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159-614: More technically, Cas9 is a RNA -guided DNA endonuclease enzyme associated with the Clustered Regularly Interspaced Short Palindromic Repeats ( CRISPR ) adaptive immune system in Streptococcus pyogenes . S. pyogenes utilizes CRISPR to memorize and Cas9 to later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking for sites complementary to

318-484: A 1993 Nobel to Philip Sharp and Richard Roberts . Catalytic RNA molecules ( ribozymes ) were discovered in the early 1980s, leading to a 1989 Nobel award to Thomas Cech and Sidney Altman . In 1990, it was found in Petunia that introduced genes can silence similar genes of the plant's own, now known to be a result of RNA interference . At about the same time, 22 nt long RNAs, now called microRNAs , were found to have

477-455: A 40% lifetime risk of death from cirrhosis or hepatocellular carcinoma . Of those infected between the age of one to six, 70% will clear the infection. Hepatitis D (HDV) can occur only with a concomitant hepatitis B infection, because HDV uses the HBV surface antigen to form a capsid . Co-infection with hepatitis D increases the risk of liver cirrhosis and liver cancer. Polyarteritis nodosa

636-479: A Cas9 to edit their genome. This is because they can attach foreign DNA, that does not affect them, into their genome. Another way that these cells defy Cas9 is by process of restriction modification (RM) system. When a bacteriophage enters a bacteria or archaea cell it is targeted by the RM system. The RM system then cuts the bacteriophages DNA into separate pieces by restriction enzymes and uses endonucleases to further destroy

795-496: A RuvC-like nuclease domain. These HNH and RuvC-like nuclease domains are responsible for cleavage of the complementary/target and non-complementary/non-target DNA strands, respectively. Despite low sequence similarity, the sequence similar to RNase H has a RuvC fold (one member of RNase H family) and the HNH region folds as T4 Endo VII (one member of HNH endonuclease family). Wild-type S. pyogenes Cas9 requires magnesium (Mg) cofactors for

954-425: A certain amount of time, the message degrades into its component nucleotides with the assistance of ribonucleases . Transfer RNA (tRNA) is a small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites for amino acid attachment and an anticodon region for codon recognition that binds to

1113-485: A classical example of an epidemiological study, proved that contaminated lymph was the source of the outbreak. Later, numerous similar outbreaks were reported following the introduction, in 1909, of hypodermic needles that were used, and, more importantly, reused, for administering Salvarsan for the treatment of syphilis . The largest recorded outbreak of hepatitis B was the infection of up to 330,000 American soldiers during World War II. The outbreak has been blamed on

1272-578: A day of birth. The hepatitis B vaccine was the first vaccine capable of preventing cancer, specifically liver cancer. Most vaccines are given in three doses over a course of days. A protective response to the vaccine is defined as an anti-HBs antibody concentration of at least 10 mIU/ml in the recipient's serum. The vaccine is more effective in children and 95 percent of those vaccinated have protective levels of antibody. This drops to around 90% at 40 years of age and to around 75 percent in those over 60 years. The protection afforded by vaccination

1431-413: A defense system, all three phases must be functional. Stage 1: CRISPR spacer integration. Protospacers and protospacer-associated motifs (shown in red) are acquired at the "leader" end of a CRISPR array in the host DNA. The CRISPR array is composed of spacer sequences (shown in colored boxes) flanked by repeats (black diamonds). This process requires Cas1 and Cas2 (and Cas9 in type II), which are encoded in

1590-448: A general programmable DNA binding RNA-Protein complex. The interaction of dCas9 with target dsDNA is so tight that high molarity urea protein denaturant can not fully dissociate the dCas9 RNA-protein complex from dsDNA target. dCas9 has been targeted with engineered single guide RNAs to transcription initiation sites of any loci where dCas9 can compete with RNA polymerase at promoters to halt transcription. Also, dCas9 can be targeted to

1749-476: A guide RNA composed of two disparate RNAs that associate – the CRISPR RNA (crRNA), and the trans-activating crRNA ( tracrRNA ). Cas9 targeting has been simplified through the engineering of a chimeric single guide RNA (chiRNA). Scientists have suggested that Cas9-based gene drives may be capable of editing the genomes of entire populations of organisms. In 2015, Cas9 was used to modify the genome of human embryos for

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1908-419: A higher risk. Immunosuppressive drugs favor increased HBV replication while inhibiting cytotoxic T cell function in the liver. The risk of reactivation varies depending on the serological profile; those with detectable HBsAg in their blood are at the greatest risk, but those with only antibodies to the core antigen are also at risk. The presence of antibodies to the surface antigen, which are considered to be

2067-485: A marker of immunity, does not preclude reactivation. Treatment with prophylactic antiviral drugs can prevent the serious morbidity associated with HBV disease reactivation. At least 296 million people, or 3.8% of the world's population, had chronic HBV infection as of 2019. Another 1.5 million cases of acute HBV infection also occurred that year. Regional prevalences across the globe range from around 7.5% in Africa to 0.5% in

2226-596: A more recent origin for all HBV genotypes. The evolution of HBV in humans was shown to reflect known events of human history such as the first peopling of the Americas during the late Pleistocene and the Neolithic transition in Europe. Ancient DNA studies have also showed that some ancient hepatitis viral strains still infect humans, while other strains became extinct. The earliest record of an epidemic caused by hepatitis B virus

2385-682: A negative charge each, making RNA a charged molecule (polyanion). The bases form hydrogen bonds between cytosine and guanine, between adenine and uracil and between guanine and uracil. However, other interactions are possible, such as a group of adenine bases binding to each other in a bulge, or the GNRA tetraloop that has a guanine–adenine base-pair. The chemical structure of RNA is very similar to that of DNA , but differs in three primary ways: Like DNA, most biologically active RNAs, including mRNA , tRNA , rRNA , snRNAs , and other non-coding RNAs , contain self-complementary sequences that allow parts of

2544-494: A normal immune system who were vaccinated. Only rare chronic infections have been documented. Vaccination is particularly recommended for high risk groups including: health workers, people with chronic kidney failure , and men who have sex with men. Both types of the hepatitis B vaccine, the plasma-derived vaccine (PDV) and the recombinant vaccine (RV) are of similar effectiveness in preventing infection in both healthcare workers and chronic kidney failure groups. One difference

2703-644: A nucleoprotein called a ribosome. The ribosome binds mRNA and carries out protein synthesis. Several ribosomes may be attached to a single mRNA at any time. Nearly all the RNA found in a typical eukaryotic cell is rRNA. Transfer-messenger RNA (tmRNA) is found in many bacteria and plastids . It tags proteins encoded by mRNAs that lack stop codons for degradation and prevents the ribosome from stalling. The earliest known regulators of gene expression were proteins known as repressors and activators – regulators with specific short binding sites within enhancer regions near

2862-547: A nucleus, also contain nucleic acids. The role of RNA in protein synthesis was suspected already in 1939. Severo Ochoa won the 1959 Nobel Prize in Medicine (shared with Arthur Kornberg ) after he discovered an enzyme that can synthesize RNA in the laboratory. However, the enzyme discovered by Ochoa ( polynucleotide phosphorylase ) was later shown to be responsible for RNA degradation, not RNA synthesis. In 1956 Alex Rich and David Davies hybridized two separate strands of RNA to form

3021-544: A number of RNA-dependent RNA polymerases that use RNA as their template for synthesis of a new strand of RNA. For instance, a number of RNA viruses (such as poliovirus) use this type of enzyme to replicate their genetic material. Also, RNA-dependent RNA polymerase is part of the RNA interference pathway in many organisms. Many RNAs are involved in modifying other RNAs. Introns are spliced out of pre-mRNA by spliceosomes , which contain several small nuclear RNAs (snRNA), or

3180-457: A pathogen and determine which molecular parts to extract, inactivate, and use in a vaccine. Small molecules with conventional therapeutic properties can target RNA and DNA structures, thereby treating novel diseases. However, research is scarce on small molecules targeting RNA and approved drugs for human illness. Ribavirin, branaplam, and ataluren are currently available medications that stabilize double-stranded RNA structures and control splicing in

