text:
So lately (as in the past 2 days) i've been getting these sharp pains in my stomach. Its kinda like a shooting pain, it hurts for a moment then its gone. I still feel my baby kick regularly and i have no bleeding, but should i be concerned?
Sharp Pain...ouuuchh
"Nobody will ever know the strength of my love for you, after all you're the only one who knows what my heart sounds like from the inside"
Comments (6)
*Cole 2/8/10* *Jake 3/26/12*
I had this on and off yesterday. The pain was right around my belly button. So far this morning I haven't felt it and last night I didn't feel anything while sleeping. I read somewhere that there is a big growth spurt between 21 and 23 weeks, so I was guessing it was stretching. Either that or gas! If I keep feeling it today I might call my ob just to be sure and to ease my mind. My baby was keeping me awake last night kicking away too, so I'm guessing it was nothing.
Karen full-time working wife to Jason 3/12/05 mom to Travis 5/5/09 and Tanner 4/22/12
I've had those too. Almost like a shit cramp. Lol.
Lizz (31) and Adrian (37) married 9/25/10.
Furkids Ludo (pitbull x) and Pica (shepherd x).
Adia Gwen or Raiden (Rye-den) Gabriel due 4/25/12!
I am also having the same sharp pain.
Proud Mommy to Jordyn Demond!!! April 2, 2012
I asked my midwife about this on Tuesday actually. She said that there is a nerve there that is about as thick as a pencil. As the belly grows to accommodate the baby, this nerve gets stretched out. By the end of the pregnancy it will be flat. The pain I'm feeling is it getting stretched b/c apparently nerves don't like that too much.
April 2012 - Kaija
Aug. 2015 - Nora


text:
Bay roan or rabicano?
1 Attachment(s)
So I've always thought my QH was a bay roan....but I've always noticed he doesnt "roan out" as much as a lot of roans I've seen. I tell my friend he's a wanna-b roan haha! Recently I had someone tell me hes actually a Rabicano, I'd never heard of that before, but from pics it does look more like him...What do you experts say? Its like he gets patches of it...I don't have a good pic of his chest but thats like the most normal roan looking place lol
Brown roan.
1 Attachment(s)
That's him trying to be brown
Posted via Mobile Device
1 Attachment(s)
Ha so I wasn't right about either! I was always told he was a bay roan till someone told me he was a rabicano because of the white above his tail! Well nice to know for sure now
Bay and brown are often confused as they are modifications of the same gene (agouti). The way to tell if he's bay or brown is that brown lightens the soft points of the horse (muzzle, inside of elbow and flank, under eyes).
Posted via Mobile Device
The roan/rabicano confusion in your case comes from the fact that roans tend to get more solid colored in appearance with their winter coats. In a very similar situation, my BOs used to own a mare registered blue roan. They got her in the winter and I disagreed, saying she was a brown rabicano because that was exactly that she looked like, but I forgot to account for the winter roan look. She was also a brown roan.
1 Attachment(s)
Oo, I see! He actually doesn't change TOO much in the winter, little darker but for the most part same graying, some patches tend to come and go though lol The pic with the yellow thing wrapped on him its his winterish coat, I don't remember exactly what month but it was winter, then the pic with his shipping boots was in april, so spring coat.
1 Attachment(s)
Hm thats from Jan, terrible pic to judge by haha but his chest doesnt have as much roan so hes def darker, can't find a good picture :( Guess I don't notice him getting darker since I see him so much but when I look at pics he is darker just still has a few goofy spots haha
Do you think he'll ever get more "roany" looking, lol...or always just kinda have his patches and light roaning
You have to remember too that with Rabicano, the "roaning" originates from the flank and spreads as compared to normal roan which covers the body (except the head and legs). Plus rabicano's coon tails are generally more "barred" looking rather than just a bunch of white.
|All times are GMT -4. The time now is 04:04 PM.
Powered by vBulletin® Version 3.8.5
Copyright ©2000 - 2016, Jelsoft Enterprises Ltd.
Search Engine Friendly URLs by vBSEO 3.6.0

subset: commoncrawl


text:
Imperial guards by vega218Watch
©2011-2016 vega218
holy shit the armor detail
Amazing art, bravo!
Gorgeous concept.
I like how you drew images that seem to come from The Romance of the Three Kingdoms with a great deal of personal touch but still keeping the basic elements there. Very nice, I like it.
i love it very much but If u dont mind i would like to bring your attention to the sword . The buttom of it i think is bit too bright (light on metal part) so it look like it bend wrong way. But its only me )
were did u think this up????? its awsome
wow! nice coloring.
Those are some intense guards ><


text:
Summary: CRISPR associated protein
Pfam includes annotations and additional family information from a range of different sources. These sources can be accessed via the tabs below.
This is the Wikipedia entry entitled "CRISPR". More...
The Wikipedia text that you see displayed here is a download from Wikipedia. This means that the information we display is a copy of the information from the Wikipedia database. The button next to the article title ("Edit Wikipedia article") takes you to the edit page for the article directly within Wikipedia. You should be aware you are not editing our local copy of this information. Any changes that you make to the Wikipedia article will not be displayed here until we next download the article from Wikipedia. We currently download new content on a nightly basis.
Does Pfam agree with the content of the Wikipedia entry ?
Pfam has chosen to link families to Wikipedia articles. In some case we have created or edited these articles but in many other cases we have not made any direct contribution to the content of the article. The Wikipedia community does monitor edits to try to ensure that (a) the quality of article annotation increases, and (b) vandalism is very quickly dealt with. However, we would like to emphasise that Pfam does not curate the Wikipedia entries and we cannot guarantee the accuracy of the information on the Wikipedia page.
Editing Wikipedia articles
Before you edit for the first time
Wikipedia is a free, online encyclopedia. Although anyone can edit or contribute to an article, Wikipedia has some strong editing guidelines and policies, which promote the Wikipedia standard of style and etiquette. Your edits and contributions are more likely to be accepted (and remain) if they are in accordance with this policy.
You should take a few minutes to view the following pages:
How your contribution will be recorded
Anyone can edit a Wikipedia entry. You can do this either as a new user or you can register with Wikipedia and log on. When you click on the "Edit Wikipedia article" button, your browser will direct you to the edit page for this entry in Wikipedia. If you are a registered user and currently logged in, your changes will be recorded under your Wikipedia user name. However, if you are not a registered user or are not logged on, your changes will be logged under your computer's IP address. This has two main implications. Firstly, as a registered Wikipedia user your edits are more likely seen as valuable contribution (although all edits are open to community scrutiny regardless). Secondly, if you edit under an IP address you may be sharing this IP address with other users. If your IP address has previously been blocked (due to being flagged as a source of 'vandalism') your edits will also be blocked. You can find more information on this and creating a user account at Wikipedia.
If you have problems editing a particular page, contact us at email@example.com and we will try to help.
Contact us
The community annotation is a new facility of the Pfam web site. If you have problems editing or experience problems with these pages please contact us.
CRISPR Edit Wikipedia article
CRISPRs (clustered regularly interspaced short palindromic repeats) are DNA loci containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a virus.[2]
CRISPRs are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea.[3][4]
CRISPRs are often associated with cas genes that code for proteins related to CRISPRs. The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages[5][6] and provides a form of acquired immunity. CRISPR spacers recognize and cut these exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.[2]
Since 2013, the CRISPR/Cas system has been used for gene editing (adding, disrupting or changing the sequence of specific genes) and gene regulation in species throughout the tree of life.[7] By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location.
It may be possible to use CRISPR to build RNA-guided gene drives capable of altering the genomes of entire populations.[8]
- 1 History
- 2 Locus structure
- 3 Mechanism
- 4 Evolution and diversity
- 5 Bioinformatic identification of CRISPRs in genomes and metagenomes
- 6 Evolutionary significance
- 7 Applications
- 8 Functions
- 9 Automation and library support
- 10 Patent
- 11 See also
- 12 References
- 13 Further reading
- 14 External links
Bacteria may incorporate foreign DNA in other circumstances and even scavenge damaged DNA from their environment.[9]
Repeats were first described in 1987 for the bacterium Escherichia coli.[10] In 2000, similar clustered repeats were identified in additional bacteria and archaea and were termed Short Regularly Spaced Repeats (SRSR).[11] SRSR were renamed CRISPR in 2002.[12] A set of genes, some encoding putative nuclease or helicase proteins, were found to be associated with CRISPR repeats (the cas, or CRISPR-associated genes).[12]
In 2005, three independent researchers showed that CRISPR spacers showed homology to several phage DNA and extrachromosomal DNA such as plasmids. This was an indication that the CRISPR/cas system could have a role in adaptive immunity in bacteria.[1] Koonin and colleagues proposed that spacers serve as a template for RNA molecules, analogously to eukaryotic cells that use a system called RNA interference.[13]
In 2007 Barrangou, Horvath (food industry scientists at Danisco) and Moineau's group at Université Laval (Canada) showed that they could alter the resistance of Streptococcus thermophilus to phage attack with spacer DNA.[13]
Doudna and Charpentier had independently been exploring CRISPR-associated proteins to learn how bacteria deploy spacers in their immune defenses. They jointly studied a simpler CRISPR system that relies on a protein called Cas9. They found that bacteria respond to an invading phage by transcribing spacers and palindromic DNA into a long RNA molecule that the cell then uses tracrRNA and Cas9 to cut it into pieces called crRNAs.[13]
Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. The team demonstrated that they could disable one or both sites while preserving Cas9's ability to home in on its target DNA. Jinek combined tracrRNA and spacer RNA into a "single-guide RNA" molecule that, mixed with Cas9, could find and cut the correct DNA targets. Jinek et al proposed that such synthetic guide RNAs might be able to be used for gene editing.[13]
CRISPR was first shown to work as a genome engineering/editing tool in human cell culture by 2012[14][15] It has since been used in a wide range of organisms including baker's yeast (S. cerevisiae),[16] zebra fish (D. rerio),[17] flies (D. melanogaster),[18] nematodes (C. elegans),[19] plants,[20] mice,[21] and several other organisms.
