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epigenetics | Can genetic material actually provide a physical description of appearance? | https://biology.stackexchange.com/questions/52410/can-genetic-material-actually-provide-a-physical-description-of-appearance | <p>A bodies genetic material provides the blueprint for your appearance. Your predisposition to be tall, freckled, blue eyed and blonde are encoded from birth.</p>
<p>However, external and environmental factors can influence the manner these instructions are carried out. For example malnutrition during childhood will a... | 434 | |
epigenetics | Why is the human body hair not uniformly colored? | https://biology.stackexchange.com/questions/16394/why-is-the-human-body-hair-not-uniformly-colored | <p>Most of my body hair is black however my lip hair is light brown/blonde the rest of my beard region is black. Since hair color is genetic what causes this?</p>
| <p>The hairs you mention are also called "<a href="http://en.wikipedia.org/wiki/Androgenic_hair" rel="nofollow">androgenic hairs</a>", meaning their growth and pigmentation is influenced by androgens. These include pubic hair, the hairs on the breast and shoulders (almost exclusively for men) and the beard.</p>
<p>It ... | 435 |
epigenetics | Why is the 3'UTR region highly methylated in most of the human genes? | https://biology.stackexchange.com/questions/1541/why-is-the-3utr-region-highly-methylated-in-most-of-the-human-genes | <p>Most of the human genes are found to be highly methylated in their 3'UTR region (0.8-0.9%). I was wondering if there is any specific reason for this?</p>
| <p>According to <a href="http://genomebiology.com/2009/10/9/R89">Choi et al. Genome Biology 2009, 10:R89</a>, DNA methylation at both coding boundaries may regulate transcription elongation and stabilize splicing by reducing the occurrences of exon skipping.</p>
<p>From the abstract:</p>
<blockquote>
<p>Here we rep... | 436 |
epigenetics | On parents' similarity | https://biology.stackexchange.com/questions/59639/on-parents-similarity | <p>I don't know if this is a real claim, I live in Mexico City and most of the people I know are more similar to their father than to their mother? Why is that? Are the x and y cromosomes somehow related to the strength of a particular gen?</p>
| 437 | |
epigenetics | Is it the case that all changes in phenotype during life are not inheritable? | https://biology.stackexchange.com/questions/884/is-it-the-case-that-all-changes-in-phenotype-during-life-are-not-inheritable | <p>This came up in a talk with a friend. I wanted to clear this doubt. I've read about it before and did again after her remark (my thoughts didn't change: her concept is Lamarck's, not Darwin's), but wanted to clarify.</p>
<p>Regarding Evolution, nothing, absolutely nothing, that a person does to herself in life can ... | <p>The assertion "You cannot change in life what will be genetically inherited in any possible way" is true, as you cannot (healthily) change the DNA in your germ cells.</p>
<p>However, the assertion "You cannot change in life what will be inherited in any possible way" is wrong, because of <a href="http://en.wikipedi... | 438 |
epigenetics | Doubt on genomic code for nucleosome positioning? | https://biology.stackexchange.com/questions/30342/doubt-on-genomic-code-for-nucleosome-positioning | <p>I was reading "<a href="http://www.wisdom.weizmann.ac.il/~/eran/NucleosomeModel.pdf" rel="nofollow noreferrer">A genomic code for nucleosome positioning</a>" (by Eran Segal et al). And I am having 2 doubts.
<img src="https://i.sstatic.net/7eyzE.png" alt="enter image description here"></p>
<p>The figure(b) in this i... | <p>Dyad is the centre of the DNA that is wrapped around the nucleosome core (It basically is the centre of symmetry of the nucleosome). It is a common practice to set it at 0 thereby making incoming DNA half, negative and outgoing DNA half, positive.</p>
<p>By oscillations the authors mean that there is a periodic rep... | 439 |
epigenetics | "Enhancers" of enhancers? | https://biology.stackexchange.com/questions/30416/enhancers-of-enhancers | <p>I am looking for examples (if any) of genomic regions which regulates the activity of enhancers, either augmenting or reducing it. Essentially some kind of enhancers (or repressors) of enhancers to make a Russian doll analogy. I know about epigenetic markings but I am really looking at an example of a DNA region dir... | <p>If you're looking for a strictly and directly DNA-DNA mediated effect (no histones, no transcription involved) I'd look for sequence effects on chromatin remodelling, modulating access of transcription factors (TFs) to their elements. Something about this might be in this paper:
<a href="http://www.ncbi.nlm.nih.gov... | 440 |
epigenetics | Is it possible to create DNA of a species that could be any animal | https://biology.stackexchange.com/questions/51557/is-it-possible-to-create-dna-of-a-species-that-could-be-any-animal | <p>I was thinking about a fiction world in which all animal (at least vertebrate) could crossbred because they share same number and layout of chromosome but differ in hereditary detail (like human skin color) and/or using epigenetic modifications.</p>
<p>Is it actually possible? And how large of DNA it could be to co... | <p>Suppose genome A generates organism oA and genome B generates organism oB. Their phenotypes pA and pB are quite different from each other.</p>
<p>Because genome A and B are not similar enough, organism oA and oB cannot sexually produce viable offspring.</p>
<p>Your question seems to boil down to: "Would it be poss... | 441 |
epigenetics | Histone Deacetylase Inhibition | https://biology.stackexchange.com/questions/93758/histone-deacetylase-inhibition | <p>So I am trying to brush up on my knowledge of HATs and HDACs.</p>
<p>I am reading the just the 1st paragraph of the background of this <a href="https://www.alzforum.org/therapeutics/amx0035" rel="nofollow noreferrer">study</a></p>
<p>I remember learning that HATs turn things on on, and HDACs turn things off.</p>
... | <p>Yes, Sodium phenylbutyrate can upregulate the transcription of silenced genes by inhibiting the activity of Histone deacetylases (HDACs).<br>
Histone acetylation performed by Histone acetyltransferases (HATs) helps in activation of genes, while deacetylation performed by HDACs is responsible for gene silencing. This... | 442 |
epigenetics | Why is polyploidy lethal for some organisms while for others is not? | https://biology.stackexchange.com/questions/935/why-is-polyploidy-lethal-for-some-organisms-while-for-others-is-not | <p><a href="http://en.wikipedia.org/wiki/Polyploid">Polyploidy</a> is the multiplication of number of chromosomal sets from 2n to 3n (triploidy), 4n (tetraploidy) and so on. It is quite common in plants, for example many crops like wheat or Brassica forms. It seems to be rarer in animals but still it is present among s... | <p>Great question, and one about which there has historically been a lot of speculation, and there is currently a lot of misinformation. I will first address the two answers given by other users, which are both incorrect but have been historically suggested by scientists. Then I will try to explain the current understa... | 443 |
epigenetics | Does the bending of a tree's trunk in the wind stimulate and strengthen root growth? | https://biology.stackexchange.com/questions/43014/does-the-bending-of-a-trees-trunk-in-the-wind-stimulate-and-strengthen-root-gro | <p>Recently Southern California experienced extreme wind velocities and afterwards the news reported over 300 trees had fallen in San Diego County. I had either heard or read somewhere that the action of the wind and bending of a tree assists to strengthen the roots ( I suppose as long as the wind is not strong enough ... | <p>In tree growth there is principle called <em>The axiom of universal stress</em> whereby the growth is in such a way as to equalise the stress across the whole of its structure. The roots, the stem and the branches are one continuous system and can not be thought of as separate.</p>
<p>On a simplistic level for a br... | 444 |
CRISPR | simple explanation of a crispr screen vs pooled crispr screen? | https://biology.stackexchange.com/questions/92506/simple-explanation-of-a-crispr-screen-vs-pooled-crispr-screen | <p>I'm familiar with how CRISPR works to make either knockdown or amplification effects for a single gene because you can make precision cuts. </p>
<p>What exactly does a pooled crispr screen do & how does it work?</p>
| <p>In the context of pooled CRISPR screen, you first design a library of gRNAs against a certain set of targets (instead of just one target). </p>
<p>The library can have the size you want, most commonly it is either targeting the kinome, or the whole genome (genome-wide).
