy uses D10A Cas9 and two sgRNAs complementary to the adjacent area on opposite strands of the target site (Figure 2B). While this induces DSBs in the target DNA, it is expected to create only single nicks in off-target locations and, therefore, result in minimal off-target mutations.By leveraging computation to reduce off-target mutations, several groups have developed webbased tools to facilitate the identification of potential CRISPR target sites and assess their potential for off-target cleavage. Examples include the CRISPR Design Tool (38) and the ZiFiT Targeter, Version 4.2 (39, 40).Applications as a Genome-editing and Genome Targeting ToolFollowing its initial demonstration in 2012 (9), the CRISPR/Cas9 system has been widely adopted. This has already been successfully used to target important genes in many cell lines and organisms, including human (34), bacteria (41), zebrafish (32), C. elegans (42), plants (34), Xenopus tropicalis (43), yeast (44), Drosophila(45), monkeys (46), rabbits (47), pigs (42), rats (48) and mice (49). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA (14, 21, 29). Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations (50). A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation (26, 51, 52), epigenetic modification (25), and microscopic visualization of specific genome loci (27).The CRISPR/Cas9 system requires only the redesign of the crRNA to change target specificity. This contrasts with other genome editing tools, including zinc finger and TALENs, where redesign of the protein-DNA interface is required. Furthermore, CRISPR/Cas9 enables rapid genome-wide interrogation of gene function by generating large gRNA libraries (51, 53) for genomic screening.The future of CRISPR/Cas9The rapid progress in developing Cas9 into a set of tools for cell and molecular biology research has been remarkable, likely due to the simplicity, high efficiency and versatility of the system. Of the designer nuclease systems currently available for precision genome engineering, the CRISPR/Cas system is by far the most user friendly. It is now also clear that Cas9’s potential reaches beyond DNA cleavage, and its usefulness for genome locus-specific recruitment of proteins will likely only be limited by our imagination.ReferencesCong L., et al. (2013) Science, 339, 819–823.Capecchi, M.R. (2005) Nat. Rev. Genet. 6, 507–512.Fire, A., et al. (1998) Nature, 391, 806–811.Elbashir, http://S.Mm, et al. (2002) Methods, 26, 199–213.Martinez, J., et al. (2003) Nucleic Acids Res. Suppl. 333.Alic, N, et al. (2012) PLoS One, 7, e45367.Miller, J., et al. (2005) Mol. Ther. 11, S35–S35.Mussolino, C., et al. (2011) Nucleic Acids Res. 39, 9283–9293.Ishino, Y., et al. (1987) J. Bacteriol. 169, 5429–5433.Barrangou, R., et al. (2007). Science, 315, 1709–1712.Jinek, M., et al. (2012) Science, 337, 816–821.Deltcheva, E., et al. (2011) Nature, 471, 602–607.Sapranauskas, R., et al. (2011) Nucleic Acids Res. 39, 9275–9282.Nishimasu, H., et al. (2014) Cell, doi:10.1016/j.cell.2014.02.001Swarts, D.C., et al. (2012) PLoS One, 7:e35888.Sternberg, S.H., et al. (2014) Nature, doi:10.1038/nature13011.Overballe-Petersen, S., et al. (2013) Proc. Natl. Acad. Sci. U.S.A. 110,19860–19865.Gong, C., et al. (2005) Nat. Struct. Mol. Biol. 12, 304–312.Davis, L., Maizels, N. (2014) Proc. Natl. Acad. Sci. U S A, 111, E924–932.Ran, F.A., et al. (2013) Cell, 154, 1380–1389.Qi, L.S., et al. (2013) Cell, 152, 1173–1183.Gasiunas, G., et al. (2012) Proc. Natl. Acad. Sci. U S A, 109, E2579–2586.Maede, M.L., et al. (2013) Nat. Methods, 10, 977–979.Gilbert, L.A., et al. (2013) Cell, 154, 442–451.Hu, J., et al. (2014) Nucleic Acids Res. doi:10.1093/nar/gku109.Perez-Pinera, P., et al. (2013) Nat. Methods, 10, 239–242.Chen, B., et al. (2013) Cell, 155, 1479–1491.Seung, W., et al. (2014) Genome Res. 24, 132–141.Miller, J.C., et al. (2011). Nat. Biotechnol. 29, 143–148.Mussolino, C., et al. (2011). Nucleic Acids Res. 39, 9283–9293.Maeder, M.L., et al. (2008) Mol. Cell, 31, 294–301.Hwang, W.Y., et al. (2013) PLoS One, 8:e68708.Feng, Z., et al. (2013) Cell Res. 23, 1229–1232.Mali, P., et al. (2013) Science, 339, 823–826.Zhou, J., et al. (2014) FEBS J. doi:10.1111/febs.12735.Fu, Y., et al. (2013) Nat. Biotechnol. 31, 822–826.Fu, Y., et al. (2014) Nat Biotechnol. doi: 10.1038/nbt.2808.Hsu, P.D., et al. (2013) Nat. Biotechnol. 31, 827–832.Sander, J.D., et al. (2007) Nucleic Acids Res. 35, W599-605.Sander, J.D., et al (2010) Nucleic Acids Res. 38, W462–468.Fabre, L., et al. (2014) PLoS Negl. Trop. Dis. 8:e2671.Hai, T., et al. (2014) Cell Res. doi: 10.1038/cr.2014.11.Guo, X., et al. (2014) Development, 141, 707–714.DiCarlo, J.E., et al. (2013) Nucleic Acids Res. 41, 4336–4343.Gratz, S.J., et al. (2014) Genetics, doi:10.1534/genetics.113.160713.Niu, Y., et al. (2014) Cell, 156, 836–843.Yang, D., et al. (2014) J. Mol. Cell Biol. 6, 97-99.Ma, Y., et al. (2014) Cell Res. 24, 122–125.Mashiko, D., et al. (2014) Dev. Growth Differ. 56, 122–129.Gratz, S.J., et al. (2013) Fly, 249.Mali, P., et al. (2013) Nat. Biotechnol. 31, 833–838.Cheng, A.W., et al. (2013) Cell Res. 23, 1163–1171.Koike-Yusa, H., et al. (2013) Nat. Biotechnol. doi: 10.1038/nbt.2800.Sander, J.D., and Joung, J.K. (2014) Nat Biotechnol. doi:10.1038/nbt.2842.From NEB expressions Issue I, 2014Article by Alex Reis, Ph.D., Bitesize BioBreton Hornblower, Ph.D., Brett Robb, Ph.D. and George Tzertzinis, Ph.D., New EnglandBiolabs, Inc.Source- CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular BiologyYou would also like to see- http://www.nature.com/nbt/journal/v32/n4/full/nbt.2842.htmlHope it helps