Additionally CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes.[22]
Libraries of tens of thousands of guide RNAs are now available.[13]
The first evidence that CRISPR can reverse disease symptoms in living animals was demonstrated in March 2014, when MIT researchers cured mice of a rare liver disorder.[23]
Gene-editing predecessors
In the early 2000s, researchers developed zinc finger nucleases, synthetic proteins whose DNA-binding domains enable them to cut DNA at specific spots. Later, synthetic nucleases called TALENs provided an easier way to target specific DNA and were predicted to surpass zinc fingers. They both depend on making custom proteins for each DNA target, a more cumbersome procedure than guide RNAs. CRISPRs are more efficient and can target more genes than these earlier techniques.[24]
Locus structure
Repeats and spacers
CRISPR loci range in size from 24 to 48 base pairs.[25] They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic.[26] Repeats are separated by spacers of similar length.[25] Some CRISPR spacer sequences exactly match sequences from plasmids and phages,[27][28][29] although some spacers match the prokaryote's genome (self-targeting spacers).[30] New spacers can be added rapidly in response to phage infection.[31]
Cas genes and CRISPR subtypes
CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. Extensive comparative genomics have identified many different cas genes; an initial analysis of 40 bacterial and archaeal genomes suggested that there may be 45 cas gene families, with only two genes, cas1 and cas2, universally present.[25] The current CRISPR classification groups cas operons into three major divisions, each with multiple subdivisions based on cas1 phylogeny and cas operon gene complement.[32] Aside from cas1 and cas2, the three major divisions have vastly different sets of constituent genes, with each of the subdivisions characterised by a 'signature gene' found exclusively in that subdivision. Many organisms contain multiple CRISPR-Cas systems suggesting that they are compatible and may even share components.[33][34] The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.
|Cas type
|Signature gene
|Single-stranded DNA nuclease (HD domain) and ATP-dependent helicase
|Subunit of the interference module
|contains a domain homologous the palm domain of nucleic acid polymerases and nucleotide cyclases
|Not Determined
|RuvC and HNH domain containing nuclease
|Not Determined
|Not Determined
|Characterized by the absence of either Csn2 or Cas4
|Homolog of Cas10d and Cse1
|Not Determined
|Not Determined
|CRISPR associated protein
crystal structure of a crispr-associated protein from thermus thermophilus
|Pfam clan
|CRISPR associated protein Cas2
crystal structure of a hypothetical protein tt1823 from thermus thermophilus
|CRISPR-associated protein Cse1
|CRISPR-associated protein Cse2
Acquisition of Spacers into CRISPR loci
Capturing invading DNA into a CRISPR locus in the form of a spacer is the first stage in the immune response. The prevalence of cas1 and cas2 was the first clue that they were involved in spacer acquisition as all CRISPRs shared the regular repeating structure. Mutation studies confirmed this hypothesis as removal of cas1 or cas2 abrogated spacer acquisition, without affecting CRISPR immune response.[36][40][41][42][43] The exact function of Cas1 and Cas2 is unknown, however a number of Cas1 proteins have been biochemically characterised and their structures resolved.[44][45][46] Cas1 proteins have very diverse amino acid sequences, however their crystal structures are strikingly similar and all purified Cas1 proteins are metal-dependent nucleases that bind to DNA in a sequence-independent manner.[33] Representative Cas2 proteins have also been characterised and possess either ssRNA [47] or dsDNA [48][49] specific endoribonuclease activity. The functional data and genetic mutation studies suggests that Cas1 and Cas2 excise fragments of invading DNA and insert them into CRISPR arrays.
Bioinformatic analysis of regions of phage genomes that were excised as spacers (termed protospacers) revealed that they were not randomly distributed in but instead were found adjacent to short (3 – 5 bp) DNA sequences termed PAMs (protospacer adjacent motifs). Analysis of CRISPR-Cas systems from the three major divisions have shown PAMs to be important for type I, type II but not type III systems during the spacer acquisition process.[28][50][51][52][53][54] In type I and type II systems, protospacers are excised at positions adjacent to a PAM sequence, with the other end of the spacer cut using a ruler mechanism inherent to the Cas1 protein, thus maintaining the regularity of the spacer size in the CRISPR array.[55][56] The conservation of the PAM sequence differs between CRISPR-Cas systems and appears to be evolutionarily linked to cas1 and the leader sequence.[54][57]
New spacers are added to a CRISPR array in a directional manner, occurring preferentially [50][51][58][59][60] but not exclusively adjacent [53][56] to the leader sequence. Analysis of the type I-E system from E. coli have demonstrated that the first direct repeat, adjacent to the leader sequence is copied, with the newly acquired spacer inserted between the first and second direct repeats.[42][55] The PAM sequence also appears to be important during spacer insertion in type I-E systems. The PAM sequence of the I-E system contains a strongly conserved final nucleotide (adjacent to the first nucleotide of the protospacer) and it has been shown that this nucleotide becomes the final base in the first direct repeat.[43][61][62] This suggests that the spacer acquisition machinery generates single stranded overhangs in the second-to-last position of the direct repeat and in the PAM during spacer insertion. However, not all CRISPR-Cas systems appear to share this mechanism as PAMs characterised in other organisms do not show the same level of conservation in the final position.[57] It is likely that in those systems, a blunt end is generated at the very end of the direct repeat and the protospacer during acquisition. Recent analysis of Sulfolobus solfataricus CRISPRs revealed further complexities to the canonical model of spacer insertion as one of its six CRISPR loci inserted new spacers randomly throughout its CRISPR array, as opposed to inserting closest to the leader sequence.[56]
It has been noted in a number of CRISPRs that they contain many spacers to the same phage. The mechanism that causes this phenomenon has recently been elucidated in the type I-E system of E. coli. A significant enhancement in spacer acquisition has been detected where there are already spacers targeting the phage, even mismatches to the protospacer. This 'priming' requires both the Cas proteins involved in acquisition and interference to interact with each other. Newly acquired spacers that result from the priming mechanism are always found on the same strand as the original spacer that caused the priming.[43][61][62] This observation has led to the hypothesis that the acquisition machinery slides along the foreign DNA after priming to find a new protospacer.[62]
Interference stage
The CRISPR immune response occurs through two steps: CRISPR-RNA (crRNA) biogenesis and crRNA-guided interference. A CRISPR array is transcribed from a promoter in the leader into a single long transcript.[36][63][64] This transcript is processed by cleavage inside the repeat sequence to form crRNAs. The mechanisms to produce mature crRNAs differ greatly between the three main CRISPR-Cas systems. In both type I-E and type I-F systems, the proteins Cas6e and Cas6f respectively, recognise stem-loops [65][66][67] created by the palindromic nature of the direct repeats.[26] These proteins cleave the primary transcript at the junction between double-stranded and single-stranded RNA, leaving an 8 nt 5ʹ-handle originating from the repeat on mature crRNAs along with a single spacer sequence. Type III systems also use Cas6, however the repeats found in type III systems do not produce stem-loops, instead cleavage occurs by the primary transcript wrapping around the Cas6 to allow cleavage 8 nt upstream of the repeat spacer junction.[68][69][70] Type II systems lack the Cas6 gene and instead utilize RNaseIII for cleavage. Functional type II systems encode an extra small RNA that is complementary to the repeat sequence, known as a trans-activating RNA (tracrRNA).[40] Transcription of the tracrRNA and the primary CRISPR transcript results in base pairing and the formation of dsRNA at the repeat sequence, which is subsequently targeted by RNaseIII to produce crRNAs. Unlike the other two systems the crRNA does not contain the full spacer but instead is truncated at one end by 10 nt.[38]
crRNAs associate with Cas proteins to form ribonucleotide complexes that recognize foreign nucleic acids. A number of phage and plasmid challenge experiments have shown that crRNAs show no preference between coding and non-coding strand, which is indicative of an RNA-guided DNA-targeting system.[6][36][43][71][72][73][74] The type I-E complex (commonly referred to as Cascade) requires five Cas proteins arranged in a 'seahorse' conformation, bound to a single crRNA that runs down the spine.[75][76] During the interference stage in type I systems the PAM sequence is recognized on the crRNA-complementary strand and is required along with crRNA annealing. In type I systems correct base pairing between the crRNA and the protospacer signals a conformational change in Cascade that recruits Cas3 for DNA degradation.
Type II systems rely on a single multifunctional protein, Cas9, for the interference step.[38] Cas9 requires both the crRNA and the tracrRNA to function and cleaves DNA using its dual HNH and RuvC/RNaseH-like endonuclease domains. Basepairing between the PAM and the phage genome is also required in type II systems, however the PAM is recognized on the same strand as the crRNA (the opposite strand to type I systems).
Type III systems, like type I require a multi-protein complex to associate with the crRNA. Biochemical and structural analyses of complexes from S. solfataricus and Pyrococcus furiosus have elucidated that six or seven cas proteins bind to crRNAs, respectively.[77][78] Surprisingly, the type III systems analysed from S. solfataricus and P. furiosus have both target the mRNA of phage/plasmids,[34][78] which may make these systems uniquely capable of targeting RNA based phage genomes.[33]
The mechanism for distinguishing self from foreign DNA during interference is built into the crRNAs and is therefore inferred to be common to all three systems. Even through the distinctive maturation process of each major type, all crRNAs contain a spacer sequence and some portion of the repeat at one or both ends. It is the partial repeat sequence that prevents the CRISPR-Cas system from targeting the chromosome as base pairing beyond the spacer sequence signals self and prevents DNA cleavage of the chromosome.[79] RNA-guided CRISPR enzymes are classified as type V restriction enzymes.
Evolution and diversity
Studies of Streptococcus thermophilus first indicated how CRISPRs drive phage and bacterial evolution. A CRISPR spacer must correspond perfectly to the sequence of the target phage gene. Phages can continue to infect their hosts where there are point mutations in the spacer.[79] Similar stringency is required in PAM or the strain will remain phage sensitive.[51][79] The basic model of CRISPR evolution is one where newly incorporated spacers drive phages to mutate their genomes creating diversity in both the phage and host populations.
CRISPR evolution has been studied using comparative genomics of many strains of S. thermophilus, Escherichia coli and Salmonella enterica. A study of 124 strains of S. thermophilus showed that 26% of all spacers were unique and that different CRISPR loci showed different rates of new spacer acquisition.[50] The results showed that particular CRISPR loci evolve more rapidly than others, which allowed the strains' phylogenetic relationships to be determined. A similar analysis of E. coli and S. enterica strains revealed that they evolved much slower than S. thermophilus. The latter's strains that had diverged 250 thousand years ago still contained the same spacer complement.[80]
CRISPR diversity was studied in multiple environmental communities using metagenomics. Analysis of two acid mine drainage biofilms showed that one of the analyzed CRISPRs contained extensive deletions and spacer additions in comparison to the other biofilm, suggesting a higher phage activity/prevalence in one community compared to the other.[59] In the oral cavity, a temporal study determined that 7-22% of spacers were shared between timepoints over 17 months within an individual and less than 2% of spacers were shared between different individuals at any single timepoint.[60] From the same environment a single strain was tracked using PCR primers specific to its CRISPR. Unlike the broad-level results of spacer presence/absence, which showed significant diversity, this CRISPR added 3 spacers over 17 months,[60] suggesting that even in an environment with significant CRISPR diversity some loci evolve slowly. CRISPRs have also been analysed from the metagenomes produced for the human microbiome project.[81] Although most CRISPRs were body-site specific, some CRISPRs within a body site are widely shared among individuals. One of these CRISPR loci originated from streptococcal species and contained ~15,000 spacers, 50% of which were unique. Similar to the targeted studies of the oral cavity, some of the CRISPRs showed little evolution between timepoints.[81]
CRISPR evolution has been studied in chemostats using S. thermophilus to explicitly examine the rate of spacer acquisition. Over a period of one week, strains of S. thermophilus acquired up to three spacers when challenged with a single phage.[82] During the same time period the phage developed a number of single nucleotide polymorphisms that became fixed in the population, suggesting that CRISPR targeting had prevented all other phage types from replicating if they did not contain these mutations.[82] Other experiments, also with S. thermophilus, showed that phages can still infect and replicate in hosts that have only one targeting spacer and that sensitive hosts can exist in environments with high phage titres.[83] The chemostat results combined with the observational studies of CRISPRs suggest many nuances to the outcome of CRISPR and phage evolution.