In this context, you would add the full libra... | 445 |
CRISPR | Is CRISPR being utilized when scientists use the CRISPR/Cas9 system to edit genes? | https://biology.stackexchange.com/questions/100421/is-crispr-being-utilized-when-scientists-use-the-crispr-cas9-system-to-edit-gene | <p>"CRISPR" and "Cas9" are different things. When a virus attacks a bacteria, the bacteria stores the viral code of the virus in CRISPR. And when the virus attacks again the Cas9 protein uses the RNA in the CRISPR to find the viral DNA and then destroy it.</p>
<p><strong>So, what is the use of CRISP... | <p><a href="https://i.sstatic.net/hdnyG.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/hdnyG.png" alt="Diagram of adaptive immunity mediated by CRISPR RNA, from https://sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engineering-technique/" /></a>
<strong>Figure 1. Process of bacterial im... | 446 |
CRISPR | Splice in with CRISPR/Cas | https://biology.stackexchange.com/questions/30839/splice-in-with-crispr-cas | <p>I need to splice a gene into a human cell genome, with highest rate possible. I mean, doesn't really matter where the gene enters, nor does it matter if some cells die as a result of this.</p>
<p>CRISPR know to knock-in genes with very high specifically, this reduce the success rate if we have a low amount of gRNA ... | <p>A paper was published about a week ago in Nature Biotechnology and adresses your question, <a href="http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3190.html" rel="nofollow">Maruyama T et al., 2015</a>. I must say I found the authors' strategy extremely clever.</p>
<p>It is not about increasing efficiency ... | 447 |
CRISPR | CRISPR-Cas Systems | https://biology.stackexchange.com/questions/34811/crispr-cas-systems | <p>In the context of the bacterial systems (not the gene editing tool), I was wondering what happens to the foreign DNA after the Cas proteins have created a new spacer. </p>
<p>It is really not clear to me, most of the documentation I have found focuses on the subsequent steps (expression and interference) and do not... | 448 | |
CRISPR | CRISPR-Cas9 knockout | https://biology.stackexchange.com/questions/91082/crispr-cas9-knockout | <p>With CRISPR-Cas9 I have conducted a targeted knockout of a DNA region encoding a certain protein (working with leukocytes). My question is, how long does it take until this protein is not detectable any more (e.g. via surface staining + flow cytometry) after the DNA has been manipulated? Or in other terms: how often... | <p>To answer your question multiple parameters which one should be taken into account. </p>
<p>There are two main sets of parameters: <strong>gene specific</strong> and <strong>cell line specific</strong>.</p>
<p>Parameters related to your specific gene of interest :</p>
<ul>
<li>Do you know the half-life of the RNA... | 449 |
CRISPR | Are Restriction Enzymes obsolete with CRISPR? | https://biology.stackexchange.com/questions/74475/are-restriction-enzymes-obsolete-with-crispr | <p>Are Restriction Enzymes obsolete with CRISPR?</p>
| <p>No.</p>
<p>While CRISPR allows you cut a piece of DNA anywhere, you need to order a guide RNA to target your desired cut site. All standard plasmids still carry traditional restriction sites, and it's often convenient to use these. Using CRISPR as a restriction enzyme is probably more expensive and more complicated... | 450 |
CRISPR | CRISPR Knock in | https://biology.stackexchange.com/questions/30460/crispr-knock-in | <p>Using the CRISPR/Cas9 technology, it is possible that after inducing a DSB with the Cas9 endonuclease guided with an RNA designed by the user and using a template DNA, get a desired Knock-In (KI) by homologous recombination.</p>
<p>I have read that for desired KI-s that are less than 200 bp, a olygonucleatide shoul... | 451 | |
CRISPR | Crispr complex in human cells? | https://biology.stackexchange.com/questions/78609/crispr-complex-in-human-cells | <p>Is the crispr (where the parts of Virus DNA is saved) section of the DNA existing in human cells aswell or is it just in bacteria cells?</p>
| <p>No analogues of the CRISPR-Cas system have been found in any eukaryotic species, including humans. So far, it appears to have evolved only in prokaryotes and archaea.</p>
<p>Reference: <a href="https://doi.org/10.1186/s13062-017-0177-2" rel="nofollow noreferrer">Evolution of RNA- and DNA-guided antivirus defense sy... | 452 |
CRISPR | Why do bacteria need the CRISPR system? | https://biology.stackexchange.com/questions/115330/why-do-bacteria-need-the-crispr-system | <p>I'm learning about CRISPR at my college.<br/> I understand that when viral DNA is inserted into a bacterial cell, the <strong>Cas1-Cas2</strong> proteins identify the <strong>PAM</strong> site in the viral DNA and then cut the protospacer from the viral DNA to make it a spacer in the CRISPR array for the adaptive im... | 453 | |
CRISPR | CRISPR/Cas9 edited E.coli on AFM? | https://biology.stackexchange.com/questions/82134/crispr-cas9-edited-e-coli-on-afm | <p>I am doing CRISPR/Cas9 experiment on <em>E. coli</em>. I am introducing recombinant plasmid BPK764 (which carries Cas9 + sgRNA designed and added later in that plasmid) into compentent <em>E.coli</em> cells which already carries another plasmid with GFP gene. sgRNA will be designed so that it corresponds to that GF... | <p>Unless you're specifically targeting genes involved in the physical morphology of the cell, I doubt you would find any differences with CRISPRed vs non-CRISPRed cells that is measurable via AFM.</p>
<p>Perhaps you should instead target genes known to contribute to the structure of the cell wall, which I would expec... | 454 |
CRISPR | Does immunity to CRISPR proteins limit their effectiveness? | https://biology.stackexchange.com/questions/94978/does-immunity-to-crispr-proteins-limit-their-effectiveness | <p>The use of crisper-cas systems is currently applied to cells cultivated in vitro. As control of the ‘off target’ effects of Crispr improves and Crispr is used in vivo, why won’t the immune system neutralize it?</p>
| <p>Usually not. the Crispr/Cas proteins can be delivered to the cell as DNA/RNA and the proteins will only exist inside the cell in low numbers.</p>
<p>Even in systems that deliver the protein from the outside <em>in vivo</em> almost always the proteins would be encapsulated in a delivery system to ensure they and any... | 455 |
CRISPR | Mutations/deletions with CRISPR | https://biology.stackexchange.com/questions/31928/mutations-deletions-with-crispr | <p>I need to stop some protein from being active and searching for some universal way to do so. In mammalians. </p>
<p>With CRISPR it is possible to knock-out the entire gene. But it's a little complicate (need two gRNA for eg.). There are also another reasons, so I need to do this with only one gRNA.</p>
<p>So I am ... | <p>You can do with a single gRNA. All that CRISPR-Cas, ZFN or TALEN systems do is to introduce a double strand break at a specific site. The DNA gets repaired via two mechanisms — non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is error prone and it may introduce indels that can compromise wit... | 456 |
CRISPR | Inheriting Modified DNA after CRISPR editing | https://biology.stackexchange.com/questions/62675/inheriting-modified-dna-after-crispr-editing | <p>If CRISPR is used to modify the DNA sequence to cure a disease - say MS in a woman - will the RNA guides also modify the sequence in her eggs so her children could be born without MS inherited from her.</p>
| <p>First off, CRISPR can't be used to cure diseases in an adult human or, more generally, for any adult vertebrate at present.</p>
<p>As you can see in <a href="http://www.nature.com/nrd/journal/v16/n2/full/nrd.2016.238.html" rel="nofollow noreferrer">this paper</a>, a major problem with CRISPR is it has to target spe... | 457 |
CRISPR | Can CRISPR also remove DNA viruses? | https://biology.stackexchange.com/questions/81849/can-crispr-also-remove-dna-viruses | <p>If I'm not mistaken only RNA viruses insert themselves into the host genome. As an example of DNA viruses, herpes viruses for example do not insert themselves in the host genome.</p>
<p>Can CRISPR cut DNA that isn't in a chromossome, like a DNA virus?</p>
| <p>Yes, this should in principle work, and a number of groups have shows that it works in cultured cells:</p>
<blockquote>
<p>We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells a... | 458 |
CRISPR | Size constraints on CRISPR guide RNA | https://biology.stackexchange.com/questions/80464/size-constraints-on-crispr-guide-rna | <p>I had a quick questions on the size limitations of a CRISPR guide. More specifically on the shorter end. Can I make a guide that is say 7-10bp and still have an active complex? I transfect using an RNP system. </p>
<p>Just curious. </p>
<p>Thanks in advance. </p>
| 459 | |
CRISPR | CRISPR-Cas9 system, DNA repair | https://biology.stackexchange.com/questions/111668/crispr-cas9-system-dna-repair | <p>As a critical stage in the CRISPR-Cas9 system, two different mechanisms of DNA repair can occur in the target DNA after RNA has been introduced: non-homologous end joining (NHEJ) and homologous recombination (HR).</p>
<p>Can these repair mechanisms be intentionally controlled or manipulated, and if so, what effects ... | <p>I would recommend reading some basic resources on current Cas9 technologies. <a href="https://www.nature.com/articles/s41434-021-00263-9" rel="nofollow noreferrer">Here is one such review on prime editing</a>, which illustrates one mechanism by which you can force cells to use a favorable precise repair mechanism. T... | 460 |
CRISPR | Linearising plasmids for CRISPR experiment | https://biology.stackexchange.com/questions/111705/linearising-plasmids-for-crispr-experiment | <p>I am currently designing a mock CRISPR knock-out experiment, and I’m wanting to insert a plasmid for selection. Using a restriction enzyme at 2750bp for cutting, would the location of the cut site then be considered position 1?</p>
<p>I’m wanting to run PCR after insertion to confirm it has been successfully transfe... | <p>No. Plasmids should generally be considered as a circular DNA rather than linear. However, no matter where you cut it, the positions don't change.</p>
<p>The position of the cut site is still the position on the plasmid at which the RE cuts. The numbering on plasmids is based on the position of a common restriction ... | 461 |
CRISPR | What is the most up-to-date CRISPR/Cas9 protocol? | https://biology.stackexchange.com/questions/45836/what-is-the-most-up-to-date-crispr-cas9-protocol | <p>I am collecting literature to start a new project on CRISPR/Cas9 gene editing. I must put together a protocol to start and am intending to use the following paper as guidance:</p>
<p>"Genome engineering using the CRISPR-Cas9 system". Nature Protocol 8(11):2281-308 · November 2013</p>
<p>Could you please indicate w... | <p>It really depends on what you want to do with it. Sticking to genome engineering in Human cells, <a href="http://www.nature.com/nprot/journal/v8/n11/full/nprot.2013.143.html" rel="nofollow noreferrer">Nature Protocol 8(11):2281-308 · November 2013</a> is the de-facto standard. One improvement I suggest is to replace... | 462 |
CRISPR | How was gene knock out done in pre CRISPR era? | https://biology.stackexchange.com/questions/100416/how-was-gene-knock-out-done-in-pre-crispr-era | <p>I am trying to understand how CRISPR has made the gene knockout or gene editing process simpler to make transgenic animals. Here is an old (pre CRISPR) flowchart from <a href="https://pubmed.ncbi.nlm.nih.gov/18077807/" rel="nofollow noreferrer">Manis, 2007</a> that shows how knockouts can be made.
