variants:
83-471: Cas12a ( C RISPR- as sociated protein 12a , previously known as Cpf1 ) is an RNA-guided endonuclease that forms an essential component of the CRISPR systems found in some bacteria and archaea . In its natural context, Cas12a targets and destroys the genetic material of viruses and other foreign mobile genetic elements , thereby protecting the host cell from infection. Like other Cas enzymes, Cas12a binds to
166-458: A protospacer adjacent motif (PAM) 5'-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase ), in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cas12a introduces a sticky-end-like DNA double- stranded break of 4 or 5 nucleotides overhang. The CRISPR-Cas12a system consist of a Cas12a enzyme and a guide RNA that finds and positions the complex at the correct spot on
249-461: A reporter or therapeutic gene. However, it was also published that the ITRs are not the only elements required in cis for the effective replication and encapsidation. A few research groups have identified a sequence designated cis-acting Rep-dependent element (CARE) inside the coding sequence of the rep gene. CARE was shown to augment the replication and encapsidation when present in cis . On
332-640: A tracrRNA (which in natural CRISPR systems must base-pair with a separate crRNA before binding to a Cas protein), instead binding a single crRNA. Both Cas12a and its guide RNA are smaller than the protein and RNA components of the Cas9 system; the crRNA of Cas12a is approximately half as long as sgRNAs used with Cas9. This reduced size renders Cas12a more suitable for applications such as in vivo delivery via adeno-associated virus (AAV), which have limited DNA packaging capacity due to their small capsids. The Cas12a-crRNA complex cleaves target DNA or RNA by identification of
415-468: A "guide" RNA (termed a crRNA , or CR ISPR RNA ) which targets it to a DNA sequence in a specific and programmable matter. In the host organism, the crRNA contains a constant region that is recognized by the Cas12a protein and a "spacer" region that is complementary to a piece of foreign nucleic acid (e.g. a portion of a phage genome) that previously infected the cell. As with Cas9 and other Cas proteins,
498-649: A CRISPR system protein, its naming, and a hidden Markov model (HMM) for its detection were provided in 2012 in a release of the TIGRFAMs database of protein families . Cas12a appears in many bacterial species. The ultimate Cas12a endonuclease that was developed into a tool for genome editing was taken from one of the first 16 species known to harbor it. Two candidate enzymes from Acidaminococcus and Lachnospiraceae display efficient genome-editing activity in human cells. CRISPR-Cas systems are separated into two classes: Class I , in which several Cas proteins associate with
581-867: A T-rich PAM and cleaves DNA via a staggered DNA DSB. In summary, important differences between Cas12a and Cas9 systems are that Cas12a: Cas12 endonucleases ultimately likely evolved from the TnpB endonuclease of IS200/IS605-family transposons. TnpB , not yet "domesticated" into the CRISPR immune system, are themselves able to perform RNA-guided cleavage using a OmegaRNA template system. Multiple aspects influence target efficiency and specificity when using CRISPR, including guide RNA design. Many design models and CRISPR-Cas software tools for optimal design of guide RNA have been developed. These include SgRNA designer, CRISPR MultiTargeter, SSFinder. In addition, commercial antibodies are available for use to detect Cas12a protein. CRISPR-Cas9
664-467: A caveat, scAAV can only encode half of the already limited capacity of AAV. Recent reports suggest that scAAV vectors are more immunogenic than single stranded adenovirus vectors, inducing a stronger activation of cytotoxic T lymphocytes . Humoral immunity instigated by infection with the wild type is thought to be common. The associated neutralising activity limits the usefulness of the most commonly used serotype AAV2 in certain applications. Accordingly,
747-504: A co-receptor activity and enable AAV to enter the cell by receptor-mediated endocytosis . These study results have been disputed by Qiu, Handa, et al. HSPG functions as the primary receptor, though its abundance in the extracellular matrix can scavenge AAV particles and impair the infection efficiency. Studies have shown that serotype 2 of the virus (AAV-2) apparently kills cancer cells without harming healthy ones. "Our results suggest that adeno-associated virus type 2, which infects
830-463: A contaminant in adenovirus preparations, was first identified as a dependoparvovirus in the 1960s in the laboratories of Bob Atchison at Pittsburgh and Wallace Rowe at NIH . Serological studies in humans subsequently indicated that, despite being present in people infected by helper viruses such as adenovirus or herpes virus, AAV itself did not cause any disease. Wild-type AAV has attracted considerable interest from gene therapy researchers due to