Bioinformatic identification of CRISPRs in genomes and metagenomes
CRISPRs are widely distributed amongst the bacteria and archaea [32] and show some sequence similarities,[26] however their most notable characteristic is their repeating spacers and direct repeats. This characteristic makes CRISPRs easily identifiable in long sequences of DNA, since the number of repeat copies decreases the likelihood of a false positive match. There are currently three programs used for CRISPR repeat identification that search for regularly interspaced repeats in long sequences: CRT,[84] PILER-CR [85] and CRISPRfinder.[86]
Analysis of CRISPRs in metagenomic data is more challenging, as CRISPR loci do not typically assemble due to their repetitive nature or through strain variation, which confuses assembly algorithms. Where there are many reference genomes available, PCR can be used to amplify CRISPR arrays and analyse spacer content.[50][60][87][88][89] However, this approach will only yield information for CRISPRs specifically targeted and for organisms with sufficient representation in public databases to design reliable PCR primers.
The alternative approach is to extract and reconstruct CRISPR arrays from shotgun metagenomic data. Identification of CRISPR arrays from metagenomic reads is computationally more difficult, particularly with second generation sequencing technologies (e.g. 454, Illumina), as the short read lengths prevent more than two or three repeat units being present in a single read. CRISPR identification in raw reads has been achieved using purely denovo identification [90] or by using direct repeat sequences in partially assembled CRISPR arrays from contigs [81] and direct repeat sequences from published genomes [91] as a hook for identifying direct repeats in individual reads.
Evolutionary significance
A bioinformatic study showed that CRISPRs are evolutionarily conserved and cluster into related types. Many show signs of a conserved secondary structure.[26]
Through the CRISPR/Cas mechanism, bacteria can acquire immunity to certain phages and thus halt further transmission of targeted phages. For this reason, CRISPR/Cas can be described as a Lamarckian inheritance mechanism.[92] Others investigated the coevolution of host and viral genomes by analysis of CRISPR sequences.[93]
Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Cas-mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts. For example, Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence.[94]
The proof-of-principle demonstration of selective engineered redirection of the CRISPR/Cas system in 2012[95] provided a first step toward realization of proposals for CRISPR-derived biotechnology:[96]
- Artificial immunization against phage by introduction of engineered CRISPR loci in industrially important bacteria, including those used in food production and large-scale fermentation
- Genome engineering at cellular or organismic level by reprogramming a CRISPR/Cas system to achieve RNA-guided genome engineering. Proof of concept studies demonstrated examples both in vitro[14][97] and in vivo[21][98][99]
- Discrimination of bacterial strains by comparison of spacer sequences
Editas Medicine, a $43 million startup, aims to develop treatments that employ CRISPR/Cas to make edits to single base pairs and larger stretches of DNA. Inherited diseases such as cystic fibrosis and sickle-cell anemia are caused by single base pair mutations; CRISPR/Cas technology has the potential to correct these errors. The "corrected" gene remains in its normal location on its chromosome, which preserves the way the cell normally activates and/or inhibits its expression.[100]
After harvesting blood cell precursors called hematopoietic stem cells from a patient's bone marrow, CRISPR gene surgery would correct the defective gene. Then the gene-corrected stem cells would be returned to the patient's marrow, which would then produce healthy red blood cells. Replacing 70% of the sickle cells would produce a cure.[24]
Before it can be used clinically, the company must be able to guarantee that only the targeted region will be affected and determine how to deliver the therapy to a patient's cells.[100]
Other pathologies potentially treatable by CRISPR include Huntington's disease, aging, schizophrenia and autism, not to mention modifying DNA in living embryos.[24]
Improved targeting is required before CRISPR can be used in medical applications. Current guide RNAs may target sequences that differ by multiple base pairs from the intended sequence.[13]
In 2014, UCSF researchers used CRISPR to create disease-free versions of induced pluripotent stem cells of beta thalassemia patients.[101]
Mouse models
CRISPR simplifies creation of mouse models and reduces the time required to a matter of weeks from months or longer. Knockdown of endogenous genes has been achieved by transfection with a plasmid that contains a CRISPR area with a spacer, which inhibits a target gene. Injecting mouse zygotes with Cas9 and two guide RNAs was able to disable two genes with 80% efficiency. So-called homology-directed repair involves using Cas9 to "nick" DNA, to introduce new gene parts to the zygote.[citation needed]
In 2014, Chinese researcher Gao Caixia filed patents on the creation of a strain of wheat that is resistant to powdery mildew. The strain lacks genes that encode proteins that repress defenses against the mildew. The researchers deleted all three copies of the genes from wheat's hexaploid genome. The strain promises to reduce or eliminate the heavy use of fungicides to control the disease. Gao used the TALENs and CRISPR gene editing tools without adding or changing any other genes. No field trials are yet planned.[102][103]
CRISPRs can add and delete base pairs at specifically targeted DNA loci. CRISPRs have been used to cut as many as five genes at once.[13]
Reversible knockdown
"CRISPRi" like RNAi, turns off genes in a reversible fashion by targeting but not cutting a site. In bacteria, the presence of Cas9 alone is enough to block transcription, but for mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA, called promoters that immediately precede the gene target.[13]
Cas9 was used to carry synthetic transcription factors (protein fragments that turn on genes) that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different spots on the gene's promoter.[13]
The genes included some tied to human diseases, including those involved in muscle differentiation, cancer, inflammation and producing fetal hemoglobin.[13]
Use by phages
Another way for bacteria to defend against phage infection is by having chromosomal islands. A subtype of chromosomal islands called phage-inducible chromosomal island (PICI) is excised from bacterial chromosome upon phage infection and can inhibit phage replication.[104] The mechanisms that induce PICI excision and how PICI inhibits phage replication are not well understood. One study showed that lytic ICP1 phage, which specifically targets Vibrio cholerae serogroup O1, has acquired a CRISPR/Cas system that targets a V. cholera PICI-like element. The system has 2 CRISPR loci and 9 Cas genes. It seems to be homologous to the 1-F system found in Yersinia pestis. Moreover, like the bacterial CRISPR/Cas system, ICP1 CRISPR/Cas can acquire new sequences, which allows the phage to co-evolve with its host.[105]
Automation and library support
Free software is available to design RNA to target any desired gene. The Addgene repository offers academics the DNA to make their own CRISPR system for $65. In 2013 Addgene distributed more than 10,000 CRISPR constructs. The facility has received CRISPR-enabling DNA sequences from 11 independent research teams.[13]
A provisional US patent application on the use of the CRISPR system for editing genes and regulating gene expression was filed by the inventors on May 12, 2012. Subsequent applications were combined and on March 6, 2014 the resulting patent application was published by the USPTO.[106] The patent rights have been assigned by the inventors to the Regents of the University of California and to the University of Vienna.
See also
- Gene drive
- Transcription activator-like effector nuclease (TALEN)
- Zinc finger nuclease
- CRISPR interference
- ^ a b c Horvath, P.; Barrangou, R. (2010). "CRISPR/Cas, the Immune System of Bacteria and Archaea". Science 327 (5962): 167–170. doi:10.1126/science.1179555. PMID 20056882.
- ^ a b c Marraffini, L. A.; Sontheimer, E. J. (2010). "CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea". Nature Reviews Genetics 11 (3): 181–190. doi:10.1038/nrg2749. PMC 2928866. PMID 20125085.
- ^ 71/79 Archaea, 463/1008 Bacteria CRISPRdb, Date: 19.6.2010
- ^ Grissa, I.; Vergnaud, G.; Pourcel, C. (2007). "The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats". BMC Bioinformatics 8: 172. doi:10.1186/1471-2105-8-172. PMC 1892036. PMID 17521438.
- ^ Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval, P.; Moineau, S.; Romero, D. A.; Horvath, P. (2007). "CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes". Science 315 (5819): 1709–1712. doi:10.1126/science.1138140. PMID 17379808.
- ^ a b Marraffini, L. A.; Sontheimer, E. J. (2008). "CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA". Science 322 (5909): 1843–1845. doi:10.1126/science.1165771. PMC 2695655. PMID 19095942. Cite error: Invalid
<ref>tag; name "pmid19095942" defined multiple times with different content (see the help page).
- ^ Mali, P; Esvelt, K. M.; Church, G. M. (2013). "Cas9 as a versatile tool for engineering biology". Nature Methods 10 (10): 957–63. doi:10.1038/nmeth.2649. PMC 4051438. PMID 24076990.
- ^ Esvelt, Kevin M; Smidler, Andrea L; Catteruccia, Flaminia; Church, George M (July 2014). "Concerning RNA-guided gene drives for the alteration of wild populations". eLife: e03401. doi:10.7554/eLife.03401. PMID 25035423.
- ^ Overballe-Petersen, S.; Harms, K.; Orlando, L. A. A.; Mayar, J. V. M.; Rasmussen, S.; Dahl, T. W.; Rosing, M. T.; Poole, A. M.; Sicheritz-Ponten, T.; Brunak, S.; Inselmann, S.; De Vries, J.; Wackernagel, W.; Pybus, O. G.; Nielsen, R.; Johnsen, P. J.; Nielsen, K. M.; Willerslev, E. (2013). "Bacterial natural transformation by highly fragmented and damaged DNA". Proceedings of the National Academy of Sciences 110 (49): 19860–5. doi:10.1073/pnas.1315278110. PMID 24248361.
"Bacteria incorporate pieces of old DNA in their own genome, scientists discover". KurzweilAI. doi:10.1073/pnas.1315278110. Retrieved 2013-11-26.
- ^ Ishino, Y.; Shinagawa, H.; Makino, K.; Amemura, M.; Nakata, A. (1987). "Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product". Journal of bacteriology 169 (12): 5429–5433. PMC 213968. PMID 3316184.
- ^ Mojica, F. J.; Díez-Villaseñor, C.; Soria, E.; Juez, G. (2000). "Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria". Molecular microbiology 36 (1): 244–246. doi:10.1046/j.1365-2958.2000.01838.x. PMID 10760181.
- ^ a b Jansen, R.; Embden, J. D.; Gaastra, W.; Schouls, L. M. (2002). "Identification of genes that are associated with DNA repeats in prokaryotes". Molecular microbiology 43 (6): 1565–1575. doi:10.1046/j.1365-2958.2002.02839.x. PMID 11952905.
- ^ a b c d e f g h i j k Pennisi, E. (2013). "The CRISPR Craze". Science 341 (6148): 833–836. doi:10.1126/science.341.6148.833. PMID 23970676.
- ^ a b Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. (2012). "A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity". Science 337 (6096): 816–821. doi:10.1126/science.1225829. PMID 22745249.
- ^ "CRISPR gene therapy: Scientists call for more public debate around breakthrough technique - Science - News". The Independent. 2013-11-07. Retrieved 2013-11-25.
- ^ Dicarlo, J. E.; Norville, J. E.; Mali, P; Rios, X; Aach, J; Church, G. M. (2013). "Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems". Nucleic Acids Research 41 (7): 4336–43. doi:10.1093/nar/gkt135. PMC 3627607. PMID 23460208.
- ^ Hwang, W. Y.; Fu, Y; Reyon, D; Maeder, M. L.; Tsai, S. Q.; Sander, J. D.; Peterson, R. T.; Yeh, J. R.; Joung, J. K. (2013). "Efficient genome editing in zebrafish using a CRISPR-Cas system". Nature Biotechnology 31 (3): 227–9. doi:10.1038/nbt.2501. PMC 3686313. PMID 23360964.
- ^ Gratz, S. J.; Cummings, A. M.; Nguyen, J. N.; Hamm, D. C.; Donohue, L. K.; Harrison, M. M.; Wildonger, J; O'Connor-Giles, K. M. (2013). "Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease". Genetics 194 (4): 1029–35. doi:10.1534/genetics.113.152710. PMC 3730909. PMID 23709638.