<a href="https://i... | 463 | |
CRISPR | How specific are CRISPR-cas9 cuts? | https://biology.stackexchange.com/questions/49106/how-specific-are-crispr-cas9-cuts | <p>CRISPR-cas9 uses a string of RNA that matches with DNA and makes a double stranded cut at that point. </p>
<p>If the RNA is just a few letters in length, the enzyme would cut DNA in many places. It would be unspecific. But if you can make the RNA string very long, it would only cut at the exact place where you want... | <p>The problem of off-targets in CRISPR/Cas is often discussed. It was shown that the system allows mismatches <a href="http://www.nature.com/nbt/journal/v31/n9/abs/nbt.2623.html" rel="nofollow">up to five basepairs</a>. For your question, if it is helpful to elongate the gRNA: it was shown that <a href="http://www.nat... | 464 |
CRISPR | Can the genetic sequences for CRISPR components be inserted into the host genome so that the cell perpetually produces CRISPR components? | https://biology.stackexchange.com/questions/111231/can-the-genetic-sequences-for-crispr-components-be-inserted-into-the-host-genome | <ol>
<li><p>Theoretically speaking, can you insert the gene sequences for cas9, sgRNA, and promoters into the host genome so that the cell perpetually produces CRISPR components?</p>
</li>
<li><p>In this scenario, I'm guessing there would be no way to have the host cell produce DNA templates for HDR?</p>
</li>
<li><p>H... | 465 | |
CRISPR | what's the difference between traditional genetic engineering and and CRISPR? | https://biology.stackexchange.com/questions/88193/whats-the-difference-between-traditional-genetic-engineering-and-and-crispr | <p>While at school, I learnt about restriction enzymes and how you could insert foreign DNA into an organism.</p>
<p>When I first heard about CRISPR I thought it was very similar except much more precise/targeted.</p>
<p>It seems however that CRISPR only allows editing (substituting nucleotides)</p>
<p>What are the ... | <p>Bacteria and archaea evolved CRISPR as part of their adaptive immune system to protect themselves from invading viruses and foreign plasmids. The defence system relies on small, non-coding RNA molecules (CRISPR RNAs/guide RNA) that in association with a CRISPR associated (Cas) protein silence foreign sequences by me... | 466 |
CRISPR | Mathematical models of gene editing using CRISPR | https://biology.stackexchange.com/questions/74171/mathematical-models-of-gene-editing-using-crispr | <p>One confusing thing I have found when reading articles about possible CRISPR based gene therapy treatments in humans is that there is vey little discussion about what percentage of your cells will actually have their DNA edited, the rate at which the editing takes place, and how to quantitatively estimate how many e... | <p>There are several probabilities that you need to take into account. </p>
<ul>
<li>The efficiency of DNA transformation of CRISPR encoding DNA and
target DNA into the host cell (varies between cell types and
transformation agent).</li>
<li>Efficiency of cutting by CRISPR at target site. (varies by DNA compaction - w... | 467 |
CRISPR | Can CRISPR-Cas9 make changes on a living organism? | https://biology.stackexchange.com/questions/66782/can-crispr-cas9-make-changes-on-a-living-organism | <p>Can CRISPR-Cas9 make changes on a living organism? What's the limit here? E.g. reversing/restoring hearing loss in a living adult mice.</p>
| <p>Answer is yes, but it depends on which kind of phenotype you're going to restore/change. As you can imagine, using crispr cas9 on an high amount of cells require one condition: edited cells need to have a clear advantage in surviving besides the previous. </p>
<p><a href="https://www.nature.com/articles/nbt.2884" r... | 468 |
CRISPR | What is ApoCas9 in the CRISPR-Cas9 system? | https://biology.stackexchange.com/questions/85949/what-is-apocas9-in-the-crispr-cas9-system | <p>I am currently reading an article about a particular assay of Cas9 nucleases. In one of the experiments, they have used ApoCas9 (Apo variants of other CRISPR nucleases) as some sort of control. </p>
<p>But the whole article they have not defined ApoCas9? I did check online of definition and activity of ApoCas9, but... | <p>In general, "apo" is used to indicate an <a href="https://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Apoenzyme_and_Holoenzyme" rel="nofollow noreferrer">apoenzyme</a> — that is the protein part of an enzyme without essential cofactor(s). </p>
<p>This is actually defined in the <strong>Experimental</stron... | 469 |
CRISPR | DIYbio - CRISPR injection sites for targeting the ABCC11 gene | https://biology.stackexchange.com/questions/81004/diybio-crispr-injection-sites-for-targeting-the-abcc11-gene | <p>I've been researching into the biohacking world where people most notable <a href="https://en.wikipedia.org/wiki/Josiah_Zayner" rel="nofollow noreferrer">Josiah Zayner</a> and <a href="https://www.bbc.com/news/world-us-canada-41990981" rel="nofollow noreferrer">Tristan Roberts</a> have used a CRISPR solution develop... | <p>Creating changes in the genome in order to get your favorable results is not always as easy as it looks. Expression of a particular gene is not necessarily bound to its existence. There are other factors (mainly proteins) that have to be in the cell at the right time to make that gene expressed. Yet a trait is not a... | 470 |
CRISPR | Functions of tracrRNA and crRNA in the CRISPR/Cas9 system | https://biology.stackexchange.com/questions/58400/functions-of-tracrrna-and-crrna-in-the-crispr-cas9-system | <p>I need some help about Crispr / Cas9.
The CRISPR/Cas9 technic consists (for bacterias) in "cut" bacteriophage's DNA, in order to make it unfunctional.