3339-409: A plant gene. This happens when dCAS9 binds to repressor domains, and in the case of the plants, deactivation of a regulatory gene such as AtCSTF64, does occur. Bacteria are another focus of the usage of dCas9 proteins as well. Since eukaryotes have a larger DNA makeup and genome; the much smaller bacteria are easy to manipulate. As a result, eukaryotes use dCas9 to inhibit RNA polymerase from continuing

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3498-425: A rapid onset of sickness with nausea , vomiting , yellowish skin , fatigue , dark urine, and abdominal pain . Symptoms during acute infection typically last for a few weeks, though some people may feel sick for up to six months. Deaths resulting from acute stage HBV infections are rare. An HBV infection lasting longer than six months is usually considered chronic. The likelihood of developing chronic hepatitis B

3657-640: A result. The infection may be entirely asymptomatic and may go unrecognized. Chronic infection with hepatitis B virus may be asymptomatic or may be associated with chronic inflammation of the liver (chronic hepatitis), leading to cirrhosis over a period of several years. This type of infection dramatically increases the incidence of hepatocellular carcinoma (HCC; liver cancer). Across Europe, hepatitis B and C cause approximately 50% of hepatocellular carcinomas. Chronic carriers are encouraged to avoid consuming alcohol as it increases their risk for cirrhosis and liver cancer. Hepatitis B virus has been linked to

3816-468: A role in the development of C. elegans . Studies on RNA interference earned a Nobel Prize for Andrew Fire and Craig Mello in 2006, and another Nobel for studies on the transcription of RNA to Roger Kornberg in the same year. The discovery of gene regulatory RNAs has led to attempts to develop drugs made of RNA, such as siRNA , to silence genes. Adding to the Nobel prizes for research on RNA, in 2009 it

3975-417: A role in the activation of the innate immune system against viral infections. In the late 1970s, it was shown that there is a single stranded covalently closed, i.e. circular form of RNA expressed throughout the animal and plant kingdom (see circRNA ). circRNAs are thought to arise via a "back-splice" reaction where the spliceosome joins a upstream 3' acceptor to a downstream 5' donor splice site. So far

4134-462: A sequence specific guide RNA molecule. Since dCas9 appears to down regulate gene expression, this action is amplified even more when it is used in conjunction with repressive chromatin modifier domains. The dCas9 protein has other functions outside of the regulation of gene expression. A promoter can be added to the dCas9 protein which allows them to work with each other to become efficient at beginning or stopping transcription at different sequences along

4293-423: A significant number of cases in the 1980s; however, this has become less common with improved sterilization. The hepatitis B viruses cannot be spread by holding hands, sharing eating utensils, kissing, hugging, coughing, sneezing, or breastfeeding. The infection can be diagnosed 30 to 60 days after exposure. The diagnosis is usually confirmed by testing the blood for parts of the virus and for antibodies against

4452-455: A specific sequence on the messenger RNA chain through hydrogen bonding. Ribosomal RNA (rRNA) is the catalytic component of the ribosomes. The rRNA is the component of the ribosome that hosts translation. Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA. Three of the rRNA molecules are synthesized in the nucleolus , and one is synthesized elsewhere. In the cytoplasm, ribosomal RNA and protein combine to form

4611-431: A specific spatial tertiary structure . The scaffold for this structure is provided by secondary structural elements that are hydrogen bonds within the molecule. This leads to several recognizable "domains" of secondary structure like hairpin loops , bulges, and internal loops . In order to create, i.e., design, RNA for any given secondary structure, two or three bases would not be enough, but four bases are enough. This

4770-411: A strand of DNA. These two proteins are specific in where they act on a gene. This is prevalent in certain types of prokaryotes when a promoter and dCas9 align themselves together to impede the ability of elongation of polymer of nucleotides coming together to form a transcribed piece of DNA. Without the promoter, the dCas9 protein does not have the same effect by itself or with a gene body. When examining

4929-459: A template for transcription of four viral mRNAs by host RNA polymerase. The largest mRNA, (which is longer than the viral genome), is used to make the new copies of the genome and to make the capsid core protein and the viral DNA polymerase . These four viral transcripts undergo additional processing and go on to form progeny virions that are released from the cell or returned to the nucleus and re-cycled to produce even more copies. The long mRNA

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5088-809: A variety of disorders. Protein-coding mRNAs have emerged as new therapeutic candidates, with RNA replacement being particularly beneficial for brief but torrential protein expression. In vitro transcribed mRNAs (IVT-mRNA) have been used to deliver proteins for bone regeneration, pluripotency, and heart function in animal models. SiRNAs, short RNA molecules, play a crucial role in innate defense against viruses and chromatin structure. They can be artificially introduced to silence specific genes, making them valuable for gene function studies, therapeutic target validation, and drug development. mRNA vaccines have emerged as an important new class of vaccines, using mRNA to manufacture proteins which provoke an immune response. Their first successful large-scale application came in

5247-458: A variety of modulatory protein domains to carry out a myriad of functions. Recently, dCas9 has been fused to chromatin remodeling proteins (HDACs/HATs) to reorganize the chromatin structure around various loci. This is important in targeting various eukaryotic genes of interest as heterochromatin structures hinder Cas9 binding. Furthermore, because Cas9 can react to heterochromatin , it is theorized that this enzyme can be further applied to studying

5406-532: A yellow fever vaccine made with contaminated human blood serum, and after receiving the vaccinations about 50,000 soldiers developed jaundice. The virus was not discovered until 1966 when Baruch Blumberg , then working at the National Institutes of Health (NIH), discovered the Australia antigen (later known to be hepatitis B surface antigen, or HBsAg) in the blood of Aboriginal Australian people. Although

5565-402: Is protein synthesis , a universal function in which RNA molecules direct the synthesis of proteins on ribosomes . This process uses transfer RNA ( tRNA ) molecules to deliver amino acids to the ribosome , where ribosomal RNA ( rRNA ) then links amino acids together to form coded proteins. It has become widely accepted in science that early in the history of life on Earth , prior to

5724-408: Is 50 to 100 times more infectious than human immunodeficiency virus (HIV) . HBV can be transmitted through several routes of infection. In vertical transmission , HBV is passed from mother to child (MTCT) during childbirth. Without intervention, a mother who is positive for HBsAg has a 20% risk of passing the infection to her offspring at the time of birth. This risk is as high as 90% if the mother

5883-510: Is a polymeric molecule that is essential for most biological functions, either by performing the function itself ( non-coding RNA ) or by forming a template for the production of proteins ( messenger RNA ). RNA and deoxyribonucleic acid (DNA) are nucleic acids . The nucleic acids constitute one of the four major macromolecules essential for all known forms of life . RNA is assembled as a chain of nucleotides . Cellular organisms use messenger RNA ( mRNA ) to convey genetic information (using

6042-423: Is a ribozyme . Each nucleotide in RNA contains a ribose sugar, with carbons numbered 1' through 5'. A base is attached to the 1' position, in general, adenine (A), cytosine (C), guanine (G), or uracil (U). Adenine and guanine are purines , and cytosine and uracil are pyrimidines . A phosphate group is attached to the 3' position of one ribose and the 5' position of the next. The phosphate groups have

6201-459: Is a member of the hepadnavirus family . The virus particle ( virion ) consists of an outer lipid envelope and an icosahedral nucleocapsid core composed of core protein . These virions are 30–42 nm in diameter. The nucleocapsid encloses the viral DNA and a DNA polymerase that has reverse transcriptase activity. The outer envelope contains embedded proteins that are involved in viral binding of, and entry into, susceptible cells. The virus

6360-722: Is also moderate, with studies placing India's infection rate between 2-4%. Countries with low HBV prevalence include Australia (0.9%), those in the WHO European Region (which average 1.5%), and most countries in North and South America (which average 0.28%). In the United States, an estimated 0.26% of the population was living with HBV infection as of 2018. Findings of HBV DNA in ancient human remains have shown that HBV has infected humans for at least ten millennia, both in Eurasia and in