913-472: A crRNA to build a functional endonuclease, and Class II , in which a single Cas endonuclease associates with a crRNA; Class II is further divided into Type II , Type V , and Type VI systems. Cas12a is identified as a Class II, Type V CRISPR-Cas system. The acronym CRISPR ( C lustered R egularly I nterspaced S hort P alindromic R epeats) refers to the invariant DNA sequences found in bacteria and archaea which encode Cas proteins and their crRNAs. Cas12a
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#1732868737911996-455: A crucial role in RNA processing, a fundamental step in gene expression. This process involves the precise cleavage of precursor RNA molecules, guided by endonucleases, to generate functional RNAs essential for various cellular functions. Endonucleases selectively cleave precursor RNAs at specific sites, defining the boundaries of functional RNA segments during RNA processing. The outcome of RNA processing
1079-549: A deoxyribose sugar with a missing base. The AP endonuclease recognizes this sugar and essentially cuts the DNA at this site and then allows for DNA repair to continue. E. coli cells contain two AP endonucleases: endonuclease IV (endoIV) and exonuclease III (exoIII) while in eukaryotes, there is only one AP endonuclease. [REDACTED] Repair of DNA in which the two complementary strands are joined by an interstrand covalent crosslink requires multiple incisions in order to disengage
1162-433: A double-strand break, further steps are required to complete the repair process. If a crosslink is not properly repaired it can block DNA replication . Exposure of bacteriophage (phage) T4 to ultraviolet irradiation induces thymine dimers in the phage DNA. The phage T4 denV gene encodes endonuclease V that catalyzes the initial steps in the repair of these UV-induced thymine dimers. Endonuclease V first cleaves
1245-721: A group of neurodegenerative autosomal recessive disorders that is caused by mutations in three of the four different subunits of the tRNA-splicing endonuclease complex. Adeno-associated virus Adeno-associated viruses ( AAV ) are small viruses that infect humans and some other primate species . They belong to the genus Dependoparvovirus , which in turn belongs to the family Parvoviridae . They are small (approximately 26 nm in diameter) replication-defective , nonenveloped viruses and have linear single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb). Several features make AAV an attractive candidate for creating viral vectors for gene therapy , and for
1328-517: A helper virus, but usually AAV can not replicate and kill a cell on its own. The minimal set of the adenoviral genes required for efficient generation, of progeny AAV particles, was discovered by Matsushita, Ellinger et al. This discovery allowed for new production methods of recombinant AAV, which do not require adenoviral co-infection of the AAV-producing cells. In the absence of helper virus or genotoxic factors, AAV DNA can either integrate into
1411-473: A number of diseases, including Leber's congenital amaurosis , hemophilia , congestive heart failure , spinal muscular atrophy , lipoprotein lipase deficiency , and Parkinson's disease . The AAV genome is built of single-stranded deoxyribonucleic acid (ss DNA ), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises ITRs at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap . The former
1494-405: A number of features. Chief amongst these was the virus's apparent lack of pathogenicity. It can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) in the human chromosome 19 . This feature makes it somewhat more predictable than retroviruses , which present the threat of a random insertion and of mutagenesis, which
1577-399: A positive-sensed AAV genome encodes overlapping sequences of three capsid proteins, VP1, VP2 and VP3, and two accessory proteins, MAAP & AAP, which start from one promoter, designated p40. The molecular weights of these proteins are 87, 72 and 62 kiloDaltons, respectively. The AAV capsid is composed of a mixture of VP1, VP2, and VP3 totaling 60 monomers arranged in icosahedral symmetry in
1660-630: A ratio of 1:1:10, with an empty mass of approximately 3.8 MDa . The crystal structure of the VP3 protein was determined by Xie, Bue, et al. The cap gene produces an additional, non-structural protein called the Assembly-Activating Protein (AAP). This protein is produced from ORF2 and is essential for the capsid-assembly process. The exact function of this protein in the assembly process and its structure have not been solved to date. All three VPs are translated from one mRNA. After this mRNA