When the RNA is transcribed (RNA which is complementary to the bacteriophage's DNA), it will be associated with crRNA and tracrRNA to form the sgRNA (single guide RNA... | <p>I have also been wondering the same thing for a while now and I think that the best answer that I could find after reading several review papers on CRISPR/Cas9 and online information, points to tracrRNA as serving the role of multiplexing with pre-crRNA which helps pre-crRNA mature to crRNA. This process activates t... | 471 |
CRISPR | In CRISPR bacteria, how does viral genomes get integrated into the spacers of CRISPR? Also, in its use, where does Cas9 cut the DNA? | https://biology.stackexchange.com/questions/40576/in-crispr-bacteria-how-does-viral-genomes-get-integrated-into-the-spacers-of-cr | <p>I've been out of Biology for about a year polishing my programming skills. I know CRISPR/Cas9 allows targeted 'cutting' of DNA via RNA-guidance. Few questions regarding this.</p>
<ol>
<li><p>Regarding to its natural phenomenon, when a virus infects a microbe with CRISPR capabilities, how does the genome get integra... | <p><a href="http://www.nature.com/nbt/journal/v32/n4/full/nbt.2842.html" rel="nofollow noreferrer">This paper</a> should clear up a lot more than anyone on here truly can about the CRISPR system.</p>
<p>CRISPR/Cas9 is very unique, even compared to other proteins purified from bacteria like Taq Poly.</p>
<p><a href="h... | 472 |
CRISPR | Can CRISPR-cas9 be used to insert a large gene? | https://biology.stackexchange.com/questions/79267/can-crispr-cas9-be-used-to-insert-a-large-gene | <p>I would like to insert a 1500 bp or longer gene to the broken site in E.coli (or may be <em>bacillus subtilis</em> which support NHEJ) after cut with Crispr-Cas9. Is this possible for both NHEJ or HDR?</p>
| 473 | |
CRISPR | why selecting cells after gene editing with CRISPR Cas9? | https://biology.stackexchange.com/questions/46094/why-selecting-cells-after-gene-editing-with-crispr-cas9 | <p>I am new to the CRISPR Cas9 genome editing system and I have the above basic question.</p>
<p>Another way to think about this: what would happen if after cells are transfected, the cultures are left to grow without any selection. Thus, if a mutation disrupts a cell cycle gene (e.g. p53), such cells will proliferate... | 474 | |
CRISPR | CRISPR guide RNA design and primer synthesis | https://biology.stackexchange.com/questions/59627/crispr-guide-rna-design-and-primer-synthesis | <p>I am trying to knockout huntingtin gene from HeLa cells (human epithelial cells). I used CRISPR explorer and benchling.com to determine the best guide RNA sequence that would bind to the target DNA sequence and cas9 could then make the cut. The gRNA I have is as follows:</p>
<p>GGAGGCCTCGGGCCGACTCG
Strand (-)</p>
<p... | <p>A quick check with BLAST returned the following result:</p>
<pre><code>Homo sapiens huntingtin (HTT), mRNA
Sequence ID: NM_002111.7Length: 13669Number of Matches: 1
Related Information
Gene-associated gene details
GEO Profiles-microarray expression data
PubChem BioAssay-bioactivity screening
Range 1: 267 to 286GenBa... | 475 |
CRISPR | What is the role of tracrRNA in CRISPR-cas9? | https://biology.stackexchange.com/questions/54690/what-is-the-role-of-tracrrna-in-crispr-cas9 | <p>From what I understand, in a CRISPR cas9 complex, gRNA is comprised of tracrRNA and crRNA. I've read that crRNA is the part which is matched to the DNA which is targeted, but what role does tracrRNA play in the process? That I'm not clear about. Also, unless this should be its own question; how is gRNA prepared w... | <p>Your two questions are related and you are correct in your supposition that the Cas9 protein associates to a specific RNA sequence. That of the tracrRNA processed with the crRNA into a gRNA. This is achieved without any other aid so could be considered to do so automatically. How specific the tracrRNA sequence need... | 476 |
CRISPR | How to edit/insert new gene after cutting with CRISPR/cas9 | https://biology.stackexchange.com/questions/50562/how-to-edit-insert-new-gene-after-cutting-with-crispr-cas9 | <p>I'm a student started who has started learning about CRISPR/Cas9. As I understand it, CRISPR/Cas9 is an enzyme that is used to cut a gene at a specific sequence. I would like to know how scientist do the next step to insert/edit a genome.</p>
<p>For example, say I have an original sequence ...AAATTT... Is it possi... | <p>Cas9 is used to create a double-stranded break (DSB) in genomic DNA. The cell can then use <a href="https://en.wikipedia.org/wiki/Homology_directed_repair" rel="nofollow noreferrer">homology directed repair (HDR)</a> to repair the break. The cell can also use <a href="https://en.wikipedia.org/wiki/Non-homologous_end... | 477 |
CRISPR | Can CRISPR knockout of a gene be referred gene inhibition? | https://biology.stackexchange.com/questions/104967/can-crispr-knockout-of-a-gene-be-referred-gene-inhibition | <p>Can gene knockout (e.g. by CRISPR) be referred to as gene inhibition? I am trying to use precise words for a manuscript. We first showed siRNA of a gene has effect X. We also found knockout of the gene has the same effect. For the MS, could I say:</p>
<p>Knockout of the gene showed effect X, confirming inhibition of... | <p>Looking to a standard source of definitions, the <a href="https://www.ebi.ac.uk/sbo/main/SBO:0000169" rel="nofollow noreferrer">Systems Biology Ontology definition of inhibition</a> is "Negative modulation of the execution of a process."</p>
<p>As such, it doesn't really make sense to talk about inhibiting... | 478 |
CRISPR | How is the type of genetic manipulation determined in CRISPR-Cas9? | https://biology.stackexchange.com/questions/29735/how-is-the-type-of-genetic-manipulation-determined-in-crispr-cas9 | <p>I've been reading up a bit on the CRISPR-Cas9 system for gene manipulation. From what I read, it introduces double-strand breaks at specific points determined by the choice of sgRNA. But how do you get from double-strand breaks to editing genes in incredibly specific ways? As far as I read you can fix point mutation... | <blockquote>
<p>But how do you get from double-strand breaks to editing genes in
incredibly specific ways?</p>
</blockquote>
<p>You should read the wikipedia page on CRISPR-Cas system. The target specificity is provided by the tracrRNA. </p>
<blockquote>
<p>How do you specify which kind of repair mechanism is i... | 479 |
CRISPR | Could multiplexed CRISPR disable the mitotic and meiotic genes of cancerous cells? | https://biology.stackexchange.com/questions/113734/could-multiplexed-crispr-disable-the-mitotic-and-meiotic-genes-of-cancerous-cell | <p>Although I believe there is a good reason -- or reasons -- why this theory, that CRISPR could disable the genes for division in cancerous cells, is incorrect, I haven't been able to find them.</p>
<p>In short, the theory is that multiplexed CRISPR could knock out the genes responsible for cellular division (mitotic ... | <p>As with all cancer therapeutics one of the biggest problems is how you get the therapeutic to kill cancer cells without also killing so many healthy cells that the therapy kills the patient.</p>
<p>For CRISPR to work you have to get the CRISPR components into the target cells. Right now this is done using relatively... | 480 |
CRISPR | Can we cure cancer with CRISPR dead Cas? | https://biology.stackexchange.com/questions/104755/can-we-cure-cancer-with-crispr-dead-cas | <p>Here's a silly idea I had this morning:</p>
<ol>
<li>Sequence a bunch of normal patient cells.</li>
<li>Sequence a bunch of tumor cells from a biopsy.</li>
<li>Find a DNA sequence that we're reasonably certain exists in the cancer cells but doesn't exist in normal cells.</li>
<li>Create a guide DNA that matches the ... | <p>There are a lot of assumptions implicit in the system you describe, all of which need to be addressed prior to any serious discussion of your method's feasibility.</p>
<blockquote>
<ol>
<li>Sequence a bunch of normal patient cells.</li>
<li>Sequence a bunch of tumor cells from a biopsy.</li>
<li>Find a DNA sequence ... | 481 |
CRISPR | Difference between micro RNA and short-interfering RNA and CRISPR Cas 9 system? | https://biology.stackexchange.com/questions/41050/difference-between-micro-rna-and-short-interfering-rna-and-crispr-cas-9-system | <p>I read this article <a href="https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/" rel="nofollow">https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/</a> and am slightly puzzled as to why the CRISPR/Cas 9 system is seen as being so revolutionary. It seems like the very same thing that ... | <p>The CRISPR Cas 9 system is used to introduce insertion or deletion in a genomic sequence not mRNA.</p>
<p><a href="https://www.addgene.org/CRISPR/guide/" rel="nofollow">https://www.addgene.org/CRISPR/guide/</a></p>
| 482 |
CRISPR | How can I, using gene therapy/CRISPR, change my eye color? | https://biology.stackexchange.com/questions/116480/how-can-i-using-gene-therapy-crispr-change-my-eye-color | <p><a href="https://knowyourmeme.com/memes/people-with-blue-eyes" rel="nofollow noreferrer">Blue eyes are unsaleable garbage.</a> How would you use CRISPR and gene editing to change a man's eye color from blue to dark brown?</p>