6519-532: Is also positive for HBeAg . Early life horizontal transmission can occur through bites, lesions, certain sanitary habits, or other contact with secretions or saliva containing HBV. Adult horizontal transmission is known to occur through sexual contact , blood transfusions and transfusion with other human blood products, re-use of contaminated needles and syringes. Breastfeeding after proper immunoprophylaxis does not appear to contribute to mother-to-child-transmission (MTCT) of HBV. Hepatitis B virus (HBV)

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6678-414: Is associated with acute viral hepatitis , an illness that begins with general ill-health, loss of appetite, nausea, vomiting, body aches, mild fever, and dark urine, and then progresses to development of jaundice . The illness lasts for a few weeks and then gradually improves in most affected people. A few people may have a more severe form of liver disease known as fulminant hepatic failure and may die as

6837-627: Is becoming a prominent tool in the field of genome editing. Cas9 has gained traction in recent years because it can cleave nearly any sequence complementary to the guide RNA. Because the target specificity of Cas9 stems from the guide RNA:DNA complementarity and not modifications to the protein itself (like TALENs and zinc fingers ), engineering Cas9 to target new DNA is straightforward. Versions of Cas9 that bind but do not cleave cognate DNA can be used to locate transcriptional activator or repressors to specific DNA sequences in order to control transcriptional activation and repression. Native Cas9 requires

6996-495: Is called enhancer RNAs .  It is not clear at present whether they are a unique category of RNAs of various lengths or constitute a distinct subset of lncRNAs.  In any case, they are transcribed from enhancers , which are known regulatory sites in the DNA near genes they regulate.  They up-regulate the transcription of the gene(s) under control of the enhancer from which they are transcribed. At first, regulatory RNA

7155-401: Is higher for those who are infected with HBV at a younger age. About 90% of those infected during or shortly after birth develop chronic hepatitis B, while less than 10% of those infected after the age of five develop chronic cases. Most of those with chronic disease have no symptoms; however, cirrhosis and liver cancer eventually develop in about 25% of those with chronic HBV. The virus

7314-479: Is initiated and mediated by the CTLs, antigen -nonspecific inflammatory cells can worsen CTL-induced immunopathology, and platelets activated at the site of infection may facilitate the accumulation of CTLs in the liver. The tests, called assays , for detection of hepatitis B virus infection involve serum or blood tests that detect either viral antigens (proteins produced by the virus) or antibodies produced by

7473-419: Is likely why nature has "chosen" a four base alphabet: fewer than four would not allow the creation of all structures, while more than four bases are not necessary to do so. Since RNA is charged, metal ions such as Mg are needed to stabilise many secondary and tertiary structures . The naturally occurring enantiomer of RNA is D -RNA composed of D -ribonucleotides. All chirality centers are located in

7632-423: Is long lasting even after antibody levels fall below 10 mIU/ml. For newborns of HBsAg-positive mothers: hepatitis B vaccine alone, hepatitis B immunoglobulin alone, or the combination of vaccine plus hepatitis B immunoglobulin, all prevent hepatitis B occurrence. Furthermore, the combination of vaccine plus hepatitis B immunoglobulin is superior to vaccine alone. This combination prevents HBV transmission around

7791-501: Is made of 180 or 240 copies of the core protein, alternatively known as hepatitis B core antigen, or HBcAg . During this 'window' in which the host remains infected but is successfully clearing the virus, IgM antibodies specific to the hepatitis B core antigen ( anti-HBc IgM ) may be the only serological evidence of disease. Therefore, most hepatitis B diagnostic panels contain HBsAg and total anti-HBc (both IgM and IgG). Shortly after

7950-723: Is made possible by hybridizing ssDNA with a PAM complement sequence to ssRNA allowing for a dsDNA-RNA PAM site for Cas9 binding. This technology makes available the ability to isolate endogenous RNA transcripts in cells without the need to induce chemical modifications to RNA or RNA tagging methods. In contrast to silencing genes, dCas9 can also be used to activate genes when fused to transcription activating factors. These factors include subunits of bacterial RNA Polymerase II and traditional transcription factors in eukaryotes. Recently, genome-wide screens of transcription activation have also been accomplished using dCas9 fusions named 'CRISPRa' for activation. RNA Ribonucleic acid ( RNA )

8109-449: Is more common in people with hepatitis B infection. A number of different tests are available to determine the degree of cirrhosis present. Transient elastography (FibroScan) is the test of choice, but it is expensive. Aspartate aminotransferase to platelet ratio index may be used when cost is an issue. Hepatitis B virus DNA remains in the body after infection, and in some people, including those that do not have detectable HBsAg,

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8268-471: Is most prevalent in Africa (affecting 7.5% of the continent's population) and in the Western Pacific region (5.9%). Infection rates are 1.5% in Europe and 0.5% in the Americas. According to some estimates, about a third of the world's population has been infected with hepatitis B at one point in their lives. Hepatitis B was originally known as "serum hepatitis". Acute infection with hepatitis B virus

8427-502: Is no different from the risk in spontaneous conception. Those at high risk of infection should be tested as there is effective treatment for those who have the disease. Groups that screening is recommended for include those who have not been vaccinated and one of the following: people from areas of the world where hepatitis B occurs in more than 2%, those with HIV, intravenous drug users, men who have sex with men, and those who live with someone with hepatitis B. Screening during pregnancy

8586-458: Is one of the smallest enveloped animal viruses. The 42 nm virions, which are capable of infecting liver cells known as hepatocytes , are referred to as "Dane particles". In addition to the Dane particles, filamentous and spherical bodies lacking a core can be found in the serum of infected individuals. These particles are not infectious and are composed of the lipid and protein that forms part of

8745-400: Is present. The DNA polymerase is encoded by gene P. Gene S is the gene that codes for the surface antigen (HBsAg). The HBsAg gene is one long open reading frame but contains three in frame "start" (ATG) codons that divide the gene into three sections, pre-S1, pre-S2, and S. Because of the multiple start codons, polypeptides of three different sizes called large (the order from surface to

8904-413: Is processed to mature mRNA. This removes its introns —non-coding sections of the pre-mRNA. The mRNA is then exported from the nucleus to the cytoplasm , where it is bound to ribosomes and translated into its corresponding protein form with the help of tRNA . In prokaryotic cells, which do not have nucleus and cytoplasm compartments, mRNA can bind to ribosomes while it is being transcribed from DNA. After

9063-405: Is recommended in the United States. Acute hepatitis B infection does not usually require treatment and most adults clear the infection spontaneously. Early antiviral treatment may be required in fewer than 1% of people, whose infection takes a very aggressive course (fulminant hepatitis) or who are immunocompromised . On the other hand, treatment of chronic infection may be necessary to reduce

9222-463: Is the most common form. Other immune-mediated hematological disorders, such as essential mixed cryoglobulinemia and aplastic anemia have been described as part of the extrahepatic manifestations of HBV infection, but their association is not as well-defined; therefore, they probably should not be considered etiologically linked to HBV. Transmission of hepatitis B virus results from exposure to infectious blood or body fluids containing blood. HBV

9381-517: Is then cleaved, however if there are mismatches between the spacer and the target DNA, or if there are mutations in the PAM, then cleavage will not be initiated. In the latter scenario, the foreign DNA is not targeted for attack by the cell, thus the replication of the virus proceeds and the host is not immune to viral infection. The interference stage can be mechanistically and temporally distinct from CRISPR acquisition and expression, yet for complete function as

9540-401: Is then transported back to the cytoplasm where the virion P protein (the DNA polymerase) synthesizes DNA via its reverse transcriptase activity. The virus is divided into four major serotypes (adr, adw, ayr, ayw) based on antigenic epitopes presented on its envelope proteins, and into eight major genotypes (A–H). The genotypes have a distinct geographical distribution and are used in tracing