- ^ Friedland, A. E.; Tzur, Y. B.; Esvelt, K. M.; Colaiácovo, M. P.; Church, G. M.; Calarco, J. A. (2013). "Heritable genome editing in C. Elegans via a CRISPR-Cas9 system". Nature Methods 10 (8): 741–3. doi:10.1038/nmeth.2532. PMC 3822328. PMID 23817069.
- ^ Jiang, W; Zhou, H; Bi, H; Fromm, M; Yang, B; Weeks, D. P. (2013). "Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice". Nucleic Acids Research 41 (20): e188. doi:10.1093/nar/gkt780. PMC 3814374. PMID 23999092.
- ^ a b Wang, H.; Yang, H.; Shivalila, C. S.; Dawlaty, M. M.; Cheng, A. W.; Zhang, F.; Jaenisch, R. (2013). "One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering". Cell 153 (4): 910–918. doi:10.1016/j.cell.2013.04.025. PMID 23643243.
- ^ Larson, M. H.; Gilbert, L. A.; Wang, X; Lim, W. A.; Weissman, J. S.; Qi, L. S. (2013). "CRISPR interference (CRISPRi) for sequence-specific control of gene expression". Nature Protocols 8 (11): 2180–96. doi:10.1038/nprot.2013.132. PMID 24136345.
- ^ "Researchers reverse a liver disorder in mice by correcting a mutated gene". PhysOrg. 30 March 2014. Retrieved 31 March 2014.
- ^ a b c Young, Susan (2014-02-11). "CRISPR and Other Genome Editing Tools Boost Medical Research and Gene Therapy's Reach | MIT Technology Review". Technologyreview.com. Retrieved 2014-04-13.
- ^ a b c Haft, D. H.; Selengut, J.; Mongodin, E. F.; Nelson, K. E. (2005). "A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes". PLoS Computational Biology 1 (6): e60. doi:10.1371/journal.pcbi.0010060. PMC 1282333. PMID 16292354. Cite error: Invalid
<ref>tag; name "pmid16292354" defined multiple times with different content (see the help page).
- ^ a b c d Kunin, V.; Sorek, R.; Hugenholtz, P. (2007). "Evolutionary conservation of sequence and secondary structures in CRISPR repeats". Genome Biology 8 (4): R61. doi:10.1186/gb-2007-8-4-r61. PMC 1896005. PMID 17442114. Cite error: Invalid
<ref>tag; name "pmid17442114" defined multiple times with different content (see the help page). Cite error: Invalid
<ref>tag; name "pmid17442114" defined multiple times with different content (see the help page).
- ^ Mojica, F. J. M.; Díez-Villaseñor, C. S.; García-Martínez, J. S.; Soria, E. (2005). "Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements". Journal of Molecular Evolution 60 (2): 174–182. doi:10.1007/s00239-004-0046-3. PMID 15791728.
- ^ a b Bolotin, A.; Quinquis, B.; Sorokin, A.; Ehrlich, S. D. (2005). "Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin". Microbiology 151 (8): 2551–2561. doi:10.1099/mic.0.28048-0. PMID 16079334. Cite error: Invalid
<ref>tag; name "pmid16079334" defined multiple times with different content (see the help page).
- ^ Pourcel, C.; Salvignol, G.; Vergnaud, G. (2005). "CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies". Microbiology 151 (3): 653–663. doi:10.1099/mic.0.27437-0. PMID 15758212.
- ^ Stern, A.; Keren, L.; Wurtzel, O.; Amitai, G.; Sorek, R. (2010). "Self-targeting by CRISPR: Gene regulation or autoimmunity?". Trends in Genetics 26 (8): 335–340. doi:10.1016/j.tig.2010.05.008. PMC 2910793. PMID 20598393.
- ^ Tyson, G. W.; Banfield, J. F. (2007). "Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses". Environmental Microbiology 10 (1): 200–207. doi:10.1111/j.1462-2920.2007.01444.x. PMID 17894817.
- ^ a b c Chylinski, K; Makarova, K. S.; Charpentier, E; Koonin, E. V. (2014). "Classification and evolution of type II CRISPR-Cas systems". Nucleic acids research 42 (10): 6091–105. doi:10.1093/nar/gku241. PMC 4041416. PMID 24728998.
- ^ a b c Wiedenheft, B; Sternberg, S. H.; Doudna, J. A. (2012). "RNA-guided genetic silencing systems in bacteria and archaea". Nature 482 (7385): 331–8. doi:10.1038/nature10886. PMID 22337052.
- ^ a b Deng, L; Garrett, R. A.; Shah, S. A.; Peng, X; She, Q (2013). "A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus". Molecular microbiology 87 (5): 1088–99. doi:10.1111/mmi.12152. PMID 23320564.
- ^ Sinkunas, T; Gasiunas, G; Fremaux, C; Barrangou, R; Horvath, P; Siksnys, V (2011). "Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system". The EMBO journal 30 (7): 1335–42. doi:10.1038/emboj.2011.41. PMC 3094125. PMID 21343909.
- ^ a b c d Aliyari, R; Ding, S. W. (2009). "RNA-based viral immunity initiated by the Dicer family of host immune receptors". Immunological reviews 227 (1): 176–88. doi:10.1111/j.1600-065X.2008.00722.x. PMC 2676720. PMID 19120484.
- ^ a b Makarova, K. S.; Aravind, L; Wolf, Y. I.; Koonin, E. V. (2011). "Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems". Biology direct 6: 38. doi:10.1186/1745-6150-6-38. PMC 3150331. PMID 21756346.
- ^ a b c Gasiunas, G.; Barrangou, R.; Horvath, P.; Siksnys, V. (2012). "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria". Proceedings of the National Academy of Sciences 109 (39): E2579–E2586. doi:10.1073/pnas.1208507109. PMC 3465414. PMID 22949671.
- ^ Chylinski, K; Le Rhun, A; Charpentier, E (2013). "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems". RNA biology 10 (5): 726–37. doi:10.4161/rna.24321. PMC 3737331. PMID 23563642.
- ^ a b Dugar, G; Herbig, A; Förstner, K. U.; Heidrich, N; Reinhardt, R; Nieselt, K; Sharma, C. M. (2013). "High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacter jejuni isolates". PLoS genetics 9 (5): e1003495. doi:10.1371/journal.pgen.1003495. PMC 3656092. PMID 23696746.
- ^ Hatoum-Aslan, A; Maniv, I; Marraffini, L. A. (2011). "Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site". Proceedings of the National Academy of Sciences of the United States of America 108 (52): 21218–22. doi:10.1073/pnas.1112832108. PMC 3248500. PMID 22160698.
- ^ a b Yosef, I; Goren, M. G.; Qimron, U (2012). "Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli". Nucleic acids research 40 (12): 5569–76. doi:10.1093/nar/gks216. PMC 3384332. PMID 22402487.
- ^ a b c d Swarts, D. C.; Mosterd, C; Van Passel, M. W.; Brouns, S. J. (2012). "CRISPR interference directs strand specific spacer acquisition". PloS one 7 (4): e35888. doi:10.1371/journal.pone.0035888. PMC 3338789. PMID 22558257.
- ^ Babu, M; Beloglazova, N; Flick, R; Graham, C; Skarina, T; Nocek, B; Gagarinova, A; Pogoutse, O; Brown, G; Binkowski, A; Phanse, S; Joachimiak, A; Koonin, E. V.; Savchenko, A; Emili, A; Greenblatt, J; Edwards, A. M.; Yakunin, A. F. (2011). "A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair". Molecular microbiology 79 (2): 484–502. doi:10.1111/j.1365-2958.2010.07465.x. PMC 3071548. PMID 21219465.
- ^ Han, D; Lehmann, K; Krauss, G (2009). "SSO1450--a CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA". FEBS letters 583 (12): 1928–32. doi:10.1016/j.febslet.2009.04.047. PMID 19427858.
- ^ Wiedenheft, B; Zhou, K; Jinek, M; Coyle, S. M.; Ma, W; Doudna, J. A. (2009). "Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense". Structure (London, England : 1993) 17 (6): 904–12. doi:10.1016/j.str.2009.03.019. PMID 19523907.
- ^ Beloglazova, N; Brown, G; Zimmerman, M. D.; Proudfoot, M; Makarova, K. S.; Kudritska, M; Kochinyan, S; Wang, S; Chruszcz, M; Minor, W; Koonin, E. V.; Edwards, A. M.; Savchenko, A; Yakunin, A. F. (2008). "A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats". The Journal of biological chemistry 283 (29): 20361–71. doi:10.1074/jbc.M803225200. PMC 2459268. PMID 18482976.
- ^ Samai, P; Smith, P; Shuman, S (2010). "Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris". Acta crystallographica. Section F, Structural biology and crystallization communications 66 (Pt 12): 1552–6. doi:10.1107/S1744309110039801. PMC 2998353. PMID 21139194.
- ^ Nam, K. H.; Ding, F; Haitjema, C; Huang, Q; Delisa, M. P.; Ke, A (2012). "Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein". The Journal of biological chemistry 287 (43): 35943–52. doi:10.1074/jbc.M112.382598. PMC 3476262. PMID 22942283.
- ^ a b c d Horvath, P.; Romero, D. A.; Coûté-Monvoisin, A. -C.; Richards, M.; Deveau, H.; Moineau, S.; Boyaval, P.; Fremaux, C.; Barrangou, R. (2007). "Diversity, Activity, and Evolution of CRISPR Loci in Streptococcus thermophilus". Journal of Bacteriology 190 (4): 1401–1412. doi:10.1128/JB.01415-07. PMC 2238196. PMID 18065539.
- ^ a b c Deveau, H.; Barrangou, R.; Garneau, J. E.; Labonté, J.; Fremaux, C.; Boyaval, P.; Romero, D. A.; Horvath, P.; Moineau, S. (2007). "Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus". Journal of Bacteriology 190 (4): 1390–1400. doi:10.1128/JB.01412-07. PMC 2238228. PMID 18065545.
- ^ Mojica, F. J. M.; Diez-Villasenor, C.; Garcia-Martinez, J.; Almendros, C. (2009). "Short motif sequences determine the targets of the prokaryotic CRISPR defence system". Microbiology 155 (3): 733–740. doi:10.1099/mic.0.023960-0. PMID 19246744.
- ^ a b Lillestøl, R. K.; Shah, S. A.; Brügger, K.; Redder, P.; Phan, H.; Christiansen, J.; Garrett, R. A. (2009). "CRISPR families of the crenarchaeal genus Sulfolobus: Bidirectional transcription and dynamic properties". Molecular Microbiology 72 (1): 259–272. doi:10.1111/j.1365-2958.2009.06641.x. PMID 19239620.
- ^ a b Shah, S. A.; Hansen, N. R.; Garrett, R. A. (2009). "Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism". Biochemical Society Transactions 37 (Pt 1): 23–28. doi:10.1042/BST0370023. PMID 19143596.
- ^ a b Díez-Villaseñor, C; Guzmán, N. M.; Almendros, C; García-Martínez, J; Mojica, F. J. (2013). "CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli". RNA biology 10 (5): 792–802. doi:10.4161/rna.24023. PMC 3737337. PMID 23445770.