1743-427: A specific DNA sequence. The nucleotide sequence recognized for cleavage by a restriction enzyme is called the restriction site . Typically, a restriction site will be a palindromic sequence about four to six nucleotides long. Most restriction endonucleases cleave the DNA strand unevenly, leaving complementary single-stranded ends. These ends can reconnect through hybridization and are termed "sticky ends". Once paired,
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#17328687379111826-539: A wave of childhood hepatitis. Although AAV2 is the most popular serotype in various AAV-based research, it has been shown that other serotypes can be more effective as gene delivery vectors. AAV9 passes the blood-brain-barrier in humans, AAV6 appears much better in infecting airway epithelial cells, AAV7 presents very high transduction rate of murine skeletal muscle cells (similar to AAV1 and AAV5), AAV8 transduce hepatocytes and AAV1 and 5 were shown to be very efficient in gene delivery to vascular endothelial cells. In
1909-517: Is VP1. Since the bigger intron is preferred to be spliced out, and since in the major splice the ACG codon is a much weaker translation initiation signal, the ratio at which the AAV structural proteins are synthesized in vivo is about 1:1:20, which is the same as in the mature virus particle. The unique fragment at the N terminus of VP1 protein was shown to possess the phospholipase A2 (PLA2) activity, which
1992-476: Is a helper virus, AAV's gene expression activates, allowing the virus to replicate using the host cell's polymerase. When the helper virus kills the host cell, the new AAV virions are released. If there is not a helper virus present, AAV exhibits lysogenic behavior. When AAV infects a cell alone, its gene expression is repressed (AAV does not replicate), and its genome is incorporated into the host genome (into human chromosome 19). In rare cases, lysis can occur without
2075-492: Is a rare, autosomal recessive disease caused by a defective UV-specific endonuclease. Patients with mutations are unable to repair DNA damage caused by sunlight. Sickle Cell anemia is a disease caused by a point mutation. The sequence altered by the mutation eliminates the recognition site for the restriction endonuclease MstII that recognizes the nucleotide sequence. tRNA splicing endonuclease mutations cause pontocerebellar hypoplasia. Pontocerebellar hypoplasias (PCH) represent
2158-549: Is activated to initiate controlled cellular disassembly. This disintegration is characterized by the cleavage of genomic DNA into specific fragments. The precise role of endonucleases in this context is to cleave the DNA at specific sites, generating fragments with defined lengths. These fragments are then packaged into apoptotic bodies, ensuring a neat and efficient removal of the dying cell without causing inflammation or damage to neighboring cells. Flap endonuclease 1 (FEN1) and Dna2 endonuclease are integral to DNA replication on
2241-432: Is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact to form a capsid with icosahedral symmetry. The inverted terminal repeat (ITR) sequences comprise 145 bases each. They were named so because of their symmetry, which was shown to be required for efficient multiplication of
2324-455: Is cut out, resulting in a reduced overall level of VP1 protein synthesis. The first AUG codon that remains in the major splice is the initiation codon for VP3 protein. However, upstream of that codon in the same open reading frame lies an ACG sequence (encoding threonine) which is surrounded by an optimal Kozak context . This contributes to a low level of synthesis of VP2 protein, which is actually VP3 protein with additional N terminal residues, as
2407-460: Is known to instigate robust humoral immunity in animal models and in the human population, where up to 80% of individuals are thought to be seropositive for AAV2. Antibodies are known to be neutralising, and for gene therapy applications these do impact vector transduction efficiency via some routes of administration. As well as persistent AAV specific antibody levels, it appears from both prime-boost studies in animals and from clinical trials that
2490-399: Is lost through cell division, since the episomal DNA is not replicated along with the host cell DNA. Random integration of AAV DNA into the host genome is detectable but occurs at very low frequency. AAVs also present very low immunogenicity , seemingly restricted to generation of neutralizing antibodies , while they induce no clearly defined cytotoxic response . This feature, along with