9699-630: Is transmitted by exposure to infectious blood or body fluids . In areas where the disease is common , infection around the time of birth or from contact with other people's blood during childhood are the most frequent methods by which hepatitis B is acquired. In areas where the disease is rare, intravenous drug use and sexual intercourse are the most frequent routes of infection . Other risk factors include working in healthcare, blood transfusions , dialysis , living with an infected person, travel in countries with high infection rates, and living in an institution. Tattooing and acupuncture led to

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9858-550: Is used as template for building the ends of eukaryotic chromosomes . Double-stranded RNA (dsRNA) is RNA with two complementary strands, similar to the DNA found in all cells, but with the replacement of thymine by uracil and the adding of one oxygen atom. dsRNA forms the genetic material of some viruses ( double-stranded RNA viruses ). Double-stranded RNA, such as viral RNA or siRNA , can trigger RNA interference in eukaryotes , as well as interferon response in vertebrates . In eukaryotes, double-stranded RNA (dsRNA) plays

10017-696: Is ~45% in types A and B but only 25–30% in types C and D. It seems unlikely that the disease will be eliminated by 2030, the goal set in 2016 by WHO. However, progress is being made in developing therapeutic treatments. In 2010, the Hepatitis B Foundation reported that 3 preclinical and 11 clinical-stage drugs were under development, based on largely similar mechanisms. In 2020, they reported that there were 17 preclinical- and 32 clinical-stage drugs under development, using diverse mechanisms. Hepatitis B virus infection may be either acute (self-limiting) or chronic (long-standing). Persons with self-limiting infection clear

10176-547: The D -ribose. By the use of L -ribose or rather L -ribonucleotides, L -RNA can be synthesized. L -RNA is much more stable against degradation by RNase . Like other structured biopolymers such as proteins, one can define topology of a folded RNA molecule. This is often done based on arrangement of intra-chain contacts within a folded RNA, termed as circuit topology . RNA is transcribed with only four bases (adenine, cytosine, guanine and uracil), but these bases and attached sugars can be modified in numerous ways as

10335-493: The RNA World theory. There are indications that the enterobacterial sRNAs are involved in various cellular processes and seem to have significant role in stress responses such as membrane stress, starvation stress, phosphosugar stress and DNA damage. Also, it has been suggested that sRNAs have been evolved to have important role in stress responses because of their kinetic properties that allow for rapid response and stabilisation of

10494-453: The amino acid sequence in the protein that is produced. However, many RNAs do not code for protein (about 97% of the transcriptional output is non-protein-coding in eukaryotes ). These so-called non-coding RNAs ("ncRNA") can be encoded by their own genes (RNA genes), but can also derive from mRNA introns . The most prominent examples of non-coding RNAs are transfer RNA (tRNA) and ribosomal RNA (rRNA), both of which are involved in

10653-575: The galactic center of the Milky Way Galaxy . RNA, initially deemed unsuitable for therapeutics due to its short half-life, has been made useful through advances in stabilization. Therapeutic applications arise as RNA folds into complex conformations and binds proteins, nucleic acids, and small molecules to form catalytic centers. RNA-based vaccines are thought to be easier to produce than traditional vaccines derived from killed or altered pathogens, because it can take months or years to grow and study

10812-414: The genetic code . There are more than 100 other naturally occurring modified nucleosides. The greatest structural diversity of modifications can be found in tRNA , while pseudouridine and nucleosides with 2'-O-methylribose often present in rRNA are the most common. The specific roles of many of these modifications in RNA are not fully understood. However, it is notable that, in ribosomal RNA, many of

10971-535: The genotype of the infecting virus or the person's heredity. The treatment reduces viral replication in the liver, thereby reducing the viral load (the amount of virus particles as measured in the blood). Response to treatment differs between the genotypes. Interferon treatment may produce an e antigen seroconversion rate of 37% in genotype A but only a 6% seroconversion in type D. Genotype B has similar seroconversion rates to type A while type C seroconverts only in 15% of cases. Sustained e antigen loss after treatment

11130-440: The nitrogenous bases of guanine , uracil , adenine , and cytosine , denoted by the letters G, U, A, and C) that directs synthesis of specific proteins. Many viruses encode their genetic information using an RNA genome . Some RNA molecules play an active role within cells by catalyzing biological reactions, controlling gene expression , or sensing and communicating responses to cellular signals. One of these active processes

11289-400: The 'e' antigen ( anti-HBe ) will arise immediately afterwards. This conversion is usually associated with a dramatic decline in viral replication. If the host is able to clear the infection, eventually the HBsAg will become undetectable and will be followed by IgG antibodies to the hepatitis B surface antigen and core antigen ( anti-HBs and anti HBc IgG ). The time between the removal of

11448-602: The 20 nucleotide spacer region of the guide RNA (gRNA). If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. In this sense, the CRISPR-Cas9 mechanism has a number of parallels with the RNA interference (RNAi) mechanism in eukaryotes. Apart from its original function in bacterial immunity, the Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double-strand breaks in DNA. These breaks can lead to gene inactivation or

11607-469: The 2006 Nobel Prize in Physiology or Medicine for discovering microRNAs (miRNAs), specific short RNA molecules that can base-pair with mRNAs. Post-transcriptional expression levels of many genes can be controlled by RNA interference , in which miRNAs , specific short RNA molecules, pair with mRNA regions and target them for degradation. This antisense -based process involves steps that first process

11766-423: The 3’ to 5’ direction, synthesizing a complementary RNA molecule with elongation occurring in the 5’ to 3’ direction. The DNA sequence also dictates where termination of RNA synthesis will occur. Primary transcript RNAs are often modified by enzymes after transcription. For example, a poly(A) tail and a 5' cap are added to eukaryotic pre-mRNA and introns are removed by the spliceosome . There are also

11925-509: The Americas. The primary method of HBV transmission and the prevalence of chronic HBV infection in specific regions often correspond with one another. In populations where HBV infection rates are 8% or higher, which are classified as high prevalence, vertical transmission (usually occurring during birth) is most common, though rates of early childhood transmission can also be significant among these populations. In 2021, 19 African countries had infection rates ranging between 8-19%, placing them in

12084-560: The Americas. This disproved the belief that hepatitis B originated in the New World and spread to Europe around 16th century. Hepatitis B virus subgenotype C4 is exclusively present in Australian aborigines, suggesting an ancient origin as much as 50,000 years old. However, analyses of ancient HBV genomes suggested that the most recent common ancestor of all known human HBV strains was dated to between 20,000 and 12,000 years ago, pointing to

12243-558: The B-form most commonly observed in DNA. The A-form geometry results in a very deep and narrow major groove and a shallow and wide minor groove. A second consequence of the presence of the 2'-hydroxyl group is that in conformationally flexible regions of an RNA molecule (that is, not involved in formation of a double helix), it can chemically attack the adjacent phosphodiester bond to cleave the backbone. The functional form of single-stranded RNA molecules, just like proteins, frequently requires

12402-506: The C-terminal end of Cas9. Cas9 undergoes distinct conformational changes between the apo, guide RNA bound, and guide RNA:DNA bound states. Cas9 recognizes the stem-loop architecture inherent in the CRISPR locus, which mediates the maturation of crRNA-tracrRNA ribonucleoprotein complex. Cas9 in complex with CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) further recognizes and degrades

12561-599: The CRISPR locus. The "Protospacer" refers to the sequence on the viral genome that corresponds to the spacer. A short stretch of conserved nucleotides exists proximal to the protospacer, which is called the protospacer adjacent motif (PAM). The PAM is a recognition motif that is used to acquire the DNA fragment. In type II, Cas9 recognizes the PAM during adaptation in order to ensure the acquisition of functional spacers. Loss of spacers and even groups of several have also been observed by Aranaz et al. 2004 and Pourcel et al. 2007. This probably occurs through homologous recombination of

12720-505: The DNA polymerase II from continuing transcription. Further explanation of how the dCas9 protein works can be found in their utilization of plant genomes by the regulation of gene production in plants to either increase or decrease certain characteristics. The CRISPR-CAS9 system has the ability to either upregulate or downregulate genes. The dCas9 proteins are a component of the CRISPR-CAS9 system and these proteins can repress certain areas of