| 483 | |
CRISPR | How to design a permanent vaccine to cholera using CRISPR? | https://biology.stackexchange.com/questions/88889/how-to-design-a-permanent-vaccine-to-cholera-using-crispr | <p>I know that <em>Vibrio Cholerae</em> infects the body through the GM1 ganglioside. So, would it be possible to engineer a CRISPR gene editing tool to prevent <em>Vibrio Cholerae</em> from getting into our cells?</p>
<p>Specifically, would it be possible to insert a piece of DNA into our genes that allow the synthes... | <blockquote>
<p>So, would it be possible to engineer a CRISPR gene editing tool to
prevent Vibrio Cholerae from getting into our cells?</p>
</blockquote>
<p>Vibrio Cholerae doesn't get into our cells.</p>
<p>Did you observe that your paper has no in vitro experiments?</p>
<p>It's going to be tough to get a cell ... | 484 |
CRISPR | How many cuts are done during CRISPR-Cas9 in one cell? | https://biology.stackexchange.com/questions/111127/how-many-cuts-are-done-during-crispr-cas9-in-one-cell | <p>In a CRISPR-Cas9 experiment, the protein cuts the site matching the cRNA part of the gRNA. My question is: How many cuts are possible if multiples sites matching the cRNA are found in the cell?</p>
<p>Especially, considering DNA is made of multiple chromosomes, if more than one chromosome have a site matching the cR... | <p>On a single molecule level, Cas9 from <em>Streptococcus pyogenes</em> cuts but does not move to other targets for a few days (<a href="https://doi.org/10.1021/jacs.7b13047" rel="nofollow noreferrer">Raper et al. 2018</a>). In contrast, Cas9 from <em>Staphylococcus aureus</em> cuts multiple targets, though also it ta... | 485 |
CRISPR | Why doesn't CAS9 cleave the original CRISPR sequence in the bacterial genome? | https://biology.stackexchange.com/questions/54696/why-doesnt-cas9-cleave-the-original-crispr-sequence-in-the-bacterial-genome | <p>CAS9 is the RNA-guided endonuclease that cleaves DNA as specified by the RNA sequence and is used to target viruses that infect bacteria. So why doesn't this RNA+endonuclease combo also cleave the original CRISPR spacer sequences (which also have the same DNA?). Is it the same reason that the bacterial genome is met... | <p>It is not the methylation status. The crRNA is not only complementary to the spacer sequence within the CRISPR array but also to the repeat sequence flanking that spacer. The additional base pairing of the sgRNA with the repeat prevents a nucleolytic cleavage by Cas9. In addition, the arrays typically do not contain... | 486 |
CRISPR | CRISPR/Cas9: What are the main differences between sgRNA and the Cr:TracrRNA ? | https://biology.stackexchange.com/questions/57503/crispr-cas9-what-are-the-main-differences-between-sgrna-and-the-crtracrrna | <p>So from what I understand, in gene editing, the CRISPR vector expresses a small RNA sequence comprised of a small guide-RNA that is complementary to your target sequence. The sgRNA comprises a 20 Bp CrRNA and a truncated (cut down) TracrRNA right ? </p>
<p>My main question is how does this truncation enable gene ed... | <p>You are correct about the components of the sgRNA.</p>
<p>However the truncation of the tracrRNA does not enable gene editing. Rather it is all that is required.</p>
<p>In bacteria the crRNA and tracrRNA are distinct molecules which must pair through the complementary palindromic repeat sequences. This is not the ca... | 487 |
CRISPR | Has anyone used Crispr/Cas to induce a knock-in in MEF cells? | https://biology.stackexchange.com/questions/19829/has-anyone-used-crispr-cas-to-induce-a-knock-in-in-mef-cells | <p>Does anyone have experience with the CRISPR/CAS9 platform performed on MEF? Or does anyone recall any relevant articles?</p>
| <p>From <a href="http://genesdev.cshlp.org/content/27/23/2602.long" rel="nofollow">this</a> paper:</p>
<blockquote>
<p>We chose to test for Cas9-driven targeted mutagenesis on the
endogenous Trp53 gene in mouse embryonic fibroblasts (MEFs), a cell
line in which the biology of p53 has been thoroughly characterize... | 488 |
CRISPR | Do users of CRISPR/Cas iterate or parallelize to try multiple guide sequences? | https://biology.stackexchange.com/questions/49133/do-users-of-crispr-cas-iterate-or-parallelize-to-try-multiple-guide-sequences | <p>I've read about on-target efficiency and off-target effects in use of CRISPR/Cas9, and about tools that suggest good guide sequences. I am wondering: how many guide sequences do typical CRISPR users try -- i.e. is there any iteration or parallelism involved?</p>
<p>I think the answer is yes for some use cases, but ... | 489 | |
CRISPR | Why is dsDNA nuclease activity by CRISPR/Cas9 only shown indirectly? | https://biology.stackexchange.com/questions/38933/why-is-dsdna-nuclease-activity-by-crispr-cas9-only-shown-indirectly | <p>In <a href="http://elifesciences.org/content/2/e00471" rel="nofollow">Jinek et al.</a>, the authors show nuclease activity of their CRISPR/Cas9 system using the so-called <em>Surveyor assay</em> method. This assay recognizes small mismatches in dsDNA which are introduced by error prone non-homologuous end-joining af... | 490 | |
CRISPR | What is the role of CRISPR-dCas9 in gRNA-dCas9 transcription regulator complexes? | https://biology.stackexchange.com/questions/76567/what-is-the-role-of-crispr-dcas9-in-grna-dcas9-transcription-regulator-complexes | <p>In <a href="https://www.cell.com/trends/biotechnology/fulltext/S0167-7799(15)00274-7?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0167779915002747%3Fshowall%3Dtrue" rel="nofollow noreferrer">this</a> paper<sup>1</sup>, I read that mutant versions of Cas proteins such as a deactivated Cas9 (dC... | <blockquote>
<p>so why can't the transcription factor be fused with the gRNA instead,
so that it can directly affect transcription when the gRNA forms
complimentary base pairs with the target gene, instead of it being on
a passive dCas9?</p>
</blockquote>
<p>The main reason for that is that both the gRNA and t... | 491 |
CRISPR | Are scientist able to correct mutiple gene defect in our body by using CRISPR | https://biology.stackexchange.com/questions/42862/are-scientist-able-to-correct-mutiple-gene-defect-in-our-body-by-using-crispr | <p>Are scientist able to correct mutiple gene defect in whole body by using CRISPR recently?</p>
<p>AS i know, it is in a beginning stage</p>
| <p>Certainly not yet - there is not yet any evidence that this can be used to correct specific deficiencies in an adult, though the in vivo applications are growing.</p>
<p>In particular, an exciting recent advance is the opportunity to better understand carcinogenesis by 'editing in' certain known mutations into a mo... | 492 |
CRISPR | When did CRISPR/Cas9 evolve and what is the likelihood that a superior system for live cell genome editing has already evolved on earth since then? | https://biology.stackexchange.com/questions/20665/when-did-crispr-cas9-evolve-and-what-is-the-likelihood-that-a-superior-system-fo | <p>I've read that CRISPR/Cas9 is currently being implemented and tested for its ability to edit genomes in live cells, and that it is supplanting other genome editing tools in labs, such as TALENs and Zinc finger nucleases. </p>
<p>I understand that there may be a few metrics used for analyzing any genome editing sys... | <p>Lots of interesting questions! Let me try to address a few of them as I don't think I am qualified to answer them all but hopefully I can get this thread started. I am a graduate student in the biophysical chemistry field and have been following a little bit of the Crispr Cas9 craze in the last couple of years. So I... | 493 |
CRISPR | Is CRISPR mediated RNA editing less specific and less efficient than DNA editing? | https://biology.stackexchange.com/questions/108213/is-crispr-mediated-rna-editing-less-specific-and-less-efficient-than-dna-editing | <p>According to <a href="https://www.sciencedirect.com/science/article/pii/S109727651830546X" rel="nofollow noreferrer">this diagram</a>, the high efficiency and the high specificity of CRISPR lies in its reversible binding with the target DNA. The Cas protein unzips the target DNA and have the gRNA to base pair with i... | 494 | |
CRISPR | What are the differences between dual vector CRISPR/Cas9 lentiviral plasmids Lenti‐Cas9‐2A‐Blast and lentiCas9-Blast? | https://biology.stackexchange.com/questions/109257/what-are-the-differences-between-dual-vector-crispr-cas9-lentiviral-plasmids-len | <p>There are multiple widely-used plasmids for using CRISPR/Cas9 with a dual lentiviral vector strategy (Cas9 & sgRNA on different vectors) in mammalian hosts with a Blasticidin selection selection marker.</p>
<p>In particular:
<a href="https://www.addgene.org/73310/" rel="nofollow noreferrer">Lenti‐Cas9‐2A‐Blast(... | <p>I see these differences in lenti-Cas9-2A (compared to lenti-Cas9):</p>
<ul>
<li>Absence of a bleomycin resistance. That just affects cloning</li>
<li>Changing from a CMV- to a RSV <strong>promoter</strong> for lentiviral mRNA production: That promoter is used for the host cells that produce your lentiviruses. They m... | 495 |