2573-417: Is of the form " Vwx yZ", where " Vwx " are, in italics, the first letter of the genus and the first two letters of the species where this restriction endonuclease may be found, for example, Escherichia coli , Eco , and Haemophilus influenzae , Hin . This is followed by the optional, non-italicized symbol "y", which indicates the type or strain identification, for example, Eco R for E. coli strains bearing
Cas12a - Misplaced Pages Continue
2656-428: Is probably required for the releasing of AAV particles from late endosomes . Muralidhar et al. reported that VP2 and VP3 are crucial for correct virion assembly. More recently, however, Warrington et al. showed VP2 to be unnecessary for the complete virus particle formation and an efficient infectivity, and also presented that VP2 can tolerate large insertions in its N terminus, while VP1 can not, probably because of
2739-472: Is sometimes followed by development of a cancer. The AAV genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency. Development of AAVs as gene therapy vectors, however, has eliminated this integrative capacity by removal of the rep and cap from the DNA of the vector. The desired gene together with a promoter to drive transcription of
2822-563: Is subject to Intellectual property disputes while CRISPR-Cas12a does not have the same issues. Endonuclease In molecular biology , endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain (namely DNA or RNA ). Some, such as deoxyribonuclease I , cut DNA relatively nonspecifically (with regard to sequence), while many, typically called restriction endonucleases or restriction enzymes , cleave only at very specific nucleotide sequences. Endonucleases differ from exonucleases , which cleave
2905-429: Is synthesized, it can be spliced in two different manners: either a longer or shorter intron can be excised resulting in the formation of two pools of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA pool. Usually, especially in the presence of adenovirus, the longer intron is preferred, so the 2.3-kb-long mRNA represents the so-called "major splice". In this form the first AUG codon , from which the synthesis of VP1 protein starts,
2988-537: Is the fact that AAV genomes are epigenetically methylated during production. Besides price, these findings might affect expression kinetics, rAAV receptor binding, trafficking, vector immunogenicity, and expression durability. Two species of AAV were recognised by the International Committee on Taxonomy of Viruses in 2013: adeno-associated dependoparvovirus A (formerly AAV-1, −2, −3 and −4) and adeno-associated dependoparvovirus B (formerly AAV-5). Until
3071-439: Is the production of functional RNA molecules, such as transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) . Endonucleases contribute to the precision of this process, ensuring the formation of mature and functional RNA species. Endonucleases like RNase P and tRNase Z (ELAC2), shape precursor tRNAs into mature, functional tRNAs, crucial for accurate translation during protein synthesis. In ribosome biogenesis, endonucleases from
3154-478: The Eco RI enzyme recognizes and cleaves the sequence 5' – GAATTC – 3'. Restriction endonucleases come in several types. A restriction endonuclease typically requires a recognition site and a cleavage pattern (typically of nucleotide bases: A, C, G, T). If the recognition site is outside the region of the cleavage pattern, then the restriction endonuclease is referred to as Type I. If the recognition sequence overlaps with
3237-505: The RNase III family , like DROSHA , play a role in processing precursor rRNAs, contributing to the assembly of functional ribosomes. DICER and DROSHA also from the RNase III family play a role in the processing pre-miRNA to functional miRNA. The endonuclease DNase1L2 also contribute prominently to the removal of DNA during the formation of hair and nails. This process is essential for
3320-590: The hairpin formed by the ITR in the self-priming act and cleave at a specific region, designated terminal resolution site, within the hairpin. They were also shown to be necessary for the AAVS1-specific integration of the AAV genome. All four Rep proteins were shown to bind ATP and to possess helicase activity. It was also shown that they upregulate the transcription from the p40 promoter (mentioned below), but downregulate both p5 and p19 promoters. The right side of
3403-410: The maturation of hair and nail structures and is crucial for the transformation of cells into durable and keratinized structures, ensuring the strength and integrity of hair and nails. Restriction endonucleases may be found that cleave standard dsDNA (double-stranded DNA), or ssDNA (single-stranded DNA), or even RNA. This discussion is restricted to dsDNA; however, the discussion can be extended to
Cas12a - Misplaced Pages Continue