12879-457: The HBsAg and the appearance of anti-HBs is called the window period . A person negative for HBsAg but positive for anti-HBs either has cleared an infection or has been vaccinated previously. Individuals who remain HBsAg positive for at least six months are considered to be hepatitis B carriers. Carriers of the virus may have chronic hepatitis B, which would be reflected by elevated serum alanine aminotransferase (ALT) levels and inflammation of

13038-732: The HNH domain is not visible. These structures suggest the conformational flexibility of HNH domain. To date, at least three crystal structures have been studied and published. One representing a conformation of Cas9 in the apo state, and two representing Cas9 in the DNA bound state. In sgRNA-Cas9 complex, based on the crystal structure, REC1, BH and PI domains have important contacts with backbone or bases in both repeat and spacer region. Several Cas9 mutants including REC1 or REC2 domains deletion and residues mutations in BH have been tested. REC1 and BH related mutants show lower or none activity compared with wild type, which indicate these two domains are crucial for

13197-768: The RNA so that it can base-pair with a region of its target mRNAs. Once the base pairing occurs, other proteins direct the mRNA to be destroyed by nucleases . Next to be linked to regulation were Xist and other long noncoding RNAs associated with X chromosome inactivation .  Their roles, at first mysterious, were shown by Jeannie T. Lee and others to be the silencing of blocks of chromatin via recruitment of Polycomb complex so that messenger RNA could not be transcribed from them. Additional lncRNAs, currently defined as RNAs of more than 200 base pairs that do not appear to have coding potential, have been found associated with regulation of stem cell pluripotency and cell division . The third major group of regulatory RNAs

13356-410: The RNA to fold and pair with itself to form double helices. Analysis of these RNAs has revealed that they are highly structured. Unlike DNA, their structures do not consist of long double helices, but rather collections of short helices packed together into structures akin to proteins. In this fashion, RNAs can achieve chemical catalysis (like enzymes). For instance, determination of the structure of

13515-469: The RNA-mediated DNA cleavage; however, Cas9 has been shown to exhibit varying levels of activity in the presence of other divalent metal ions. For instance, Cas9 in the presence of manganese (Mn) has been shown to be capable of RNA-independent DNA cleavage. The kinetics of DNA cleavage by Cas9 have been of great interest to the scientific community, as this data provides insight into the intricacies of

13674-503: The RNAs mature. Pseudouridine (Ψ), in which the linkage between uracil and ribose is changed from a C–N bond to a C–C bond, and ribothymidine (T) are found in various places (the most notable ones being in the TΨC loop of tRNA ). Another notable modified base is hypoxanthine , a deaminated adenine base whose nucleoside is called inosine (I). Inosine plays a key role in the wobble hypothesis of

13833-489: The World Health Organization recommended tenofovir or entecavir as first-line agents. Those with current cirrhosis are in most need of treatment. The use of interferon, which requires injections daily or thrice weekly, has been supplanted by long-acting PEGylated interferon , which is injected only once weekly. However, some individuals are much more likely to respond than others, and this might be because of

13992-403: The alpha-helical lobe with respect to the nuclease lobe, as well as the location of the HNH domain. The protein consists of a recognition lobe (REC) and a nuclease lobe (NUC). All regions except the HNH form tight interactions with each other and sgRNA-ssDNA complex, while the HNH domain forms few contacts with the rest of the protein. In another conformation of Cas9 complex observed in the crystal,

14151-424: The amount of HBV DNA, called the viral load , in clinical specimens. These tests are used to assess a person's infection status and to monitor treatment. Individuals with high viral loads , characteristically have ground glass hepatocytes on biopsy. Vaccines for the prevention of hepatitis B have been routinely recommended for babies since 1991 in the United States. The first dose is generally recommended within

14310-490: The appearance of the HBsAg, another antigen called hepatitis B e antigen ( HBeAg ) will appear. Traditionally, the presence of HBeAg in a host's serum is associated with much higher rates of viral replication and enhanced infectivity; however, variants of the hepatitis B virus do not produce the 'e' antigen, so this rule does not always hold true. During the natural course of an infection, the HBeAg may be cleared, and antibodies to

14469-491: The available medications can clear the infection, they can stop the virus from replicating, thus minimizing liver damage. As of 2024, there are seven medications licensed for the treatment of hepatitis B infection in the United States. These include antiviral medications lamivudine , adefovir , tenofovir disoproxil , tenofovir alafenamide , telbivudine , and entecavir , and the two immune system modulators interferon alpha-2a and PEGylated interferon alpha-2a . In 2015,

14628-506: The between-repeat material. CRISPR expression includes the transcription of a primary transcript called a CRISPR RNA (pre-crRNA), which is transcribed from the CRISPR locus by RNA polymerase. Specific endoribonucleases then cleave the pre-crRNAs into small CRISPR RNAs (crRNAs). Interference involves the crRNAs within a multi-protein complex called CASCADE, which can recognize and specifically base-pair with regions of inserting complementary foreign DNA. The crRNA-foreign nucleic acid complex

14787-414: The cas locus, which are usually located near the CRISPR array. Stage 2: CRISPR expression. Pre-crRNA is transcribed starting at the leader region by the host RNA polymerase and then cleaved by Cas proteins into smaller crRNAs containing a single spacer and a partial repeat (shown as hairpin structure with colored spacers). Stage 3: CRISPR interference. crRNA with a spacer that has strong complementarity to

14946-445: The case of the 5S rRNA of the members of the genus Halococcus ( Archaea ), which have an insertion, thus increasing its size. Messenger RNA (mRNA) carries information about a protein sequence to the ribosomes , the protein synthesis factories in the cell. It is coded so that every three nucleotides (a codon ) corresponds to one amino acid. In eukaryotic cells, once precursor mRNA (pre-mRNA) has been transcribed from DNA, it

15105-432: The cell by binding to NTCP on the surface and being endocytosed . Because the virus multiplies via RNA made by a host enzyme, the viral genomic DNA has to be transferred to the cell nucleus by host proteins called chaperones. The partially double-stranded, circular viral DNA is then made fully double stranded by HBV DNA polymerase, transforming the genome into covalently closed circular DNA (cccDNA). This cccDNA serves as

15264-402: The cell nucleus and is usually catalyzed by an enzyme— RNA polymerase —using DNA as a template, a process known as transcription . Initiation of transcription begins with the binding of the enzyme to a promoter sequence in the DNA (usually found "upstream" of a gene). The DNA double helix is unwound by the helicase activity of the enzyme. The enzyme then progresses along the template strand in

15423-414: The chromatin structure of various loci. Additionally, dCas9 has been employed in genome wide screens of gene repression. By employing large libraries of guide RNAs capable of targeting thousands of genes, genome wide genetic screens using dCas9 have been conducted. Another method for silencing transcription with Cas9 is to directly cleave mRNA products with the catalytically active Cas9 enzyme. This approach

15582-485: The coding region of loci such that inhibition of RNA Polymerase occurs during the elongation phase of transcription. In Eukaryotes, silencing of gene expression can be extended by targeting dCas9 to enhancer sequences, where dCas9 can block assembly of transcription factors leading to silencing of specific gene expression. Moreover, the guide RNAs provided to dCas9 can be designed to include specific mismatches to its complementary cognate sequence that will quantitatively weaken

15741-431: The dCas9 attaches to a form of RNA called guide-RNA, it prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism's genome. Essentially, when multiple repeat codons are produced, it elicits a response, or recruits an abundance of dCas9 to combat the overproduction of those codons and results in the shut-down of transcription. dCas9 works synergistically with gRNA and directly affects

15900-459: The detrimental strand of DNA and RNA that cause diseases and mutated strands of DNA. Cas9 has already showed promise in disrupting the effects of HIV-1. Cas9 has been shown to suppress the expression of the long terminal repeats in HIV-1. When introduced into the HIV-1 genome Cas9 has shown the ability to mutate strands of HIV-1. Cas9 has also been used in the treatment of Hepatitis B through targeting of