- ^ a b c Erdmann, S; Garrett, R. A. (2012). "Selective and hyperactive uptake of foreign DNA by adaptive immune systems of an archaeon via two distinct mechanisms". Molecular microbiology 85 (6): 1044–56. doi:10.1111/j.1365-2958.2012.08171.x. PMC 3468723. PMID 22834906.
- ^ a b Shah, S. A.; Erdmann, S; Mojica, F. J.; Garrett, R. A. (2013). "Protospacer recognition motifs: Mixed identities and functional diversity". RNA biology 10 (5): 891–9. doi:10.4161/rna.23764. PMC 3737346. PMID 23403393.
- ^ Andersson, A. F.; Banfield, J. F. (2008). "Virus Population Dynamics and Acquired Virus Resistance in Natural Microbial Communities". Science 320 (5879): 1047–1050. doi:10.1126/science.1157358. PMID 18497291.
- ^ a b Tyson, G. W.; Banfield, J. F. (2007). "Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses". Environmental Microbiology 10 (1): 200–207. doi:10.1111/j.1462-2920.2007.01444.x. PMID 17894817.
- ^ a b c d Pride, D. T.; Sun, C. L.; Salzman, J; Rao, N; Loomer, P; Armitage, G. C.; Banfield, J. F.; Relman, D. A. (2011). "Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time". Genome research 21 (1): 126–36. doi:10.1101/gr.111732.110. PMC 3012920. PMID 21149389.
- ^ a b Goren, M. G.; Yosef, I; Auster, O; Qimron, U (2012). "Experimental definition of a clustered regularly interspaced short palindromic duplicon in Escherichia coli". Journal of molecular biology 423 (1): 14–6. doi:10.1016/j.jmb.2012.06.037. PMID 22771574.
- ^ a b c Datsenko, K. A.; Pougach, K; Tikhonov, A; Wanner, B. L.; Severinov, K; Semenova, E (2012). "Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system". Nature communications 3: 945. doi:10.1038/ncomms1937. PMID 22781758.
- ^ Tang, T. H.; Bachellerie, J. P.; Rozhdestvensky, T; Bortolin, M. L.; Huber, H; Drungowski, M; Elge, T; Brosius, J; Hüttenhofer, A (2002). "Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus". Proceedings of the National Academy of Sciences of the United States of America 99 (11): 7536–41. doi:10.1073/pnas.112047299. PMC 124276. PMID 12032318.
- ^ Tang, T. H.; Polacek, N; Zywicki, M; Huber, H; Brugger, K; Garrett, R; Bachellerie, J. P.; Hüttenhofer, A (2005). "Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus". Molecular microbiology 55 (2): 469–81. doi:10.1111/j.1365-2958.2004.04428.x. PMID 15659164.
- ^ Gesner, E. M.; Schellenberg, M. J.; Garside, E. L.; George, M. M.; MacMillan, A. M. (2011). "Recognition and maturation of effector RNAs in a CRISPR interference pathway". Nature structural & molecular biology 18 (6): 688–92. doi:10.1038/nsmb.2042. PMID 21572444.
- ^ Sashital, D. G.; Jinek, M; Doudna, J. A. (2011). "An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3". Nature structural & molecular biology 18 (6): 680–7. doi:10.1038/nsmb.2043. PMID 21572442.
- ^ Haurwitz, R. E.; Jinek, M; Wiedenheft, B; Zhou, K; Doudna, J. A. (2010). "Sequence- and structure-specific RNA processing by a CRISPR endonuclease". Science 329 (5997): 1355–8. doi:10.1126/science.1192272. PMC 3133607. PMID 20829488.
- ^ Carte, J.; Wang, R.; Li, H.; Terns, R. M.; Terns, M. P. (2008). "Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes". Genes & Development 22 (24): 3489–3496. doi:10.1101/gad.1742908. PMC 2607076. PMID 19141480.
- ^ Wang, R; Preamplume, G; Terns, M. P.; Terns, R. M.; Li, H (2011). "Interaction of the Cas6 riboendonuclease with CRISPR RNAs: Recognition and cleavage". Structure (London, England : 1993) 19 (2): 257–64. doi:10.1016/j.str.2010.11.014. PMC 3154685. PMID 21300293.
- ^ Niewoehner, O; Jinek, M; Doudna, J. A. (2014). "Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases". Nucleic acids research 42 (2): 1341–53. doi:10.1093/nar/gkt922. PMC 3902920. PMID 24150936.
- ^ Garneau, J. E.; Dupuis, Marie-Ève; Villion, M; Romero, D. A.; Barrangou, R; Boyaval, P; Fremaux, C; Horvath, P; Magadán, A. H.; Moineau, S (2010). "The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA". Nature 468 (7320): 67–71. Bibcode:2010Natur.468...67G. doi:10.1038/nature09523. PMID 21048762.
- ^ Semenova, E; Jore, M. M.; Datsenko, K. A.; Semenova, A; Westra, E. R.; Wanner, B; Van Der Oost, J; Brouns, S. J.; Severinov, K (2011). "Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence". Proceedings of the National Academy of Sciences of the United States of America 108 (25): 10098–103. doi:10.1073/pnas.1104144108. PMC 3121866. PMID 21646539.
- ^ Gudbergsdottir, S; Deng, L; Chen, Z; Jensen, J. V.; Jensen, L. R.; She, Q; Garrett, R. A. (2011). "Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers". Molecular microbiology 79 (1): 35–49. doi:10.1111/j.1365-2958.2010.07452.x. PMC 3025118. PMID 21166892.
- ^ Manica, A; Zebec, Z; Teichmann, D; Schleper, C (2011). "In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon". Molecular microbiology 80 (2): 481–91. doi:10.1111/j.1365-2958.2011.07586.x. PMID 21385233.
- ^ Jore, M. M.; Lundgren, M; Van Duijn, E; Bultema, J. B.; Westra, E. R.; Waghmare, S. P.; Wiedenheft, B; Pul, U; Wurm, R; Wagner, R; Beijer, M. R.; Barendregt, A; Zhou, K; Snijders, A. P.; Dickman, M. J.; Doudna, J. A.; Boekema, E. J.; Heck, A. J.; Van Der Oost, J; Brouns, S. J. (2011). "Structural basis for CRISPR RNA-guided DNA recognition by Cascade". Nature structural & molecular biology 18 (5): 529–36. doi:10.1038/nsmb.2019. PMID 21460843.
- ^ Wiedenheft, B; Lander, G. C.; Zhou, K; Jore, M. M.; Brouns, S. J.; Van Der Oost, J; Doudna, J. A.; Nogales, E (2011). "Structures of the RNA-guided surveillance complex from a bacterial immune system". Nature 477 (7365): 486–9. doi:10.1038/nature10402. PMID 21938068.
- ^ Zhang, J; Rouillon, C; Kerou, M; Reeks, J; Brugger, K; Graham, S; Reimann, J; Cannone, G; Liu, H; Albers, S. V.; Naismith, J. H.; Spagnolo, L; White, M. F. (2012). "Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity". Molecular cell 45 (3): 303–13. doi:10.1016/j.molcel.2011.12.013. PMC 3381847. PMID 22227115.
- ^ a b Hale, C. R.; Zhao, P.; Olson, S.; Duff, M. O.; Graveley, B. R.; Wells, L.; Terns, R. M.; Terns, M. P. (2009). "RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex". Cell 139 (5): 945–956. doi:10.1016/j.cell.2009.07.040. PMC 2951265. PMID 19945378.
- ^ a b c Marraffini, L. A.; Sontheimer, E. J. (2010). "Self versus non-self discrimination during CRISPR RNA-directed immunity". Nature 463 (7280): 568–571. doi:10.1038/nature08703. PMC 2813891. PMID 20072129.
- ^ Touchon, M.; Rocha, E. P. C. (2010). Randau, Lennart, ed. "The Small, Slow and Specialized CRISPR and Anti-CRISPR of Escherichia and Salmonella". PLoS ONE 5 (6): e11126. doi:10.1371/journal.pone.0011126. PMC 2886076. PMID 20559554.
- ^ a b c Rho, M; Wu, Y. W.; Tang, H; Doak, T. G.; Ye, Y (2012). "Diverse CRISPRs evolving in human microbiomes". PLoS genetics 8 (6): e1002441. doi:10.1371/journal.pgen.1002441. PMC 3374615. PMID 22719260.
- ^ a b Sun, C. L.; Barrangou, R; Thomas, B. C.; Horvath, P; Fremaux, C; Banfield, J. F. (2013). "Phage mutations in response to CRISPR diversification in a bacterial population". Environmental microbiology 15 (2): 463–70. doi:10.1111/j.1462-2920.2012.02879.x. PMID 23057534.
- ^ Kuno, S; Sako, Y; Yoshida, T (2014). "Diversification of CRISPR within coexisting genotypes in a natural population of the bloom-forming cyanobacterium Microcystis aeruginosa". Microbiology (Reading, England) 160 (Pt 5): 903–16. doi:10.1099/mic.0.073494-0. PMID 24586036.
- ^ Bland, C; Ramsey, T. L.; Sabree, F; Lowe, M; Brown, K; Kyrpides, N. C.; Hugenholtz, P (2007). "CRISPR recognition tool (CRT): A tool for automatic detection of clustered regularly interspaced palindromic repeats". BMC bioinformatics 8: 209. doi:10.1186/1471-2105-8-209. PMC 1924867. PMID 17577412.
- ^ Edgar, R. C. (2007). "PILER-CR: Fast and accurate identification of CRISPR repeats". BMC bioinformatics 8: 18. doi:10.1186/1471-2105-8-18. PMC 1790904. PMID 17239253.
- ^ Grissa, I; Vergnaud, G; Pourcel, C (2007). "CRISPRFinder: A web tool to identify clustered regularly interspaced short palindromic repeats". Nucleic acids research 35 (Web Server issue): W52–7. doi:10.1093/nar/gkm360. PMC 1933234. PMID 17537822.
- ^ Pride, D. T.; Salzman, J; Relman, D. A. (2012). "Comparisons of clustered regularly interspaced short palindromic repeats and viromes in human saliva reveal bacterial adaptations to salivary viruses". Environmental microbiology 14 (9): 2564–76. doi:10.1111/j.1462-2920.2012.02775.x. PMC 3424356. PMID 22583485.
- ^ Held, N. L.; Herrera, A; Whitaker, R. J. (2013). "Reassortment of CRISPR repeat-spacer loci in Sulfolobus islandicus". Environmental microbiology. doi:10.1111/1462-2920.12146. PMID 23701169.
- ^ Held, N. L.; Herrera, A; Cadillo-Quiroz, H; Whitaker, R. J. (2010). "CRISPR associated diversity within a population of Sulfolobus islandicus". PloS one 5 (9). doi:10.1371/journal.pone.0012988. PMC 2946923. PMID 20927396.
- ^ Skennerton, C. T.; Imelfort, M; Tyson, G. W. (2013). "Crass: Identification and reconstruction of CRISPR from unassembled metagenomic data". Nucleic acids research 41 (10): e105. doi:10.1093/nar/gkt183. PMC 3664793. PMID 23511966.
- ^ Stern, A; Mick, E; Tirosh, I; Sagy, O; Sorek, R (2012). "CRISPR targeting reveals a reservoir of common phages associated with the human gut microbiome". Genome research 22 (10): 1985–94. doi:10.1101/gr.138297.112. PMC 3460193. PMID 22732228.