3486-441: The platelet-derived growth factor receptor. There have been many efforts to engineer and improve new AAV variants for both clinical and research purposes. Such modifications include new tropisms to target specific tissues, and modified surface residues to evade detection by the immune system. Beyond opting for particular strains of recombinant AAV (rAAV) to target particular cells, researchers have also explored AAV pseudotyping,
3569-511: The "left side" of the genome there are two promoters called p5 and p19, from which two overlapping messenger ribonucleic acids ( mRNAs ) of different length can be produced. Each of these contains an intron which can be either spliced out or not. Given these possibilities, four various mRNAs, and consequently four various Rep proteins with overlapping sequence can be synthesized. Their names depict their sizes in kilodaltons (kDa): Rep78, Rep68, Rep52 and Rep40. Rep78 and 68 can specifically bind
3652-491: The 1990s, virtually all AAV biology was studied using AAV serotype 2. However, AAV is highly prevalent in humans and other primates and several serotypes have been isolated from various tissue samples. Serotypes 2, 3, 5, and 6 were discovered in human cells, AAV serotypes 1, 4, and 7–11 in nonhuman primate samples. As of 2006 there have been 11 AAV serotypes described, the 11th in 2004. AAV capsid proteins contain 12 hypervariable surface regions, with most variability occurring in
3735-473: The AAV DNA combined with generation of a fully assembled, deoxyribonuclease -resistant AAV particles. With regard to gene therapy, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural ( cap ) and packaging ( rep ) proteins can be delivered in trans . With this assumption many methods were established for efficient production of recombinant AAV (rAAV) vectors containing
3818-425: The AAV capsid in vitro , it appears that there may be a cytotoxic T lymphocyte response to AAV vectors. Cytotoxic responses would imply the involvement of CD4+ T helper cells in the response to AAV and in vitro data from human studies suggests that the virus may indeed induce such responses, including both Th1 and Th2 memory responses. A number of candidate T cell stimulating epitopes have been identified within
3901-478: The AAV capsid protein VP1, which may be attractive targets for modification of the capsid if the virus is to be used as a vector for gene therapy. There are several steps in the AAV infection cycle, from infecting a cell to producing new infectious particles: Some of these steps may look different in various types of cells, which, in part, contributes to the defined and quite limited native tropism of AAV. Replication of
3984-425: The AAV genome. The feature of these sequences that gives them this property is their ability to form a hairpin , which contributes to so-called self-priming that allows primase -independent synthesis of the second DNA strand. The ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome (19th chromosome in humans) and rescue from it, as well as for efficient encapsidation of
4067-675: The B-cell memory is also strong. In seropositive humans, circulating IgG antibodies for AAV2 appear to be primarily composed of the IgG1 and IgG2 subclasses, with little or no IgG3 or IgG4 present. The cell-mediated response to the virus and to vectors is poorly characterised, and has been largely ignored in the literature as recently as 2005. Clinical trials using an AAV2-based vector to treat haemophilia B seem to indicate that targeted destruction of transduced cells may be occurring. Combined with data that shows that CD8+ T-cells can recognise elements of
4150-476: The PLA2 domain presence. Recent discoveries made through use of high-throughput 'omics approaches include the fact that AAV capsids are post-translationally modified (PTM) during production such as acetylation, methylation, phosphorylation, deamidation, O-GlycNAcylation and SUMOylation throughout capsid proteins VP1, VP2 and VP3. These PTMs differ depending on the manufacturing production platform. Another such discovery
4233-576: The RuvC domain of Cas9, and a zinc finger -like domain. Unlike Cas9, Cas12a does not have an HNH endonuclease domain, and the N-terminal region of Cas12a does not have an alpha-helical recognition lobe as seen in Cas9. The Cas12a loci encode Cas1 , Cas2 and Cas4 proteins more similar to types I and III than from type II systems. Database searches suggest the abundance of Cas12a-family proteins in many bacterial species. Also unlike Cas9, Cas12a does not require
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#17328687379114316-450: The ability to infect quiescent cells present their dominance over adenoviruses as vectors for human gene therapy . Use of the virus does present some disadvantages. The cloning capacity of the vector is relatively limited and most therapeutic genes require the complete replacement of the virus's 4.8 kilobase genome. Large genes are, therefore, not suitable for use in a standard AAV vector. Options are currently being explored to overcome