16059-435: The development of membranous glomerulonephritis (MGN). Symptoms outside of the liver are present in 1–10% of HBV-infected people and include serum-sickness–like syndrome , acute necrotizing vasculitis ( polyarteritis nodosa ), membranous glomerulonephritis, and papular acrodermatitis of childhood ( Gianotti–Crosti syndrome ). The serum-sickness–like syndrome occurs in the setting of acute hepatitis B , often preceding

16218-466: The development of therapeutic treatments to cure chronic hepatitis B, as well as preventing its transmission and using vaccines to prevent new infections. An estimated 296 million people, or 3.8% of the global population, had chronic hepatitis B infections as of 2019. Another 1.5 million developed acute infections that year, and 820,000 deaths occurred as a result of HBV. Cirrhosis and liver cancer are responsible for most HBV-related deaths. The disease

16377-478: The disease recurs. Although rare, reactivation is seen most often following alcohol or drug use, or in people with impaired immunity. HBV goes through cycles of replication and non-replication. Approximately 50% of overt carriers experience acute reactivation. Males with baseline ALT of 200 UL/L are three times more likely to develop a reactivation than people with lower levels. Although reactivation can occur spontaneously, people who undergo chemotherapy have

16536-444: The earliest forms of life (self-replicating molecules) could have relied on RNA both to carry genetic information and to catalyze biochemical reactions—an RNA world . In May 2022, scientists discovered that RNA can form spontaneously on prebiotic basalt lava glass , presumed to have been abundant on the early Earth . In March 2015, DNA and RNA nucleobases , including uracil , cytosine and thymine , were reportedly formed in

16695-475: The effects of repression of transcription further, H3K27, an amino acid component of a histone, becomes methylated through the interaction of dCas9 and a peptide called FOG1. Essentially, this interaction causes gene repression on the C + N terminal section of the amino acid complex at the specific junction of the gene, and as a result, terminates transcription. dCas9 also proves to be efficient when it comes to altering certain proteins that can create diseases. When

16854-486: The end of 2021. To further prevent infection, the WHO recommends testing all donated blood for hepatitis B before using it for transfusion. Using antiviral prophylaxis to prevent mother-to-child transmission is also recommended, as is following safe sex practices, including the use of condoms . In 2016, the WHO set a goal of eliminating viral hepatitis as a threat to global public health by 2030. Achieving this goal would require

17013-674: The ends of certain of long terminal repeats in the Hepatitis B viral genome. Cas9 has been used to repair the mutations causing cataracts in mice. CRISPR-Cas systems are divided into three major types (type I, type II, and type III) and twelve subtypes, which are based on their genetic content and structural differences. However, the core defining features of all CRISPR-Cas systems are the cas genes and their proteins: cas1 and cas2 are universal across types and subtypes, while cas3 , cas9, and cas10 are signature genes for type I, type II, and type III, respectively. Adaptation involves recognition and integration of spacers between two adjacent repeats in

17172-460: The ends of the (−) sense strand and the ends are rejoined. There are four known genes encoded by the genome, called C, X, P, and S. The core protein is coded for by gene C (HBcAg), and its start codon is preceded by an upstream in-frame AUG start codon from which the pre-core protein is produced. HBeAg is produced by proteolytic processing of the pre-core protein. In some rare strains of the virus known as hepatitis B virus precore mutants , no HBeAg

17331-834: The evolution and transmission of the virus. Differences between genotypes affect the disease severity, course and likelihood of complications, and response to treatment and possibly vaccination. There are two other genotypes I and J but they are not universally accepted as of 2015. The diversity of genotypes is not shown equally in the world. For example, A, D, and E genotypes have been seen in Africa prevalently while B and C genotypes are observed in Asia as widespread. Genotypes differ by at least 8% of their sequence and were first reported in 1988 when six were initially described (A–F). Two further types have since been described (G and H). Most genotypes are now divided into subgenotypes with distinct properties. Hepatitis B virus primarily interferes with

17490-414: The evolution of DNA and possibly of protein-based enzymes as well, an " RNA world " existed in which RNA served as both living organisms' storage method for genetic information —a role fulfilled today by DNA, except in the case of RNA viruses —and potentially performed catalytic functions in cells—a function performed today by protein enzymes, with the notable and important exception of the ribosome, which

17649-456: The first crystal of RNA whose structure could be determined by X-ray crystallography. The sequence of the 77 nucleotides of a yeast tRNA was found by Robert W. Holley in 1965, winning Holley the 1968 Nobel Prize in Medicine (shared with Har Gobind Khorana and Marshall Nirenberg ). In the early 1970s, retroviruses and reverse transcriptase were discovered, showing for the first time that enzymes could copy RNA into DNA (the opposite of

17808-620: The first time. To survive in a variety of challenging, inhospitable habitats that are filled with bacteriophages , bacteria and archaea have evolved methods to evade and fend off predatory viruses . This includes the CRISPR system of adaptive immunity. In practice, CRISPR/Cas systems act as self-programmable restriction enzymes. CRISPR loci are composed of short, palindromic repeats that occur at regular intervals composed of alternate CRISPR repeats and variable CRISPR spacers between 24 and 48 nucleotides long. These CRISPR loci are usually accompanied by adjacent CRISPR-associated (cas) genes. In 2005, it

17967-483: The form of COVID-19 vaccines during the COVID-19 pandemic . Hepatitis B Hepatitis B is an infectious disease caused by the hepatitis B virus (HBV) that affects the liver ; it is a type of viral hepatitis . It can cause both acute and chronic infection . Many people have no symptoms during an initial infection. For others, symptoms may appear 30 to 180 days after becoming infected and can include

18126-423: The function of circRNAs is largely unknown, although for few examples a microRNA sponging activity has been demonstrated. Research on RNA has led to many important biological discoveries and numerous Nobel Prizes . Nucleic acids were discovered in 1868 by Friedrich Miescher , who called the material 'nuclein' since it was found in the nucleus . It was later discovered that prokaryotic cells, which do not have

18285-592: The functions of the liver by replicating in hepatocytes . A functional receptor is NTCP . There is evidence that the receptor in the closely related duck hepatitis B virus is carboxypeptidase D . The virions bind to the host cell via the preS domain of the viral surface antigen and are subsequently internalized by endocytosis. HBV-preS-specific receptors are expressed primarily on hepatocytes; however, viral DNA and proteins have also been detected in extrahepatic sites, suggesting that cellular receptors for HBV may also exist on extrahepatic cells. During HBV infection,

18444-551: The genes to be regulated.   Later studies have shown that RNAs also regulate genes. There are several kinds of RNA-dependent processes in eukaryotes regulating the expression of genes at various points, such as RNAi repressing genes post-transcriptionally , long non-coding RNAs shutting down blocks of chromatin epigenetically , and enhancer RNAs inducing increased gene expression. Bacteria and archaea have also been shown to use regulatory RNA systems such as bacterial small RNAs and CRISPR . Fire and Mello were awarded

18603-449: The hepatitis B vaccine is between 98% and 100% effective in preventing infection. The vaccine is administered in several doses; after an initial dose, two or three more vaccine doses are required at a later time for full effect. The World Health Organization (WHO) recommends infants receive the vaccine within 24 hours after birth when possible. National programs have made the hepatitis B vaccine available for infants in 190 countries as of

18762-518: The high prevalence category. High prevalence of HBV also exists in Mongolia . In moderate prevalence areas where 2–7% of the population is chronically infected, the disease is predominantly spread horizontally, often among children, but also vertically. China's HBV infection rate is at the higher end of the moderate prevalence classification with an infection rate of 6.89% as of 2019. HBV prevalence in India

18921-506: The host immune response causes both hepatocellular damage and viral clearance. Although the innate immune response does not play a significant role in these processes, the adaptive immune response, in particular virus-specific cytotoxic T lymphocytes (CTLs), contributes to most of the liver injury associated with HBV infection. CTLs eliminate HBV infection by killing infected cells and producing antiviral cytokines , which are then used to purge HBV from viable hepatocytes. Although liver damage