- ^ Koonin, E. V.; Wolf, Y. I. (2009). "Is evolution Darwinian or/and Lamarckian?". Biology Direct 4: 42. doi:10.1186/1745-6150-4-42. PMC 2781790. PMID 19906303.
- ^ Heidelberg, J. F.; Nelson, W. C.; Schoenfeld, T.; Bhaya, D. (2009). Ahmed, Niyaz, ed. "Germ Warfare in a Microbial Mat Community: CRISPRs Provide Insights into the Co-Evolution of Host and Viral Genomes". PLoS ONE 4 (1): e4169. doi:10.1371/journal.pone.0004169. PMC 2612747. PMID 19132092.
- ^ Sampson, T. R.; Saroj, S. D.; Llewellyn, A. C.; Tzeng, Y. L.; Weiss, D. S. (2013). "A CRISPR/Cas system mediates bacterial innate immune evasion and virulence". Nature 497 (7448): 254–7. doi:10.1038/nature12048. PMC 3651764. PMID 23584588.
- ^ Hale, C. R.; Majumdar, S.; Elmore, J.; Pfister, N.; Compton, M.; Olson, S.; Resch, A. M.; Glover Cv, C. V. C.; Graveley, B. R.; Terns, R. M.; Terns, M. P. (2012). "Essential Features and Rational Design of CRISPR RNAs that Function with the Cas RAMP Module Complex to Cleave RNAs". Molecular Cell 45 (3): 292–302. doi:10.1016/j.molcel.2011.10.023. PMC 3278580. PMID 22227116.
- ^ Sorek, R.; Kunin, V.; Hugenholtz, P. (2008). "CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea". Nature Reviews Microbiology 6 (3): 181–186. doi:10.1038/nrmicro1793. PMID 18157154.
- ^ Gasiunas, G.; Barrangou, R.; Horvath, P.; Siksnys, V. (2012). "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria". Proceedings of the National Academy of Sciences 109 (39): E2579–E2586. doi:10.1073/pnas.1208507109. PMC 3465414. PMID 22949671.
- ^ Cong, L.; Ran, F. A.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P. D.; Wu, X.; Jiang, W.; Marraffini, L. A.; Zhang, F. (2013). "Multiplex Genome Engineering Using CRISPR/Cas Systems". Science 339 (6121): 819–823. doi:10.1126/science.1231143. PMC 3795411. PMID 23287718.
- ^ Mali, P.; Yang, L.; Esvelt, K. M.; Aach, J.; Guell, M.; Dicarlo, J. E.; Norville, J. E.; Church, G. M. (2013). "RNA-Guided Human Genome Engineering via Cas9". Science 339 (6121): 823–826. doi:10.1126/science.1232033. PMC 3712628. PMID 23287722.
Hou, Z.; Zhang, Y.; Propson, N. E.; Howden, S. E.; Chu, L. -F.; Sontheimer, E. J.; Thomson, J. A. (2013). "Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis". Proceedings of the National Academy of Sciences 110 (39): 15644. doi:10.1073/pnas.1313587110.
- ^ a b Young, Susan. "Biotech Startup Editas Medicine Wants to Cure Grievous Genetic Diseases with New Genome Editing Technology | MIT Technology Review". Technologyreview.com. Retrieved 2013-11-30.
- ^ "Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac".
- ^ Talbot, David (2014-07-19). "Beijing Researchers Use Gene Editing to Create Disease-Resistant Wheat | MIT Technology Review". Technologyreview.com. Retrieved 2014-07-23.
- ^ Wang, Yanpeng (2014). "Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew". Nature Biotechnology. doi:10.1038/nbt.2969.
- ^ Novick, R. P.; Christie, G. E.; Penadés, J. R. (2010). "The phage-related chromosomal islands of Gram-positive bacteria". Nature Reviews Microbiology 8 (8): 541–551. doi:10.1038/nrmicro2393. PMC 3522866. PMID 20634809.
- ^ Seed, K. D.; Lazinski, D. W.; Calderwood, S. B.; Camilli, A. (2013). "A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity". Nature 494 (7438): 489–491. doi:10.1038/nature11927. PMC 3587790. PMID 23446421.
- ^ "Methods And Compositions For Rna-directed Target Dna Modification And For Rna-directed Modulation Of Transcription". Freshpatents.com. Retrieved 2014-04-13.
Further reading
- Sander, J. D.; Joung, J. K. (2014). "CRISPR-Cas systems for editing, regulating and targeting genomes". Nature Biotechnology 32 (4): 347–55. doi:10.1038/nbt.2842. PMID 24584096.
- Terns, R. M.; Terns, M. P. (2014). "CRISPR-based technologies: Prokaryotic defense weapons repurposed". Trends in Genetics 30 (3): 111–8. doi:10.1016/j.tig.2014.01.003. PMID 24555991.
- Westra, E. R.; Buckling, A.; Fineran, P. C. (2014). "CRISPR–Cas systems: Beyond adaptive immunity". Nature Reviews Microbiology. doi:10.1038/nrmicro3241.
- Horvath, P.; Romero, D. A.; Coûté-Monvoisin, A. -C.; Richards, M.; Deveau, H.; Moineau, S.; Boyaval, P.; Fremaux, C.; Barrangou, R. (2007). "Diversity, Activity, and Evolution of CRISPR Loci in Streptococcus thermophilus". Journal of Bacteriology 190 (4): 1401–1412. doi:10.1128/JB.01415-07. PMC 2238196. PMID 18065539.
- Deveau, H.; Barrangou, R.; Garneau, J. E.; Labonté, J.; Fremaux, C.; Boyaval, P.; Romero, D. A.; Horvath, P.; Moineau, S. (2007). "Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus". Journal of Bacteriology 190 (4): 1390–1400. doi:10.1128/JB.01412-07. PMC 2238228. PMID 18065545.
- Andersson, A. F.; Banfield, J. F. (2008). "Virus Population Dynamics and Acquired Virus Resistance in Natural Microbial Communities". Science 320 (5879): 1047–1050. doi:10.1126/science.1157358. PMID 18497291.
- Hale, C.; Kleppe, K.; Terns, R. M.; Terns, M. P. (2008). "Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus". RNA 14 (12): 2572–2579. doi:10.1261/rna.1246808. PMC 2590957. PMID 18971321.
- Carte, J.; Wang, R.; Li, H.; Terns, R. M.; Terns, M. P. (2008). "Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes". Genes & Development 22 (24): 3489–3496. doi:10.1101/gad.1742908. PMC 2607076. PMID 19141480.
- Shah, S. A.; Hansen, N. R.; Garrett, R. A. (2009). "Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism". Biochemical Society Transactions 37 (Pt 1): 23–28. doi:10.1042/BST0370023. PMID 19143596.
- Lillestøl, R. K.; Shah, S. A.; Brügger, K.; Redder, P.; Phan, H.; Christiansen, J.; Garrett, R. A. (2009). "CRISPR families of the crenarchaeal genus Sulfolobus: Bidirectional transcription and dynamic properties". Molecular Microbiology 72 (1): 259–272. doi:10.1111/j.1365-2958.2009.06641.x. PMID 19239620.
- Mojica, F. J. M.; Diez-Villasenor, C.; Garcia-Martinez, J.; Almendros, C. (2009). "Short motif sequences determine the targets of the prokaryotic CRISPR defence system". Microbiology 155 (3): 733–740. doi:10.1099/mic.0.023960-0. PMID 19246744.
- Van Der Ploeg, J. R. (2009). "Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages". Microbiology 155 (6): 1966–1976. doi:10.1099/mic.0.027508-0. PMID 19383692.
- Hale, C. R.; Zhao, P.; Olson, S.; Duff, M. O.; Graveley, B. R.; Wells, L.; Terns, R. M.; Terns, M. P. (2009). "RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex". Cell 139 (5): 945–956. doi:10.1016/j.cell.2009.07.040. PMC 2951265. PMID 19945378.
- Van Der Oost, J.; Brouns, S. J. J. (2009). "RNAi: Prokaryotes Get in on the Act". Cell 139 (5): 863–865. doi:10.1016/j.cell.2009.11.018. PMID 19945373.
- Marraffini, L. A.; Sontheimer, E. J. (2010). "Self versus non-self discrimination during CRISPR RNA-directed immunity". Nature 463 (7280): 568–571. doi:10.1038/nature08703. PMC 2813891. PMID 20072129.
- Karginov, F. V.; Hannon, G. J. (2010). "The CRISPR System: Small RNA-Guided Defense in Bacteria and Archaea". Molecular Cell 37 (1): 7–19. doi:10.1016/j.molcel.2009.12.033. PMC 2819186. PMID 20129051.
- Pul, Ü.; Wurm, R.; Arslan, Z.; Geissen, R.; Hofmann, N.; Wagner, R. (2010). "Identification and characterization ofE. ColiCRISPR-caspromoters and their silencing by H-NS". Molecular Microbiology 75 (6): 1495–1512. doi:10.1111/j.1365-2958.2010.07073.x. PMID 20132443.
- Diez-Villasenor, C.; Almendros, C.; Garcia-Martinez, J.; Mojica, F. J. M. (2010). "Diversity of CRISPR loci in Escherichia coli". Microbiology 156 (5): 1351–1361. doi:10.1099/mic.0.036046-0. PMID 20133361.
- Deveau, H. L. N.; Garneau, J. E.; Moineau, S. (2010). "CRISPR/Cas System and Its Role in Phage-Bacteria Interactions". Annual Review of Microbiology 64: 475–493. doi:10.1146/annurev.micro.112408.134123. PMID 20528693.
- Koonin, E. V.; Makarova, K. S. (2009). "CRISPR-Cas: An adaptive immunity system in prokaryotes". F1000 Biology Reports 1: 95. doi:10.3410/B1-95. PMC 2884157. PMID 20556198.
- Touchon, M.; Rocha, E. P. C. (2010). Randau, Lennart, ed. "The Small, Slow and Specialized CRISPR and Anti-CRISPR of Escherichia and Salmonella". PLoS ONE 5 (6): e11126. doi:10.1371/journal.pone.0011126. PMC 2886076. PMID 20559554.
External links
- E-CRISP.org A comprehensive software for CRISPR gRNA design
- CRISPR Design Tool
- CRISPR Design Tool with scoring algorithms
- Tool for finding CRISPRs
- Tool for finding CRISPR targets in other nucleic acids
- Rfam page for the CRISPR entries
This page is based on a Wikipedia article. The text is available under the Creative Commons Attribution/Share-Alike License.
This tab holds the annotation information that is stored in the Pfam database. As we move to using Wikipedia as our main source of annotation, the contents of this tab will be gradually replaced by the Wikipedia tab.
CRISPR associated protein Provide feedback
This domain forms an anti-parallel beta strand structure with flanking alpha helical regions.
This tab holds annotation information from the InterPro database.
InterPro entry IPR010179
The CRISPR-Cas system is a prokaryotic defense mechanism against foreign genetic elements. The key elements of this defense system are the Cas proteins and the CRISPR RNA.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are a family of DNA direct repeats separated by regularly sized non-repetitive spacer sequences that are found in most bacterial and archaeal genomes [PUBMED:17442114]. CRISPRs appear to provide acquired resistance against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).
The defense reaction is divided into three stages. In the adaptation stage, the invader DNA is cleaved, and a piece of it is selected to be integrated as a new spacer into the CRISPR locus, where it is stored as an identity tag for future attacks by this invader. During the second stage (the expression stage), the CRISPR RNA (pre-crRNA) is transcribed and subsequently processed into the mature crRNAs. In the third stage (the interference stage), Cas proteins, together with crRNAs, identify and degrade the invader [PUBMED:17379808, PUBMED:16545108, PUBMED:21699496].