4399-417: The brain, most AAV serotypes show neuronal tropism, while AAV5 also transduces astrocytes. AAV6, a hybrid of AAV1 and AAV2, also shows lower immunogenicity than AAV2. Serotypes can differ with the respect to the receptors they are bound to. For example, AAV4 and AAV5 transduction can be inhibited by soluble sialic acids (of different form for each of these serotypes), and AAV5 was shown to enter cells via
4482-512: The cleavage sequence, then the restriction endonuclease restriction enzyme is Type II. Endonucleases play a role in many aspects of biological life. Below are a couple examples of processes where endonucleases play a crucial role. Endonucleases play a role in DNA repair. AP endonuclease , specifically, catalyzes the incision of DNA exclusively at AP sites, and therefore prepares DNA for subsequent excision, repair synthesis and DNA ligation. For example, when depurination occurs, this lesion leaves
4565-481: The creation of isogenic human disease models . Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell. In the native virus, however, integration of virally carried genes into the host genome does occur. Integration can be important for certain applications, but can also have unwanted consequences. Recent human clinical trials using AAV for gene therapy in
4648-407: The cut site; these and other differences may make it more suitable for certain applications. Beyond its use in basic research , CRISPR-Cas12a could have applications in the treatment of genetic illnesses and in implementing gene drives . CRISPR-Cas12a was found by searching a published database of bacterial genetic sequences for promising bits of DNA. Its identification through bioinformatics as
4731-401: The double helix to cleave target DNA. CRISPR-Cas12a systems activity has three stages: Cas9 requires two RNA molecules to cut DNA while Cas12a needs one. The proteins also cut DNA at different places, offering researchers more options when selecting an editing site. Cas9 cuts both strands in a DNA molecule at the same position, leaving behind blunt ends . Cas12a leaves one strand longer than
4814-671: The drug resistance transfer factor RTF-1, Eco B for E. coli strain B, and Hin d for H. influenzae strain d . Finally, when a particular type or strain has several different restriction endonucleases, these are identified by Roman numerals, thus, the restriction endonucleases from H. influenzae strain d are named Hin dI, Hin dII, Hin dIII, etc. Another example: " Hae II" and " Hae III" refer to bacterium Haemophilus aegyptius (strain not specified), restriction endonucleases number II and number III, respectively. The restriction enzymes used in molecular biology usually recognize short target sequences of about 4 – 8 base pairs. For instance,
4897-559: The efficacy of AAV-mediated gene therapy. Host immune response has been shown to respond to the AAV vectors, the transduced cells, and the transduced proteins. The immune response can be subdivided into two categories: innate and adaptive, the latter of which is divided into humoral and cell-mediated. The innate immune response to the AAV vectors has been characterised in animal models. Intravenous administration in mice causes transient production of pro-inflammatory cytokines and some infiltration of neutrophils and other leukocytes into
4980-413: The ends of recognition sequences instead of the middle ( endo ) portion. Some enzymes known as " exo-endonucleases ", however, are not limited to either nuclease function, displaying qualities that are both endo- and exo-like. Evidence suggests that endonuclease activity experiences a lag compared to exonuclease activity. Restriction enzymes are endonucleases from eubacteria and archaea that recognize
5063-662: The following: In addition, research is now underway to construct synthetic or artificial restriction endonucleases, especially with recognition sites that are unique within a genome. Restriction endonucleases or restriction enzymes typically cleave in two ways: blunt-ended or sticky-ended patterns. An example of a Type I restriction endonuclease. Furthermore, there exist DNA/RNA non-specific endonucleases , such as those that are found in Serratia marcescens , which act on dsDNA, ssDNA, and RNA. Below are tables of common prokaryotic and eukaryotic endonucleases. Xeroderma pigmentosa
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#17328687379115146-429: The gene is inserted between the inverted terminal repeats (ITRs) that aid in concatemer formation in the nucleus after the single-stranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA. AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact for the life of the host cell. In dividing cells, AAV DNA