19080-498: The host. Interpretation of these assays is complex. The hepatitis B surface antigen ( HBsAg ) is most frequently used to screen for the presence of this infection. It is the first detectable viral antigen to appear during infection. However, early in an infection, this antigen may not be present and it may be undetectable later in the infection as it is being cleared by the host. The infectious virion contains an inner "core particle" enclosing viral genome. The icosahedral core particle

19239-451: The incoming foreign DNA begins a cleavage event (depicted with scissors), which requires Cas proteins. DNA cleavage interferes with viral replication and provides immunity to the host. The interference stage can be functionally and temporarily distinct from CRISPR acquisition and expression (depicted by white line dividing the cell). dCas9 , also referred to as endonuclease deficient Cas9 can be utilized to edit gene expression when applied to

19398-433: The infection spontaneously within weeks to months. Children are less likely than adults to clear the infection. More than 95% of people who become infected as adults or older children will stage a full recovery and develop protective immunity to the virus. However, this drops to 30% for younger children, and only 5% of newborns that acquire the infection from their mother at birth will clear the infection. This population has

19557-414: The inside: pre-S1, pre-S2, and S ), middle (pre-S2, S), and small (S) are produced. There is a myristyl group, which plays an important role in infection, on the amino-terminal end of the preS1 part of the large (L) protein. In addition to that, N terminus of the L protein have virus attachment and capsid binding sites. Because of that, the N termini of half of the L protein molecules are positioned outside

19716-425: The interaction of dCas9 for its programmed cognate sequence allowing a researcher to tune the extent of gene silencing applied to a gene of interest. This technology is similar in principle to RNAi such that gene expression is being modulated at the RNA level. However, the dCas9 approach has gained much traction as there exist less off-target effects and in general larger and more reproducible silencing effects through

19875-610: The introduction of heterologous genes through non-homologous end joining and homologous recombination respectively in many laboratory model organisms. Research on the development of various cas9 variants has been a promising way of overcoming the limitation of the CRISPR-Cas9 genome editing . Some examples include Cas9 nickase (Cas9n), a variant that induces single-stranded breaks (SSBs) or variants recognizing different PAM sequences . Alongside zinc finger nucleases and transcription activator-like effector nuclease (TALEN) proteins, Cas9

20034-461: The introns can be ribozymes that are spliced by themselves. RNA can also be altered by having its nucleotides modified to nucleotides other than A , C , G and U . In eukaryotes, modifications of RNA nucleotides are in general directed by small nucleolar RNAs (snoRNA; 60–300 nt), found in the nucleolus and cajal bodies . snoRNAs associate with enzymes and guide them to a spot on an RNA by basepairing to that RNA. These enzymes then perform

20193-472: The laboratory under outer space conditions, using starter chemicals such as pyrimidine , an organic compound commonly found in meteorites . Pyrimidine , like polycyclic aromatic hydrocarbons (PAHs), is one of the most carbon-rich compounds found in the universe and may have been formed in red giants or in interstellar dust and gas clouds. In July 2022, astronomers reported massive amounts of prebiotic molecules , including possible RNA precursors, in

20352-483: The liver, if they are in the immune clearance phase of chronic infection. Carriers who have seroconverted to HBeAg negative status, in particular those who acquired the infection as adults, have very little viral multiplication and hence may be at little risk of long-term complications or of transmitting infection to others. However, it is possible for individuals to enter an "immune escape" with HBeAg-negative hepatitis. PCR tests have been developed to detect and measure

20511-511: The membrane and the other half positioned inside the membrane. The function of the protein coded for by gene X is not fully understood but it is associated with the development of liver cancer. It stimulates genes that promote cell growth and inactivates growth regulating molecules. The life cycle of hepatitis B virus is complex. Hepatitis B is one of a few known pararetroviruses : non- retroviruses that still use reverse transcription in their replication process. The virus gains entry into

20670-498: The newborn infant. No randomized control trial has been conducted to assess the effects of hepatitis B vaccine during pregnancy for preventing infant infection. All those with a risk of exposure to body fluids such as blood should be vaccinated, if not already. Testing to verify effective immunization is recommended and further doses of vaccine are given to those who are not sufficiently immunized. In 10- to 22-year follow-up studies there were no cases of hepatitis B among those with

20829-401: The nucleotide modification. rRNAs and tRNAs are extensively modified, but snRNAs and mRNAs can also be the target of base modification. RNA can also be methylated. Like DNA, RNA can carry genetic information. RNA viruses have genomes composed of RNA that encodes a number of proteins. The viral genome is replicated by some of those proteins, while other proteins protect the genome as

20988-439: The onset of jaundice. The clinical features are fever, skin rash , and polyarteritis . The symptoms often subside shortly after the onset of jaundice but can persist throughout the duration of acute hepatitis B . About 30–50% of people with acute necrotizing vasculitis (polyarteritis nodosa) are HBV carriers. HBV-associated nephropathy has been described in adults but is more common in children. Membranous glomerulonephritis

21147-693: The physiological state. Bacterial small RNAs generally act via antisense pairing with mRNA to down-regulate its translation, either by affecting stability or affecting cis-binding ability. Riboswitches have also been discovered. They are cis-acting regulatory RNA sequences acting allosterically . They change shape when they bind metabolites so that they gain or lose the ability to bind chromatin to regulate expression of genes. Archaea also have systems of regulatory RNA. The CRISPR system, recently being used to edit DNA in situ , acts via regulatory RNAs in archaea and bacteria to provide protection against virus invaders. Synthesis of RNA typically occurs in

21306-405: The post-transcriptional modifications occur in highly functional regions, such as the peptidyl transferase center and the subunit interface, implying that they are important for normal function. Messenger RNA (mRNA) is the type of RNA that carries information from DNA to the ribosome , the sites of protein synthesis ( translation ) in the cell cytoplasm. The coding sequence of the mRNA determines

21465-489: The process of transcription of genetic material. Cas9 features a bi-lobed architecture with the guide RNA nestled between the alpha-helical lobe (blue) and the nuclease lobe (cyan, orange, and gray). These two lobes are connected through a single bridge helix. There are two nuclease domains located in the multi-domain nuclease lobe, the RuvC (gray) which cleaves the non-target DNA strand, and the HNH nuclease domain (cyan) that cleaves

21624-928: The process of translation. There are also non-coding RNAs involved in gene regulation, RNA processing and other roles. Certain RNAs are able to catalyse chemical reactions such as cutting and ligating other RNA molecules, and the catalysis of peptide bond formation in the ribosome ; these are known as ribozymes . According to the length of RNA chain, RNA includes small RNA and long RNA. Usually, small RNAs are shorter than 200  nt in length, and long RNAs are greater than 200  nt long. Long RNAs, also called large RNAs, mainly include long non-coding RNA (lncRNA) and mRNA . Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA , transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). There are certain exceptions as in

21783-448: The reaction. The cleavage efficiency of Cas9 depends on numerous factors. A key requirement is the presence of a valid PAM at the non-target strand 3 nucleotides downstream from the cleavage site. The canonical PAM sequence for S. Pyogenes Cas9 is NGG, but alternative motifs are tolerated with lower cleavage activity. The most efficient alternative PAM motifs for the wild-type S. Pyogenes Cas9 are NAG and NGA. The sequence composition at

21942-408: The reaction. While the cleavage of DNA by RNA-bound Cas9 has been shown to be relatively rapid ( k ≥ 700 s), the release of the cleavage products is very slow ( t 1/2 = ln(2)/ k ≈ 43–91 h), essentially rendering Cas9 a single- turnover enzyme. Additional studies regarding the kinetics of Cas9 have shown engineered Cas9 to be effective in reducing off-target effects by modifying the rate of

22101-508: The ribosome—an RNA-protein complex that catalyzes the assembly of proteins—revealed that its active site is composed entirely of RNA. An important structural component of RNA that distinguishes it from DNA is the presence of a hydroxyl group at the 2' position of the ribose sugar . The presence of this functional group causes the helix to mostly take the A-form geometry , although in single strand dinucleotide contexts, RNA can rarely also adopt