The CRISPR-Cas systems have been sorted into three major classes. In CRISPR-Cas types I and III, the mature crRNA is generally generated by a member of the Cas6 protein family. Whereas in system III the Cas6 protein acts alone, in some class I systems it is part of a complex of Cas proteins known as Cascade (CRISPR-associated complex for antiviral defense). The Cas6 protein is necessary for crRNA production whereas the additional Cas proteins that form the Cascade complex are needed for crRNA stability [PUBMED:24459147].
This entry represents the Cse3 (CRISPR/Cas Subtype Ecoli protein 3) family of Cas proteins. The Thermus thermophilus HB8 family member has been crystallised and found to have a structure consisting of two domains with opposing parallel beta-sheets, known as a beta-sheet platform [PUBMED:16672237]. This structure is similar to those found in the sex-lethal protein and poly(A)-binding protein and is consistent with an RNA-binding function.
Domain organisation
Below is a listing of the unique domain organisations or architectures in which this domain is found. More...
The graphic that is shown by default represents the longest sequence with a given architecture. Each row contains the following information:
- the number of sequences which exhibit this architecture
a textual description of the architecture, e.g. Gla, EGF x 2, Trypsin.
This example describes an architecture with one
Gladomain, followed by two consecutive
EGFdomains, and finally a single
- a link to the page in the Pfam site showing information about the sequence that the graphic describes
- the UniProt description of the protein sequence
- the number of residues in the sequence
- the Pfam graphic itself.
Note that you can see the family page for a particular domain by clicking on the graphic. You can also choose to see all sequences which have a given architecture by clicking on the Show link in each row.
Finally, because some families can be found in a very large number of architectures, we load only the first fifty architectures by default. If you want to see more architectures, click the button at the bottom of the page to load the next set.
Loading domain graphics...
Pfam Clan
This family is a member of clan RAMPS-Cas5-like (CL0362), which has the following description:
This group of families is one of several protein families that are always found associated with prokaryotic CRISPRs, themselves a family of clustered regularly interspaced short palindromic repeats, DNA repeats found in nearly half of all bacterial and archaeal genomes. These DNA repeat regions have a remarkably regular structure: unique sequences of constant size, called spacers, sit between each pair of repeats [1]. It has been shown that the CRISPRs are virus-derived sequences acquired by the host to enable them to resist viral infection. The Cas proteins from the host use the CRISPRs to mediate an antiviral response. After transcription of the CRISPR, a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation [2]. Cas5 contains an endonuclease motif, whose inactivation leads to loss of resistance, even in the presence of phage-derived spacers [3].
The clan contains the following 7 members:Cas6 Cas_Cas5d Cas_Cas6 Cas_Cmr3 CRISPR_assoc DUF2276 RAMPs
We store a range of different sequence alignments for families. As well as the seed alignment from which the family is built, we provide the full alignment, generated by searching the sequence database (reference proteomes) using the family HMM. We also generate alignments using four representative proteomes (RP) sets, the UniProtKB sequence database, the NCBI sequence database, and our metagenomics sequence database. More...
There are various ways to view or download the sequence alignments that we store. We provide several sequence viewers and a plain-text Stockholm-format file for download.
Alignment types
We make a range of alignments for each Pfam-A family:
- seed
- the curated alignment from which the HMM for the family is built
- full
- the alignment generated by searching the sequence database using the HMM
- Representative Proteomes (RPs) at 15%, 35%, 55% and 75% co-membership thresholds
- UniProt
- alignment generated by searching the UniProtKB sequence database using the family HMM
- alignment generated by searching the NCBI sequence database using the family HMM
- meta
- alignment generated by searching the metagenomics sequence database using the family HMM
You can see the alignments as HTML or in three different sequence viewers:
- jalview
- a Java applet developed at the University of Dundee. You will need Java installed before running jalview
- an HTML page showing the whole alignment.Please note: full Pfam alignments can be very large. These HTML views are extremely large and often cause problems for browsers. Please use either jalview or the Pfam viewer if you have trouble viewing the HTML version
- PP/Heatmap
- an HTML-based representation of the alignment, coloured according to the posterior-probability (PP) values from the HMM. As for the standard HTML view, heatmap alignments can also be very large and slow to render.
You can download (or view in your browser) a text representation of a Pfam alignment in various formats:
- Selex
- Stockholm
You can also change the order in which sequences are listed in the alignment, change how insertions are represented, alter the characters that are used to represent gaps in sequences and, finally, choose whether to download the alignment or to view it in your browser directly.
You may find that large alignments cause problems for the viewers and the reformatting tool, so we also provide all alignments in Stockholm format. You can download either the plain text alignment, or a gzipped version of it.
View options
We make a range of alignments for each Pfam-A family. You can see a description of each above. You can view these alignments in various ways but please note that some types of alignment are never generated while others may not be available for all families, most commonly because the alignments are too large to handle.
|Representative proteomes
1Cannot generate PP/Heatmap alignments for seeds; no PP data available
Key: available, not generated, — not available.
Format an alignment
Download options
We make all of our alignments available in Stockholm format. You can download them here as raw, plain text files or as gzip-compressed files.
|Representative proteomes
|Raw Stockholm
You can also download a FASTA format file containing the full-length sequences for all sequences in the full alignment.
HMM logo
HMM logos is one way of visualising profile HMMs. Logos provide a quick overview of the properties of an HMM in a graphical form. You can see a more detailed description of HMM logos and find out how you can interpret them here. More...
If you find these logos useful in your own work, please consider citing the following article:
This page displays the phylogenetic tree for this family's seed alignment. We use FastTree to calculate neighbour join trees with a local bootstrap based on 100 resamples (shown next to the tree nodes). FastTree calculates approximately-maximum-likelihood phylogenetic trees from our seed alignment.
Note: You can also download the data file for the tree.
Curation and family details
This section shows the detailed information about the Pfam family. You can see the definitions of many of the terms in this section in the glossary and a fuller explanation of the scoring system that we use in the scores section of the help pages.
|Seed source:
|Previous IDs:
|Mistry J
|Number in seed:
|Number in full:
|Average length of the domain:
|218.60 aa
|Average identity of full alignment:
|24 %
|Average coverage of the sequence by the domain:
|97.26 %
HMM information
|HMM build commands:
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 11927849 -E 1000 --cpu 4 HMM pfamseq
|Model details:
|Model length:
|Family (HMM) version:
|download the raw HMM for this family
Species distribution
Sunburst controlsShow
Weight segments by...
Change the size of the sunburst
Colour assignments
|Other sequences
|Unclassified sequence
Align selected sequences to HMM
Generate a FASTA-format file
Clear selection
Currently selected:
- 0 sequences
- 0 species
This visualisation provides a simple graphical representation of the distribution of this family across species. You can find the original interactive tree in the adjacent tab. More...
This chart is a modified "sunburst" visualisation of the species tree for this family. It shows each node in the tree as a separate arc, arranged radially with the superkingdoms at the centre and the species arrayed around the outermost ring.
How the sunburst is generated
The tree is built by considering the taxonomic lineage of each sequence that has a match to this family. For each node in the resulting tree, we draw an arc in the sunburst. The radius of the arc, its distance from the root node at the centre of the sunburst, shows the taxonomic level ("superkingdom", "kingdom", etc). The length of the arc represents either the number of sequences represented at a given level, or the number of species that are found beneath the node in the tree. The weighting scheme can be changed using the sunburst controls.
In order to reduce the complexity of the representation, we reduce the number of taxonomic levels that we show. We consider only the following eight major taxonomic levels:
- superkingdom
- kingdom
- phylum
- class
- order
- family
- genus
- species
Colouring and labels
Segments of the tree are coloured approximately according to their superkingdom. For example, archeal branches are coloured with shades of orange, eukaryotes in shades of purple, etc. The colour assignments are shown under the sunburst controls. Where space allows, the name of the taxonomic level will be written on the arc itself.
As you move your mouse across the sunburst, the current node will be highlighted. In the top section of the controls panel we show a summary of the lineage of the currently highlighed node. If you pause over an arc, a tooltip will be shown, giving the name of the taxonomic level in the title and a summary of the number of sequences and species below that node in the tree.
Anomalies in the taxonomy tree
There are some situations that the sunburst tree cannot easily handle and for which we have work-arounds in place.
Missing taxonomic levels
Some species in the taxonomic tree may not have one or more of the main eight levels that we display. For example, Bos taurus is not assigned an order in the NCBI taxonomic tree. In such cases we mark the omitted level with, for example, "No order", in both the tooltip and the lineage summary.
Unmapped species names
The tree is built by looking at each sequence in the full alignment for the family. We take the name of the species given by UniProt and try to map that to the full taxonomic tree from NCBI. In some cases, the name chosen by UniProt does not map to any node in the NCBI tree, perhaps because the chosen name is listed as a synonym or a misspelling in the NCBI taxonomy.
So that these nodes are not simply omitted from the sunburst tree, we group them together in a separate branch (or segment of the sunburst tree). Since we cannot determine the lineage for these unmapped species, we show all levels between the superkingdom and the species as "uncategorised".
Since we reduce the species tree to only the eight main taxonomic levels, sequences that are mapped to the sub-species level in the tree would not normally be shown. Rather than leave out these species, we map them instead to their parent species. So, for example, for sequences belonging to one of the Vibrio cholerae sub-species in the NCBI taxonomy, we show them instead as belonging to the species Vibrio cholerae.
Too many species/sequences
For large species trees, you may see blank regions in the outer layers of the sunburst. These occur when there are large numbers of arcs to be drawn in a small space. If an arc is less than approximately one pixel wide, it will not be drawn and the space will be left blank. You may still be able to get some information about the species in that region by moving your mouse across the area, but since each arc will be very small, it will be difficult to accurately locate a particular species.
Tree controlsHide
Download tree
- Save a text representation
Selected sequences
- (Uncheck all)
- graphically
- as an alignment
- sequence accessions
- sequences in FASTA format
The tree shows the occurrence of this domain across different species. More...
Species trees
We show the species tree in one of two ways. For smaller trees we try to show an interactive representation, which allows you to select specific nodes in the tree and view them as an alignment or as a set of Pfam domain graphics.
Unfortunately we have found that there are problems viewing the interactive tree when the it becomes larger than a certain limit. Furthermore, we have found that Internet Explorer can become unresponsive when viewing some trees, regardless of their size. We therefore show a text representation of the species tree when the size is above a certain limit or if you are using Internet Explorer to view the site.
If you are using IE you can still load the interactive tree by clicking the "Generate interactive tree" button, but please be aware of the potential problems that the interactive species tree can cause.
Interactive tree
For all of the domain matches in a full alignment, we count the number that are found on all sequences in the alignment. This total is shown in the purple box.
We also count the number of unique sequences on which each domain is found, which is shown in green. Note that a domain may appear multiple times on the same sequence, leading to the difference between these two numbers.
Finally, we group sequences from the same organism according to the NCBI code that is assigned by UniProt, allowing us to count the number of distinct sequences on which the domain is found. This value is shown in the pink boxes.