5229-404: The glycosylic bond on the 5’ side of a pyrimidine dimer and then catalyzes cleavage of the DNA phosphodiester bond that originally linked the two nucleotides of the dimer. Subsequent steps in the repair process involve removal of the dimer remnants and repair synthesis to fill in the resulting single-strand gap using the undamaged strand as template. During apoptosis, Apoptotic endonuclease DFF40
5312-451: The lagging strand, participating in crucial processes such as primer removal and Okazaki fragment processing. Endonucleases are actively involved in processing these fragments by cleaving the phosphodiester bonds between them. This process is integral to the seamless synthesis and joining of Okazaki fragments, contributing to the overall continuity of the newly replicated DNA strand. Endonucleases, more specifically endoribonuclease , play
5395-448: The limited coding capacity: Because of AAV's specialized gene therapy advantages, researchers have created an altered version of AAV termed self-complementary adeno-associated virus (scAAV) . Whereas AAV packages a single strand of DNA and must wait for its second strand to be synthesized, scAAV packages two shorter strands that are complementary to each other. By avoiding second-strand synthesis, scAAV can express more quickly, although as
5478-492: The liver, which seems to sequester a large percentage of the injected viral particles. Both soluble factor levels and cell infiltration appear to return to baseline within six hours. By contrast, more aggressive viruses produce innate responses lasting 24 hours or longer. In-vivo studies indicate that AAV vectors interact with the Toll-like receptor (TLR)9- and TLR2-MyD88 pathways to trigger the innate immune response by stimulating
5561-400: The majority of clinical trials under way involve delivery of AAV2 into the brain, a relatively immunologically privileged organ. In the brain, AAV2 is strongly neuron-specific. As of 2019, AAV vectors have been used in over 250 clinical trials worldwide, approximately 8.3% of virus-vectored gene-therapy trials. Recently, promising results have been obtained from Phase 1 and Phase 2 trials for
5644-529: The majority of the population but has no known ill effects, kills multiple types of cancer cells yet has no effect on healthy cells," said Craig Meyers, a professor of immunology and microbiology at the Penn State College of Medicine in Pennsylvania in 2005. This could lead to a new anti-cancer agent. In March 2023, a series of Nature papers linked infection of adeno-associated virus 2 (AAV2) to
5727-476: The more famous endonucleases is Cas9 . Ultimately, there are three categories of restriction endonucleases that relatively contribute to the cleavage of specific sequences. The types I and III are large multisubunit complexes that include both the endonucleases and methylase activities. Type I can cleave at random sites of about 1000 base pairs or more from the recognition sequence and it requires ATP as source of energy. Type II behaves slightly differently and
5810-403: The most extensively examined so far. AAV2 presents natural tropism towards skeletal muscles , neurons , vascular smooth muscle cells and hepatocytes . Three cell receptors have been described for AAV2: heparan sulfate proteoglycan (HSPG), a V β 5 integrin and fibroblast growth factor receptor 1 (FGFR-1). The first functions as a primary receptor, while the latter two have
5893-462: The other, creating sticky ends . The sticky ends have different properties than blunt ends during non-homologous end joining or homologous repair of DNA, which confers certain advantages to Cas12a when attempting gene insertions, compared to Cas9. Although the CRISPR-Cas9 system can efficiently disable genes, it is challenging to insert genes or generate a knock-in. Cas12a lacks tracrRNA , utilizes
5976-710: The phosphodiester bonds of the fragments can be joined by DNA ligase . There are hundreds of restriction endonucleases known, each attacking a different restriction site. The DNA fragments cleaved by the same endonuclease can be joined regardless of the origin of the DNA. Such DNA is called recombinant DNA ; DNA formed by the joining of genes into new combinations. Restriction endonucleases ( restriction enzymes ) are divided into three categories, Type I, Type II, and Type III, according to their mechanism of action. These enzymes are often used in genetic engineering to make recombinant DNA for introduction into bacterial, plant, or animal cells, as well as in synthetic biology . One of