CRISPR | Is it possible to insert DNA without cutting the recognition site with CRISPR/Cas9? | https://biology.stackexchange.com/questions/30347/is-it-possible-to-insert-dna-without-cutting-the-recognition-site-with-crispr-ca | <p>We are looking for a way to insert DNA into a genome, but we would like to do it in a way that the recognition site stay intact to be able to add again DNA at the same location. Do you know if it is possible or if it is already the way CRISPR/Cas9 does it? Apparently multiple systems were already engineered but the ... | <p>Look into <a href="http://en.wikipedia.org/wiki/Site-specific_recombination" rel="nofollow noreferrer">site-specific recombination</a>. You can use a site-specific recombinase, specifically an integrase, that can insert a sequence of DNA at a certain attachment site. You can add an identical attachment site into t... | 496 |
CRISPR | What´s the role or function of the homologous arms in a donor template in a knockout/knock edition via Crispr-cas9? | https://biology.stackexchange.com/questions/51857/what%c2%b4s-the-role-or-function-of-the-homologous-arms-in-a-donor-template-in-a-knoc | <p>I have to make an exposition in my university about Crispr-cas9 edition and I have some questions about the method. In the knock out/knock in technique is used a plasmid containing the DNA that codifies for the Cas9 protein and guideRNA and a <strong>Donor template</strong> that has a gene for puromicine resistance,... | 497 | |
CRISPR | In this illustration about CRISPR function, what do these objects mean (image provided)? | https://biology.stackexchange.com/questions/38706/in-this-illustration-about-crispr-function-what-do-these-objects-mean-image-pr | <p>In the paper on CRISPRs, the following figure is shown:
<a href="https://i.sstatic.net/gnwUp.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/gnwUp.png" alt="enter image description here"></a></p>
<p>I added the light-green boxes.</p>
<p>What do the red arrows and the line with a filled circle on the... | <p>The arrow direction denote transcription direction and its location denotes the transcription start site. The circle is probably the transcription terminator (I cannot access this article at the moment, but this is what it should mean).</p>
<p>The purple boxes refer to the protospacers which are derived from foreig... | 498 |
CRISPR | Can a gene be inactivated using CRISPR if it is not in the interspace of short palindromic repeats? | https://biology.stackexchange.com/questions/69273/can-a-gene-be-inactivated-using-crispr-if-it-is-not-in-the-interspace-of-short-p | <p>I have recently studied how CRISPR works but there is something that I do not understand at all. I have heard a lot of people claiming that with this method it is possible to modify any genome by inactivating, activating, removing or adding genes. However, as far as I have understood, DNA regions can only be modifie... | <p>When people talk about genome editing with CRISPR, they are really talking about using CRISPR associated nucleases like Cas9 and Cpf1. These nucleases are useful since their sequence specificity is determined by a guide RNA and they can therefore be used to cut at specific sites in the genome. This actually has noth... | 499 |
CRISPR | Would viral diversity result in a change in the effectiveness of CRISPR systems in a population of bacteria, within a closed system? | https://biology.stackexchange.com/questions/107756/would-viral-diversity-result-in-a-change-in-the-effectiveness-of-crispr-systems | <p>I have here my hypothesis, does this make scientific sense? Assume this situation is occurring in a closed environment with only bacteria and bacteriophages.</p>
<p>The effectiveness of CRISPR/Cas9, being an adaptive defence system, is heavily reliant on the phages that it, or its ancestor has previously hosted. Thi... | 500 | |
CRISPR | Can we change the Eye/Hair color by knocking out the OCA2, HERC2 and MC1R genes using CRISPR in an adult human? | https://biology.stackexchange.com/questions/96043/can-we-change-the-eye-hair-color-by-knocking-out-the-oca2-herc2-and-mc1r-genes | <p>This paper seems to describe the use of a plasmid delivered by a gene gun to depigment rat skin;</p>
<blockquote>
<p><a href="https://www.nature.com/articles/3302264" rel="nofollow noreferrer">https://www.nature.com/articles/3302264</a>
Published: 27 May 2004
Seeing the gene therapy: application of gene gun techniqu... | <p>Let's first breakdown this question</p>
<blockquote>
<p><strong>What is CRISPR?</strong></p>
</blockquote>
<ul>
<li>CRISPR is a group of DNA sequences that play a key role in the antiviral defense system of prokaryotic organisms such as bacteria and archaea.</li>
<li>They are derived from DNA fragments of bacterioph... | 501 |
CRISPR | CRISPR/Cas9: How can inserted DNA be used as a donor for the homology-directed repair if Cas9 only creates blunt ends? | https://biology.stackexchange.com/questions/111256/crispr-cas9-how-can-inserted-dna-be-used-as-a-donor-for-the-homology-directed-r | <p>The CRISPR/Cas9 method allows new genes to be inserted. After Cas9 cuts the Target-DNA, it can use a homologous piece of DNA as a donor template for homology-directed repair. But HDR only occurs when there are sticky ends, and Cas9's cuts end in blunt ends.</p>
<p>What did I miss?</p>
| <p>According to <a href="https://blog.addgene.org/crispr-101-homology-directed-repair" rel="nofollow noreferrer">this Addgene blog</a>:</p>
<blockquote>
<p>There are four different HDR pathways to repair DSBs. Here are three
central steps of the HDR pathways:</p>
<ol>
<li><p>The 5’-ended DNA strand is resected at the ... | 502 |
CRISPR | Crispr-Cas9 method and nobel | https://biology.stackexchange.com/questions/70708/crispr-cas9-method-and-nobel | <p>Since i had my first cell class at university i have heard about Cripsr Cas9 method. But I am quite surprised about one fact. Why actually wasnt rewarded by Nobel price? Is it something like Einsteins relativity (to early to reward it)?</p>
| <p>My guess is that some people (likely Jennifer Doudna, Emmanuelle Charpentier and Feng Zhang) will eventually be awarded a Nobel Prize for the discovery of CRISPR and the development of its applications for genome editing, because it really is a major advance. But for now, <a href="https://en.wikipedia.org/wiki/CRISP... | 503 |
CRISPR | How is the Guide RNA created for Crispr? | https://biology.stackexchange.com/questions/66646/how-is-the-guide-rna-created-for-crispr | <p>It seems that to modify DNA a guide RNA and Cripr are introduced.</p>
<p>But I'm unable to understand how the Guide RNA is made or created.</p>
<p>Is it a simple method which can be done with simple lab equipment or is it complicated?</p>
<p>What is the equipment need to create it?</p>
| 504 | |
CRISPR | How does Cas9 interact with CRISPR? | https://biology.stackexchange.com/questions/38915/how-does-cas9-interact-with-crispr | <p>I read that Cas9 protein along with guided RNA binds at a specific DNA fragment of foreign organism integrated in a host organism DNA. To make the host immune to virus infection Cas9 along with gRNA which is complementary to viral DNA attaches to it host DNA by unbinding it and then cuts the DNA at this site thereby... | <p>We are currently using CRISPR in the lab to modify cells. Like @AMR said, it was modified from the <em>In Vivo</em> immune system to be used in the lab in DNA editing field. The Addgene website features both <a href="https://www.addgene.org/crispr/reference/history/" rel="nofollow">historical background</a> and an <... | 505 |
CRISPR | if an SNP were edited using CRISPR What are the chances that, absent artificial selection, wild type alleles would reemerge? | https://biology.stackexchange.com/questions/96379/if-an-snp-were-edited-using-crispr-what-are-the-chances-that-absent-artificial | <p>I am researching a fatty acid amide hydrolase (FAAH) SNP RS324420 and FAAH out microdeletion that together lead to reduced pain sensitivity and reduced anxiety (<a href="https://www.sciencedirect.com/science/article/pii/S0028390807002146" rel="nofollow noreferrer">Moreira et al 2008</a>).</p>
<blockquote>
<p>The cau... | 506 | |
CRISPR | Does ribonuclease processing of pre-crRNAs happen co-transcriptionally? | https://biology.stackexchange.com/questions/104533/does-ribonuclease-processing-of-pre-crrnas-happen-co-transcriptionally | <p>I understand CRISPR-mediated bacterial immunity to occur in the following simplified steps:</p>
<ol>
<li>A CRISPR array is transcribed from promoters in the leader sequence to yield a precursor CRISPR RNA (pre-crRNA).</li>
<li>The pre-crRNA is processed by ribonucleases (<em>e.g.</em> Cas6 in Type III systems) to cr... | <p>I originally posted this question in an attempt to understand a pattern I observed in my sequencing data. In the time since, I've formalized my interpretation as part of a manuscript, <a href="https://www.biorxiv.org/content/10.1101/2022.04.22.489220v1" rel="nofollow noreferrer">now posted on <em>bioRxiv</em></a>.</... | 507 |