We use the NCBI species tree to group organisms according to their taxonomy and this forms the structure of the displayed tree. Note that in some cases the trees are too large (have too many nodes) to allow us to build an interactive tree, but in most cases you can still view the tree in a plain text, non-interactive representation. Those species which are represented in the seed alignment for this domain are highlighted.
You can use the tree controls to manipulate how the interactive tree is displayed:
- show/hide the summary boxes
- highlight species that are represented in the seed alignment
- expand/collapse the tree or expand it to a given depth
- select a sub-tree or a set of species within the tree and view them graphically or as an alignment
- save a plain text representation of the tree
Please note: for large trees this can take some time. While the tree is loading, you can safely switch away from this tab but if you browse away from the family page entirely, the tree will not be loaded.
There is 1 interaction for this family. More...
We determine these interactions using iPfam, which considers the interactions between residues in three-dimensional protein structures and maps those interactions back to Pfam families. You can find more information about the iPfam algorithm in the journal article that accompanies the website.
For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the CRISPR_assoc domain has been found. There are 20 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein seqence.
Loading structure mapping...


text:
Prices, specials, features and availability subject to change.
3510 North Pine Grove is a modern, elevator-equipped 5-story Lakeview Chicago apartment building. Spacious, natural light-filled units feature ceiling fans, wall-to-wall windows and large closets. The building has assigned covered parking and an onsite laundry facility, and benefits from a great location in one of Chicago's most exciting neighborhoods. You'll love being close to the lakefront, Lincoln Park and public transportation. Video surveillance and a controlled access entry system are in place to ensure your security. Emergency maintenance is available 24 hours a day for your convenience. Studio and 1-bedroom units are available. Cats are welcome.
Book Online And Save Up To $500
12 months
Cats Allowed. Call for service animal policy.
112 units
Check Availabilityor call
Just a quite peaceful place to live. Good location that is close to bus, lake, grocery stores, shops and Wrig Read Moreleyville.
January 18, 2014
I had a great time living at this location. Not to far away from the bus or the train. Only had a few issues Read More in the apartment throughout the year, and the amazing and friendly maintenance man fixed them right away. No trouble with neighbors, overall great experience.
December 14, 2013
Always some stink in the corridor.... please keep public area clean... could we re-build the exit of gar Read Moreage to main to road, especially for winter ?
September 01, 2015
so far good service
August 17, 2015
All of my electrical sockets were painted over before I moved in. Floors are really noisy. Can hear conversati Read Moreons that are happening in neighboring apartments. Not enough washing machines and dryers, and are also expensive to use. Elevators don't work most of the time. My unit was fairy priced compared to similar buildings in the area, and the location is amazing. I never feel unsafe in the neighborhood.
August 13, 2015
Monday-Friday: 9:00am-7:00pm
Saturday: 9:00am-5:00pm
Sunday: 10:00am-4:00pm
Planned Property Management Company
Don't have a Rent.com account? Create one.
Creating an account allows you to acccess your saved and contacted properties on any device. Already have an account?
Log In
We are experiencing technical difficulties at the moment. Please try again later.


text:
KONA EXPRESS HOT BEVERAGE FLASK AND DUFFEL (Item #W81929)
now $37.98
This Kona Express just made transporting your hot beverage easier! Simply prepare your coffee, tea, or hot coco as you normally would, pour into flask, and place the flask in the duffel. The duffel is made of durable 600D polyester with an adjustable shoulder strap for easy carrying. The 24oz included flask is vacuum sealed stainless steel, so the exterior stays cool to the touch, and has a push button pour top along with a 6oz insulated cup with screw top lid. Enjoy a warm beverage any time, anywhere! Duffel Color: Black with brown accents. Dimensions: 3.5" x 3.5" x 13".
Tell a friend:
- Pin It
Enter your email address below to let a friend know about this great product!
Recommended Products
non stick oven liner
Your Price: $9.98
toucan automatic can opener
Your Price: $19.98
dump crock pot meals
Your Price: $9.98
lady micro steam cleaner
Your Price: $9.98
smokeless stovetop indoor bbq grill
Your Price: $29.98
There are no FAQ for this product. Have a question? Click here!


text:
Submited: 5 years old
Views: 1061023
Copyright © 2006 - 2016 DeviantClip.com - All Rights Reserved.
Support / Contact
Webmasters Area
Site Map
All actors and models appearing on this website are 18 years or older.
Section 2257
The Biggest Baddest Black Cock - Not any girl can handle a cock that fucking large. This bitch had to open her lips extra wide for that cock to fit in her mouth and her pussy.


text:
Best Public Pool (2008)
Hamilton Fish Recreational Center
"The water's a little cloudy," says a lifeguard at the HAMILTON FISH RECREATIONAL CENTER, "but it's really the cleanest public pool in Manhattan." The city has spent millions renovating this historic pool, which opened during the Great Depression and was a training ground for the U.S. Olympic team before they competed in Helsinki in 1952. Now big-bellied men waddle around the brick deck and bronze their guts in the sun. Pregnant women are in suits, too. In the four-feet-deep, colossal pool a lot of traffic: fluttering feet, multiple splash wars, simultaneous games of chicken, young men with fuzzy mustaches who dive underwaterhold their breathand try to pinch bikini butts. "Stop," the girls say, then giggle. The water stays crisp, about 70 degrees, and the maximum capacity is around 670 swimmers, the lifeguard says, but on a recent scorcher they broke 700. So, any floaters? "Not yet," he says, then clasps his hands together in prayer.
More Arts & Entertainment Awards
Best Underground MovementThe Roundabout and the Atlantic
Best Museum to See on Your Lunch BreakNew Museum
Best MixTape ExchangeSound Fix's Fix Tape Exchange
Best Octogenarian Impresario on the BoweryTony Amato
Best Arts Venue Conjuring Avenue A in the ?80sGlassLands Gallery
Best Theater ConcessionsArs Nova
Best Country RadioColumbia University's WKCR
Best Jazz DJ?sPhil Schaap
Best Oldies Not By OldiesThe Fabulous Soul Shakers
Best Way to Ring in the New Year with a Rock LegendPatti Smith Group�s Bowery Ballroom NYE Shows


text:
The Abyss
The Hellish Productions of 6 Great Movies
- By David Christopher Bell
- November 4, 2013
All this week, Film School Rejects presents a daily dose of our favorite articles from the archive. Originally published in November 2011, David C. Bell explores some of the toughest roads to the big screen for a score of great movies. Most films tend to be technological and logistical nightmares...
8 Movies Where Humans Totally Botched Meeting Aliens
- By David Christopher Bell
- September 11, 2013
There seems to be some kind of popular misconception that if aliens were to land on our planet, they'd somehow want to beat us up. In reality, it's probably going to be the other way around. Want proof? How about the fact that we assume they'd do it to us....
What We Can Learn From The Curious Boy Who Just Went Deeper Than Any Person on Earth Has Before
- By Scott Beggs
- @scottmbeggs
- March 28, 2012
In 1989, James Cameron explored the fictional deep in The Abyss, and when he dove nearly 7 miles into the Mariana Trench, he unfortunately didn't meet any gooey alien-like beings, but he did beat a solo dive record and figuratively witnessed a different world inside our own. But hasn't that...
Movie News After Dark: X-Men's First Class Trailer, Puppets of The Abyss and Superman Classic
- By Neil Miller
- @rejects
- February 9, 2011
What is Movie News After Dark? This is a question that I am almost never asked, but I will answer it for you anyway. Movie News After Dark is FSR's newest late-night secretion, a column dedicated to all of the news stories that slip past our daytime editorial staff and...
James Cameron Takes You To The Depths of the Vintage Trailer of the Day
- By Scott Beggs
- @scottmbeggs
- February 5, 2011
Every day, come rain or shine or internet tubes breaking, Film School Rejects showcases a trailer from the past. It's the most original adventure of the summer of 1989. After Terminator, James Cameron chose to hop in a submarine and head down to the only uncharted territory on the planet....
Culture Warrior: Digital Creatures
- By Landon Palmer
- @landonspeak
- September 7, 2009
This week's Culture Warrior explains why puppet Yoda is far superior to digital Yoda....


text:
Guest,Dismiss Notice
If you need help getting to know Xenforo, please see our guide here. If you have feedback or questions, please post those here.
Happy Days: The Complete Series: The Ultimate Edition?
Discussion in 'TV on DVD and Blu-ray' started by classictvfan91, Apr 16, 2012.
Page 2 of 3
Page 2 of 3


text:
- Published: Uhrichsville, OH : Barbour Pub., c2004.
- Year Published: 2004
- Description: 218 p. ; 22 cm.
- Language: English
- Format: Book
ISBN/Standard Number
- 9781602608900 (pbk.)
- 1602608903 (pbk.)
Recently Listed On
Login to add tags
Share This
Kelly's chance
by Brunstetter, Wanda E.
There are currently 2 available
Where To Find It
Call number: Fiction
Available Copies: Downtown 2nd Floor, Malletts Adult
Additional Details
Life for Kelly McGregor is a daily drudge of driving her overbearing father's mules along Pennsylvania's Lehigh Canal. She dreams of one day owning an art gallery where her own drawings and paintings are on display. But these dreams don't include marriage . . . not after seeing what her father has done to her mother. How then can Mike Cooper, a general store owner, make her realize he is different than her father and wants to support her artistic talent? Will Kelly learn that dreams can walk hand in hand with a love created by God?
Reviews & Summaries
Community Reviews
No reviews have been written yet. You could be the first!
Login to write a review of your own.


text:
PUPPY VORE TRIBUTE by b1kWatch
©2008-2016 b1k
This is a tribute to animation on You Tube called puppy vore. This is to pay tribute to him cause he showed me that I'm not the only onw who like furry weightgain's! He also lived my dreams & did a dog vore which doesn't happen very often (except in Bugs Buuny Wood Chuck rabbit. If you know of any other dog vores in cartons let me know, okay?) Anyway I hope you like this. Especially you aldiakaroofus! It's good theres people like you in the world!
Dog gone, nope, boy gone!
I love this pic dudeXD
yeah i saw the video a it was horrible it was bloody wrong your thing is great but that was groesem
Uuum thanks. It would of been better without the gore but these days anything that's weird n gruesome sells.
very nice. I liked the clip when I saw it about a year ago... but I didn't enjoy the blood parts. you took the parts that were my favorite and drew them.
Hee... I like the squishy tummy. But could I ask what you mean't with "He also lived my dreams & did a dog vore which doesn't happen very often (except in Bugs Buuny Wood Chuck rabbit."?
I am having trouble finding anything like that anywhere on the 'net.
I am having trouble finding anything like that anywhere on the 'net.
I try to draw dog vore's at home. I felt I was the only one but showed that I'm not alone. Wood Chuck rabbit is a Bugs Bunny cartoon where he incounters a giant dog who eats him while he's in a apple. Bugs was oky. He didn't get swallowed like the apple did. It was on You Tube. I would love to see more dog vore cause when they lick you it fells there tasting you but it fells so nice it couldn't be bad.
Bad puppy. lol
Dear god! Who doesn't love this cartoon? I love it! keep up the great work!
I do, I heard too many stories of dogs eating infants too enjoy this...
Hey great your back on deviant again! Did you see my other new pics?
Aw, that's sweet of you, thank you!
It's amazing that it's so simple, yet it's more entertaining than say...transformers 2007. You really put the magic of cartoons back in my life. Sorry for fighting with you about Chuck Jones (God Rest his soul).