6059-401: The practice of creating hybrids of certain AAV strains to approach an even more refined target. The hybrid is created by taking a capsid from one strain and the genome from another strain. For example: Other efforts to engineer and improve new AAV variants have involved the ancestral reconstruction of virus variants to generate new vectors with enhanced properties for clinical applications and
6142-536: The production of interferons. It's shown that mice deficient in TLR9 are more receptive to AAV treatment and demonstrate higher levels of transgene expression Due to previous natural infection, many people have preexisting neutralizing antibodies (NAbs) against AAV's, which can significantly hinder its application in gene therapy. Even though AAV's are highly variable among wild-type and synthetic variants, antibody recognition sites may be conserved evolutionarily. The virus
6225-472: The programmable DNA-targeting activity of Cas12a makes it a useful tool for biotechnology and biological research applications. By modifying the spacer sequence in the crRNA, researchers can target Cas12a to specific DNA sequences, allowing for highly targeted modifications of DNA . Cas12a is distinguished from Cas9 by a its single RuvC endonuclease active site, its 5' protospacer adjacent motif preference, and its formation of sticky rather than blunt ends at
6308-473: The retina have shown promise. In March 2023, a series of Nature papers detected high titres of adeno-associated virus 2 (AAV2), alongside adenovirus and herpesvirus, in samples from a wave of childhood hepatitis. One paper suggested that AAV2 co-infection may contribute to more serious liver disease than infection with only adeno- or herpesviruses and that the causal link remains to be established. The adeno-associated virus (AAV), previously thought to be
6391-435: The strands and remove the damage. Incisions are required on both sides of the crosslink and on both strands of the duplex DNA. In mouse embryonic stem cells, an intermediate stage of crosslink repair involves production of double-strand breaks. MUS81 / EME1 is a structure specific endonuclease involved in converting interstrand crosslinks to double-strand breaks in a DNA replication-dependent manner. After introduction of
6474-413: The study of AAV biology. AAV is of particular interest to gene therapists due to its apparent limited capacity to induce immune responses in humans, a factor which should positively influence vector transduction efficiency while reducing the risk of any immune-associated pathology . AAV is not considered to have any known role in disease. However, host immune system response and immune tolerance reduce
6557-420: The threefold proximal peaks, but the parvovirus genome in general presents highly conserved replication and structural genes across serotypes. All of the known serotypes can infect cells from multiple diverse tissue types. Tissue specificity is determined by the capsid serotype and pseudotyping of AAV vectors to alter their tropism range will likely be important to their use in therapy. Serotype 2 (AAV2) has been
6640-415: The virus can also vary in one cell type, depending on the cell's current cell cycle phase. The characteristic feature of the adeno-associated virus is a deficiency in replication and thus its inability to multiply in unaffected cells. Adeno-associated virus spreads by co-infecting a cell with a helper virus. The first helper virus that was described as providing successful generation of new AAV particles,
6723-440: Was first isolated by Hamilton Smith in 1970. They are simpler versions of the endonucleases and require no ATP in their degradation processes. Some examples of type II restriction endonucleases include Bam HI, Eco RI, Eco RV, Hin dIII, and Hae III. Type III, however, cleaves the DNA at about 25 base pairs from the recognition sequence and also requires ATP in the process. The commonly used notation for restriction endonucleases
6806-452: Was originally known as Cpf1 , an abbreviation of CRISPR and two genera of bacteria where it appears, Prevotella and Francisella . It was renamed in 2015 after a broader rationalization of the names of Cas ( C RISPR as sociated) proteins to correspond to their sequence homology . The Cas12a protein contains a mixed alpha/beta domain, a RuvC -like endonuclease domain (broken into two non-contiguous segments, RuvC-I and RuvC-II) similar to
6889-432: Was the adenovirus, from which the AAV name originated. It was then shown that AAV replication can be facilitated by selected proteins derived from the adenovirus genome, by other viruses such as HSV or vaccinia, or by genotoxic agents, such as UV irradiation or hydroxyurea . Depending on the presence or absence of a helper virus, the life cycle of AAV follows either a lytic or lysogenic pathway, respectively. If there
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