CRISPR | CRISPR/Cas for editing the human genome | https://biology.stackexchange.com/questions/45655/crispr-cas-for-editing-the-human-genome | <p>I know, that the CRISP/Cas approach for "cutting" the human genome is not completely suitable if we can't say not suitable at all. Because we have many repeats and this approach can bring to our genome additional breaks in DNA and after DNA-repair it causes unwanted mutations. If so, why scientist still continue to ... | <p>Repeats aren't <em>the</em> problem with CRISPR. They stop you from editing repeats and repeat-like sequences, but in principle they can be worked around through careful design of guide RNA. CRISPR techniques right now do have problems with off target effects, but these problems may yet be solved. The CRISP/Cas appr... | 508 |
CRISPR | What software do I need to read-write cas9? and .dna files? | https://biology.stackexchange.com/questions/89402/what-software-do-i-need-to-read-write-cas9-and-dna-files | <p>I am still a complete beginner to crispr and I am still trying to learn what it is and how to actually use it. I now realise that you have to order the crispr components after you have actually designed them yourself in software that lets you design and alter the components. </p>
<p>Am I correct so far?</p>
<p>I a... | <p>You have a long journey ahead. This guy spent about 4 years to learn what he is doing <a href="https://www.youtube.com/watch?v=5Rv6aMdKY40" rel="nofollow noreferrer">here</a>.</p>
<p>He is using Snapgene software</p>
| 509 |
CRISPR | Webtool to design guide RNA (gRNA) for use with CRISPR-AsCpf1? | https://biology.stackexchange.com/questions/59027/webtool-to-design-guide-rna-grna-for-use-with-crispr-ascpf1 | <p><strong>My goals are to use a free webtool to:</strong></p>
<ol>
<li><em>Identify guide RNAs</em> (direct-repeat sequence followed by the targeting sequence) appropriate for use with <em>AsCpf1</em> in order <em>to target a specific segment of genomic DNA</em>.</li>
<li><em>Estimate efficiency and specificity</em> ... | <p>Most online tools will only help you to design gRNAs for Cas9 because it is the one that is most commonly used. I found an online suite called <a href="http://www.rgenome.net/be-designer/" rel="nofollow noreferrer">RGEN tools</a> that also has an option for AsCpf1. </p>
<p>However, if you don't find it satisfactory... | 510 |
CRISPR | Crispr/CAS9 genome editing is actually processed at which phase of Cell cycle? | https://biology.stackexchange.com/questions/73812/crispr-cas9-genome-editing-is-actually-processed-at-which-phase-of-cell-cycle | <p>all,</p>
<p>Is it only happens in certain phases, like S, G1,...or it can happen any time....or maybe, it has some perferences.</p>
<p>Put it in another word, does it going to processe edit if the cell is not growing ?</p>
<p>Thanks for any helps or comments.</p>
<p>Best</p>
<p>Bill Zhang</p>
| 511 | |
CRISPR | Does using CRISPR/Cas9 knockout need to add donor DNA in the process? | https://biology.stackexchange.com/questions/94021/does-using-crispr-cas9-knockout-need-to-add-donor-dna-in-the-process | <p>I am new to this technology and don't quite understand how it works. Hope someone can give some suggestions! Thank you.</p>
| <p><em>Short Answer:</em>
No, for CRISPR/Cas9 knockout you do not need to add donor DNA.</p>
<p><em>Little bit more detail:</em>
CRISPR/Cas9 allows you to cut at a given position (defined by the <a href="https://en.wikipedia.org/wiki/Guide_RNA" rel="nofollow noreferrer">gRNA</a>). This will lead to double strand brea... | 512 |
CRISPR | Can CRISPR be used in editing primary cells? How is the transfection efficiency? | https://biology.stackexchange.com/questions/94044/can-crispr-be-used-in-editing-primary-cells-how-is-the-transfection-efficiency | <p>Sometimes people wanna use primary cells to do gene-editing because the cells normally have more interesting characteristics, but can we really do so?</p>
| <p>The most efficient CRISPR-based editing of primary cells that I've read about used a technology called <a href="https://en.wikipedia.org/wiki/Prime_editing" rel="nofollow noreferrer">prime editing</a>, developed in 2019.</p>
<p><a href="http://blumberg-lab.bio.uci.edu/biod145-w2020/required%20reading/anzalone-2019_... | 513 |
CRISPR | Where does tracrRNA comes from? | https://biology.stackexchange.com/questions/65776/where-does-tracrrna-comes-from | <p>I'm talking about CRISPR system. I know the crRNA is transcribed from the palindromic repeat and the "spacer" but I don't know where the tracrRNA comes from.</p>
| <p>Usually the tracrRNA is a part of the CRISPR locus and is encoded in the the vicinity of the CRISPR array (e.g. upstream or downstream of the cas genes or the array).</p>
<p><a href="http://www.genome-engineering.org/crispr/wp-content/uploads/2013/01/crispr_processing1.jpg" rel="nofollow noreferrer">http://www.geno... | 514 |
CRISPR | What impact do genetic engineering techniques have on seed breeders? | https://biology.stackexchange.com/questions/67100/what-impact-do-genetic-engineering-techniques-have-on-seed-breeders | <p>In research of seed breeding, I'm trying to understand the impact of genetic engineering techniques like CRISPR (This is the main one as I understand) on traditional seed breeders. Through searching on the internet, I believe to have found the following two types of impact:</p>
<ol>
<li>research: increased speed of... | 515 | |
CRISPR | How to find the enhancer region of a specific gene? | https://biology.stackexchange.com/questions/88850/how-to-find-the-enhancer-region-of-a-specific-gene | <p>I am new to these concepts in biology and need some help understanding. My main concern is how to find the enhancer of my gene of interest, specifically the sequence of the enhancer. I am working on a project where I will be attempting to use CRISPR- mediated deletion of the promoter region and enhancer region in my... | <p>There are several ways to identify enhancers:</p>
<p>(1) Chip seq / chip-chip recognize transcription factor binding sites,</p>
<p>(2) Enhancer specific factors can also be used to identify enhancers, such as EP300, the binding sites of EP300 are often used to predict enhancers;</p>
<p>(3) RNA polymerase II can b... | 516 |
CRISPR | Is Cas9 unique in it's ability to act in response to a specific DNA sequence? | https://biology.stackexchange.com/questions/52480/is-cas9-unique-in-its-ability-to-act-in-response-to-a-specific-dna-sequence | <p>As a computer scientist, I'm interested in the ability of the Cas9 protein to function as an if-gate for DNA. It opens up so many questions for me. Right now the protein works as "if the sequence matches, cleave", but perhaps you could customize it to do "if the sequence matches, [do arbitrary action]". However, I'd... | <p>There are two classes of proteins that I can think of off the bat that are DNA sequence-specific. First are <a href="https://en.wikipedia.org/wiki/Restriction_enzyme" rel="nofollow">restriction enzymes</a>, which recognize a specific (usually short) sequence of DNA and cleave it, sometimes through both strands at th... | 517 |
CRISPR | When gene editing are both chromosomes in a pair changed? | https://biology.stackexchange.com/questions/80008/when-gene-editing-are-both-chromosomes-in-a-pair-changed | <p>Sorry for the possibly confused question, my knowledge of genetics is limited to medical training only but I have a question.</p>
<p>Are gene editing techniques such as CRISPR used on both of the chromosomes in a pair or just on one of them?</p>
<p>Some of the general searching I have done suggests that it can hit... | <p>Gene editing using CRISPR-Cas9 can result in both heterozygous and homozygous conditions. It is just the event of probability. But even if u get heterozygous line, making it homozygous is not very difficult. You just have to cross two heterozygous line and screen for homozygous line through markers. </p>
| 518 |
CRISPR | Mutated cell proliferation | https://biology.stackexchange.com/questions/64247/mutated-cell-proliferation | <p>Reading Jennifer Doudna's fascinating book on CRISPR. So she describes rare cases where a mutation in a single cell removes the gene responsible for a genetic disease. The cell proliferates and the disease is cured. What I don't understand is--aren't all the aberrant cells also still dividing and passing on the harm... | <p>There are some diseases in which a minority population of normal cells can rescue the organism, even in the presence of a majority of mutant cells. Sort of like a group vacation to Kazakhstan with one friend who can speak Kazakh. Very different experience from a vacation where none of you speak Kazakh. </p>
<p>Ta... | 519 |
CRISPR | How are spacer sequences created in a prokaryotic genome? | https://biology.stackexchange.com/questions/111138/how-are-spacer-sequences-created-in-a-prokaryotic-genome | <p>The CRISPR/Cas defense mechanism uses spacer sequences between palindromic repeats to search for the sequence to cut by an endonuclease. But how are these spacers created?</p>
<p>Let's take Bacteriophages, for example, which insert and integrate their genome into the prokaryotic genome. How does this infected cell g... | 520 | |
