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47

Short answer The article in particular that you reference is discussing the possibility of using a mechanism called gene drive. The concept of gene drive breaks the normal "rules" of inheritance and allows a gene to spread much more rapidly than normal in a population. Longer answer Gene drive depends on the idea of a selfish gene. There are naturally ...


26

The image was not in the DNA as such, only as an abstract representation that could be converted into an image from knowledge of the code. Briefly, they encoded the image into DNA, using a couple of different strategies in which DNA represented pixels -- either with a single DNA base representing a pixel, or with a triplet representing a pixel. Knowing the ...


16

Just to add what might have been missing in the beautiful answer by @iayork. I just want to give a more simple picture of the encoding done in the E. coli DNA. First for the rigid strategy in which 4 pixel colors were each specified by a different base, suppose we have a sequence: AAGCCCTGGTCAGCT Ignore the first AAG and start with C. Now, each base of ...


6

Since a few people asked why the AAG triplet is avoided in the code, I thought I'd add this in addition to the other answers. The interesting part of this research is not necessarily the image encoding but rather how they utilized the CRISPR system to integrate the encoding DNA into the genome. It may be a surprise to some that the image is not encoded in ...


6

A paper was published about a week ago in Nature Biotechnology and adresses your question, Maruyama T et al., 2015. I must say I found the authors' strategy extremely clever. It is not about increasing efficiency by reducing specificity, but simply increasing efficiency (which is your ultimate goal anyway). What the authors did was to inhibit nonhomologous ...


5

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 means of cleavage. Twenty nucleotides at the ...


4

If you are talking about the nuclease functionality of Cas9, then you aren't adding the Cas9 gene into the genome, you are transfecting it on a plasmid. Usually there is either some form of permeabilization done to the cells, or the construct is inserted into a viral vector in order to transfect the cells in tissue culture. In multicellular animals, you ...


4

The problem of off-targets in CRISPR/Cas is often discussed. It was shown that the system allows mismatches up to five basepairs. For your question, if it is helpful to elongate the gRNA: it was shown that truncating the RNA enhances the specificity more than elongating (see also here). So what can we do? Well, there are different methods to improve the ...


4

Yes, this should in principle work, and a number of groups have shows that it works in cultured cells: 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 against HSV-1 infection.... Combined treatment of cells with ...


4

The heads and tails, in this paper, refer to the orientation of sgRNA binding sites. If there are two tandem sites in the same orientation then they are referred to as head-to-tail (end of the first site followed by the beginning of the second site). It is also apparent from the excerpt that you have included in your question. Head-to-head and tail-to-tail ...


3

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 nothing to do with the repeats themselves. See ...


3

It means that these enzymes need a metal ion as a co-factor for their function. This metal ion is typically bound somewhere near or in the active center and helps stabilizing transition states. Without these metals, these enzymes cannot function properly, that's why chelating agents are used in protein or DNA preparations to inhibit their function. ...


3

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 PAM sequences. Here you will find a nice ...


3

There are two classes of proteins that I can think of off the bat that are DNA sequence-specific. First are restriction enzymes, which recognize a specific (usually short) sequence of DNA and cleave it, sometimes through both strands at the same spot (blunt ends) and sometimes leaving an overhang or "sticky end". The other class is transcription factors. ...


3

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 with gene function (frameshifts etc). While HR ...


3

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 always the result of one gene being ...


3

Herpesviruses maintain themselves during the latent state as "episomes", circular DNA elements, with no capsid, that are in the nucleus but that are not integrated into the host genome. Host enzymes replicate these episomes, and different viruses have evolved different strategies for ensuring that the episomes are passed on to newly-divided cells. After ...


2

This paper should clear up a lot more than anyone on here truly can about the CRISPR system. CRISPR/Cas9 is very unique, even compared to other proteins purified from bacteria like Taq Poly. The reason is due to how complex it is actually induce this protein. Essentially you are transfecting a plasmid with the Cas9 protein and its guide RNA (gRNA) on the ...


2

Could we use this technology to completely eradicate from the world all species of mosquito that prey on humans? Yes, implemented correctly, a gene drive has this capability. Also, could we accurately predict the extent of the resulting ecological disruption so we could decide if it was worth it? The current scientific and political consensus is ...


2

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 the crRNA. I'm not exactly sure how this ...


2

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 needs to be is an interesting and often ...


2

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). http://www.genome-engineering.org/crispr/wp-content/uploads/2013/01/crispr_processing1.jpg


2

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. Take for example hemophilia A. Cells have a mutant ...


2

Zika doesn't alter the host genome at all. Since Zika can alter the genome of a baby to cause deformities Zika virus is incapable of altering the host genome. The exact mechanism by which Zika virus infection causes microcephaly is still unclear, but basically it depletes the neural stem cells at a point when the brain is developing, meaning brain ...


2

So, I'll assume that you are talking about gene editing in a laboratory (e.g. using a technique like CRISPR). Theoretically, there are almost no limits to what you can do - or at least try. Fundamentally DNA is the same in all living (and non-living) things on earth, so you can absolutely take a gene from a plant and "paste" it into a human cell/genome (...


2

The two "classic" papers cited in terms of Crispr/Cas9 discovery include the Doudna/Charpentier initial discovery, and the Feng Zhang application to gene editing. There is an ongoing, famous patent dispute as to whom really discovered the Crispr/Cas9 gene editing technology. As a quick timeline that better answers your question, a good publication is Cell ...


2

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? The main reason for that is that both the gRNA and the dCas9-TF (transcription factor) combination can (each) be encoded ...


2

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. Reference: Evolution of RNA- and DNA-guided antivirus defense systems in prokaryotes and eukaryotes: common ancestry vs convergence


1

No. 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 than using traditional restriction digest. ...


1

There are several probabilities that you need to take into account. The efficiency of DNA transformation of CRISPR encoding DNA and target DNA into the host cell (varies between cell types and transformation agent). Efficiency of cutting by CRISPR at target site. (varies by DNA compaction - which varies by site and cell type, and guide RNA) Number and ...


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