22260-425: The risk of cirrhosis and liver cancer. Chronically infected individuals with persistently elevated serum alanine aminotransferase , a marker of liver damage, and HBV DNA levels are candidates for therapy. Treatment lasts from six months to a year, depending on medication and genotype. Treatment duration when medication is taken by mouth, however, is more variable and usually longer than one year. Although none of

22419-412: The sgRNA recognition at repeat sequence and stabilization of the whole complex. Although the interactions between spacer sequence and Cas9 as well as PI domain and repeat region need further studies, the co-crystal demonstrates clear interface between Cas9 and sgRNA. Previous sequence analysis and biochemical studies have posited that Cas9 contains two nuclease domains: an McrA-like HNH nuclease domain and

22578-433: The short length-strand). The negative-sense (non-coding) is complementary to the viral mRNA . The viral DNA is found in the nucleus soon after infection of the cell . The partially double-stranded DNA is rendered fully double-stranded by completion of the (+) sense strand and removal of a protein molecule from the (−) sense strand and a short sequence of RNA from the (+) sense strand. Non-coding bases are removed from

22737-709: The strands of DNA. This poses a problem to Cas9 editing because the RM system also targets the foreign genes added by the Cas9 process. Due to the unique ability of Cas9 to bind to essentially any complement sequence in any genome , researchers wanted to use this enzyme to repress transcription of various genomic loci . To accomplish this, the two crucial catalytic residues of the RuvC and HNH domain can be mutated to alanine abolishing all endonuclease activity of Cas9. The resulting protein coined 'dead' Cas9 or 'dCas9' for short, can still tightly bind to dsDNA. This catalytically inactive Cas9 variant has been used for both mechanistic studies into Cas9 DNA interrogative binding and as

22896-482: The surface of the virion, which is called the surface antigens ( HBsAg ), and is produced in excess during the life cycle of the virus. The genome of HBV is made of circular DNA , but it is unusual because the DNA is not fully double-stranded . One end of the full length strand is linked to the HBV DNA polymerase . The genome is 3020–3320 nucleotides long (for the full-length strand) and 1700–2800 nucleotides long (for

23055-688: The target DNA site complementary to the 20 nucletode spacer region of the gRNA also affects cleavage efficiency. The most relevant nucleotide composition properties that impact efficiency are those in the PAM-proximal region. Free energy changes of nucleic acids are also highly relevant in defining cleavage activity. Guide RNAs that bind to the DNA forming a duplex that falls into a restricted range of binding free energy changes that excludes extremely weak or stable bindings generally perform efficiently. Stable guide RNA folding conformations can also impair cleavage. Most archaea and bacteria stubbornly refuse to allow

23214-461: The target dsDNA. In the co-crystal structure shown here, the crRNA-tracrRNA complex is replaced by a chimeric single-guide RNA (sgRNA, in red) which has been proved to have the same function as the natural RNA complex. The sgRNA base paired with target ssDNA is anchored by Cas9 as a T-shaped architecture. This crystal structure of the DNA-bound Cas9 enzyme reveals distinct conformational changes in

23373-464: The target strand of DNA. The RuvC domain is encoded by sequentially disparate sites that interact in the tertiary structure to form the RuvC cleavage domain (See right figure). A key feature of the target DNA is that it must contain a protospacer adjacent motif (PAM) consisting of the three-nucleotide sequence- NGG. This PAM is recognized by the PAM-interacting domain (PI domain, orange) located near

23532-455: The time of birth in 86% to 99% of cases. Tenofovir given in the second or third trimester can reduce the risk of mother to child transmission by 77% when combined with hepatitis B immunoglobulin and the hepatitis B vaccine, especially for pregnant women with high hepatitis B virus DNA levels. However, there is not sufficient evidence that the administration of hepatitis B immunoglobulin alone during pregnancy, might reduce transmission rates to

23691-426: The transcription binding site of the desired section of a gene. The optimal function of dCas9 is attributed to its mode of action. Gene expression is inhibited when nucleotides are no longer added to the RNA chain and therefore terminating elongation of that chain, and as a result affects the transcription process. This process occurs when dCas9 is mass-produced so it is able to affect the most genes at any given time via

23850-417: The use of dCas9 compared to RNAi screens. Furthermore, because the dCas9 approach to gene silencing can be quantitatively controlled, a researcher can now precisely control the extent to which a gene of interest is repressed allowing more questions about gene regulation and gene stoichiometry to be answered. Beyond direct binding of dCas9 to transcriptionally sensitive positions of loci, dCas9 can be fused to

24009-423: The usual route for transmission of genetic information). For this work, David Baltimore , Renato Dulbecco and Howard Temin were awarded a Nobel Prize in 1975. In 1976, Walter Fiers and his team determined the first complete nucleotide sequence of an RNA virus genome, that of bacteriophage MS2 . In 1977, introns and RNA splicing were discovered in both mammalian viruses and in cellular genes, resulting in

24168-467: The virus particle moves to a new host cell. Viroids are another group of pathogens, but they consist only of RNA, do not encode any protein and are replicated by a host plant cell's polymerase. Reverse transcribing viruses replicate their genomes by reverse transcribing DNA copies from their RNA; these DNA copies are then transcribed to new RNA. Retrotransposons also spread by copying DNA and RNA from one another, and telomerase contains an RNA that

24327-492: The virus. It is one of five main hepatitis viruses: A , B, C , D , and E . During an initial infection, care is based on a person's symptoms. In those who develop chronic disease, antiviral medication such as tenofovir or interferon may be useful; however, these drugs are expensive. Liver transplantation is sometimes recommended for cases of cirrhosis or hepatocellular carcinoma . Hepatitis B infection has been preventable by vaccination since 1982. As of 2022,

24486-556: Was awarded for the elucidation of the atomic structure of the ribosome to Venki Ramakrishnan , Thomas A. Steitz , and Ada Yonath . In 2023 the Nobel Prize in Physiology or Medicine was awarded to Katalin Karikó and Drew Weissman for their discoveries concerning modified nucleosides that enabled the development of effective mRNA vaccines against COVID-19. In 1968, Carl Woese hypothesized that RNA might be catalytic and suggested that

24645-475: Was developed from bacterial genome systems, it can be used to target the genetic material in viruses. The use of the enzyme Cas9 can be a solution to many viral infections. Cas9 possesses the ability to target specific viruses by the targeting of specific strands of the viral genetic information. More specifically the Cas9 enzyme targets certain sections of the viral genome that prevents the virus from carrying out its normal function. Cas9 has also been used to disrupt

24804-407: Was discovered by three separate groups that the spacer regions were homologous to foreign DNA elements, including plasmids and viruses. These reports provided the first biological evidence that CRISPRs might function as an immune system. Cas9 has been used often as a genome-editing tool. Cas9 has been used in recent developments in preventing viruses from manipulating hosts' DNA. Since the CRISPR-Cas9

24963-481: Was made by Lurman in 1885. An outbreak of smallpox occurred in Bremen in 1883 and 1,289 shipyard employees were vaccinated with lymph from other people. After several weeks, and up to eight months later, 191 of the vaccinated workers became ill with jaundice and were diagnosed with serum hepatitis. Other employees who had been inoculated with different batches of lymph remained healthy. Lurman's paper, now regarded as

25122-480: Was noticed among the health worker group: the RV intramuscular route was significantly more effective compared with the RV intradermal route of administration. In assisted reproductive technology , sperm washing is not necessary for males with hepatitis B to prevent transmission, unless the female partner has not been effectively vaccinated. In females with hepatitis B, the risk of transmission from mother to child with IVF

25281-405: Was thought to be a eukaryotic phenomenon, a part of the explanation for why so much more transcription in higher organisms was seen than had been predicted. But as soon as researchers began to look for possible RNA regulators in bacteria, they turned up there as well, termed as small RNA (sRNA). Currently, the ubiquitous nature of systems of RNA regulation of genes has been discussed as support for

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