CRISPR | Are there any techniques for manufacturing exosomes in the lab? | https://biology.stackexchange.com/questions/65788/are-there-any-techniques-for-manufacturing-exosomes-in-the-lab | <p>I'm interested in the idea of using exosomes as an alternative transfection agent / delivery mechanism for gene editing applications (CRISPR/cas9, etc). </p>
<p>Information on the matter is sparse.. Does anyone know of any practical techniques for manufacturing exosomes in the lab?</p>
| 521 | |
CRISPR | How could a species be engineered to go extinct? | https://biology.stackexchange.com/questions/71545/how-could-a-species-be-engineered-to-go-extinct | <p>Non-biology background here.</p>
<p>I read this very interesting article: <a href="https://www.wired.com/story/crispr-eradicate-invasive-species/" rel="noreferrer">https://www.wired.com/story/crispr-eradicate-invasive-species/</a></p>
<p>However I am having a hard time wrapping my head around something:</p>
<p>Fr... | <p><strong>Short answer</strong></p>
<p>The article in particular that you reference is discussing the possibility of using a mechanism called <a href="https://en.wikipedia.org/wiki/Gene_drive" rel="noreferrer">gene drive</a>. The concept of gene drive breaks the normal "rules" of inheritance and allows a gene to spre... | 522 |
CRISPR | Where can I obtain Sputnik virophage samples? | https://biology.stackexchange.com/questions/90146/where-can-i-obtain-sputnik-virophage-samples | <p>I'm working on a project where I'm trying to use virophages as viral vectors in introducing CRISPR-Cas proteins into other viruses. I was wondering where I might be able to find replication-defective Sputnik virophages or other virophages for this kind of experiment?</p>
| 523 | |
CRISPR | Heat shock vs electroporation | https://biology.stackexchange.com/questions/73150/heat-shock-vs-electroporation | <p>I've been transforming E. coli via heat shock in order to insert oligonucleotides (around 50 nt); however, none of my experiments have given positive results so far. I begin to question the efficiency of chemical transformation, especially for short DNA fragments. Is there such a notable difference between chemical ... | <p>How do you detect a potenially positive transfection? Short fragments might have proper teriary structures which may make them behave differently than you would expect. Also, noncircular DNA and RNA is targeted much faster for degradation in most cells if the right head or tail signals are missing.</p>
<p>To your q... | 524 |
CRISPR | What obstacle(s) would be most prevalent in an attempt to use virotherapy to inject Cas into cells to target a cancerous mutation? | https://biology.stackexchange.com/questions/97604/what-obstacles-would-be-most-prevalent-in-an-attempt-to-use-virotherapy-to-inj | <p>Let's say a human cell mutates cancerously, we identify the mutated gene sequence and use CRISPR to mutate the patient's cells to include some variation of the cas genes and spacer sequences matching the mutated cancerous sequence.</p>
<p>Then design a virus to "infect" cells (cancerous and healthy) with t... | 525 | |
CRISPR | A reliable source of Cas9? | https://biology.stackexchange.com/questions/71189/a-reliable-source-of-cas9 | <p>I live in Egypt, and I do want to perform CRISPR-Cas9 genome modification, but there are no reliable sources of Cas9. I can order some Cas9 online from the US, but not much. So... my question is, is there any way I can replicate a small amount of Cas9 to get a bigger amount? Or maybe extract some from S. Pyogens? I'... | 526 | |
CRISPR | Can the "Cas9"(involved on the Crispr-cas9 mechanism) be considered as a restriction enzyme? | https://biology.stackexchange.com/questions/52360/can-the-cas9involved-on-the-crispr-cas9-mechanism-be-considered-as-a-restric | <p>I have many questions about the Cas9 enzyme. When the Cas9-guideRNA (crRNA and transcrRNA) complex is attached to the DNA sequence, what is the type of bond that cuts the Cas9? Does the Cas9 degrade all the sequence of DNA complementary to the guide RNA?</p>
<p>If the question before is "Not", then how is the seque... | <p>Cas9 is an endo-deoxyribonuclease (<a href="http://www.uniprot.org/uniprot/Q99ZW2" rel="nofollow">UniProt-Q99ZW2</a>). It also has a 3'-5' exonuclease activity by which it trims the DNA a little bit, from the cut site (but not too much).</p>
<p>Although, Cas9 is an endonuclease and is evolved as a mechanism of immu... | 527 |
CRISPR | Could we eradicate mosquitoes? | https://biology.stackexchange.com/questions/58604/could-we-eradicate-mosquitoes | <p>Researchers have proposed the application of CRISPR/Cas9 and <a href="https://en.wikipedia.org/wiki/Gene_drive" rel="nofollow noreferrer">gene drive</a> to genetically alter wild mosquito populations such that they don't transmit malaria. The government of New Zealand has announced a <a href="https://en.wikipedia.or... | <blockquote>
<p>Could we use this technology to completely eradicate from the world
all species of mosquito that prey on humans?</p>
</blockquote>
<p>Yes, implemented correctly, a gene drive has this capability.</p>
<blockquote>
<p>Also, could we accurately predict the extent of the resulting
ecological disru... | 528 |
CRISPR | What is the difference between DNA vs RNA Editing in the context of gene therapy? | https://biology.stackexchange.com/questions/84567/what-is-the-difference-between-dna-vs-rna-editing-in-the-context-of-gene-therapy | <p>As a someone with beginner knowledge on biology, I have come across the terms "RNA editing".</p>
<p>Take this paper for example : <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793859/" rel="nofollow noreferrer">RNA Editing with CRISPR-Cas13</a></p>
<p>From my understanding, DNA -> RNA -> Proteins</p>
<p>... | <p>There are several differences, both mechanistically and functionally.</p>
<ol>
<li>DNA edits are heritable by the daughter cells and are therefore permanent whereas RNA edits are not.</li>
<li>The type of Cas protein used in the editing process for DNA and RNA are different. </li>
<li>In the paper that you linked t... | 529 |
CRISPR | What happens to the second strand in single-stranded / prime editing? | https://biology.stackexchange.com/questions/114552/what-happens-to-the-second-strand-in-single-stranded-prime-editing | <p>Articles about ’prime’ CRISPR editing generally state that a major advantage is that it only affects one strand…</p>
<p>…But what happens to the second, complementary strand of the genome? Doesn’t it have to eventually change to match the first?</p>
<p>Or is that edited portion of the genome forever ‘open’, which w... | <p><em>Of course, both DNA strands must be changed.</em></p>
<p>The way this is done can be found,for examlple, in the Wikipedia article <a href="https://en.wikipedia.org/wiki/Prime_editing" rel="nofollow noreferrer">“Prime editing”</a>. In brief, there have been several modifications of the main approach to this, whic... | 530 |
CRISPR | How bacteria respond to toxic viral proteins? | https://biology.stackexchange.com/questions/110620/how-bacteria-respond-to-toxic-viral-proteins | <p>The lysis-lysogeny state of bacteriophage lambda is well known. Under certain conditions, the phage will enter the lysogenic state after infection of a bacterium. Then, after a while, the phage switches to the lytic state and breaks the bacterium. I forgot the details but the phage must expressed some proteins toxic... | <p>I don't know of any direct defenses available to bacteria once a prophage genome has been integrated. If a prophage has induced and carried out its program to the point that lysis proteins are being produced, it is far too late for the host cell.</p>
<p>However, an indirect defense for the bacteria is to simply surv... | 531 |
CRISPR | Are exosomes useful as a transfection or delivery mechanism in gene editing? | https://biology.stackexchange.com/questions/65786/are-exosomes-useful-as-a-transfection-or-delivery-mechanism-in-gene-editing | <p>The use of viruses as transfection or delivery agents for gene editing (CRISPR/cas9, etc) is well known. However, one problem with using viruses to deliver DNA into cells is the possibility of triggering an adverse immune response.</p>
<p>Cells already use exosomes to transport various cargo in and out of cells. Do... | 532 | |
CRISPR | Bacteriophages and their role in genetic editing? | https://biology.stackexchange.com/questions/62854/bacteriophages-and-their-role-in-genetic-editing | <p>I know how plasmids and restriction enzymes work to change the dna of a bacteria cell, but I do not really understand how a bacteriophage works to edit the genome of a cell. Is it related to crispr since its a virus which inserted genetics in bacteria? Also, is it during the lysogenic or lytic stage that the bacteri... | <p>As explained in the Wikipedia page that you have linked to, <strong>transduction</strong> is the name given to a particular phenomenon whereby phage can be used to move bacterial DNA between cells. </p>
<p>Depending upon the phage, some aberrant or infrequent process can result in the packaging of host cell DNA int... | 533 |
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