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    I have been watching all sorts of videos and been reading articles and stuff online for hours (not exaggerating),Im trying to write an article about it but I don't get how it works exactly, from what I understood so far is: there is a guiding RNA which binds to DNA so the cas9 protein knows where to go and where to cut the dna, then the dna can repair itself or a gene can be inserted?Im not sure fi this is right and I don't understand the other details and what actually happens when the cas 9 cuts the dna and I don't quite understand how they discovered it, from what I understand about how they discovered it is by studying bacteria, when viruses inject their dna something along the lines of the bacteria getting some copy of this dna and then next time the virus attacks with the dna the rna identifies it and cas 9 cuts it.Im very muddled about how all of this works and its back story, is there any tsrian who knows how it works?
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    (Original post by Anonymous1502)
    I have been watching all sorts of videos and been reading articles and stuff online for hours (not exaggerating),Im trying to write an article about it but I don't get how it works exactly, from what I understood so far is: there is a guiding RNA which binds to DNA so the cas9 protein knows where to go and where to cut the dna, then the dna can repair itself or a gene can be inserted?Im not sure fi this is right and I don't understand the other details and what actually happens when the cas 9 cuts the dna and I don't quite understand how they discovered it, from what I understand about how they discovered it is by studying bacteria, when viruses inject their dna something along the lines of the bacteria getting some copy of this dna and then next time the virus attacks with the dna the rna identifies it and cas 9 cuts it.Im very muddled about how all of this works and its back story, is there any tsrian who knows how it works?
    It can be used to insert genetic sequences into a locus of choice. To understand how you really need to understand gene targeting by homologous recombination, and to understand that, you need to understand homologous recombination. You can find material to cover this in more depth online but I'll try explain at the most simple level.

    DNA damage is pretty bad for cells, especially double-strand breaks (DSBs) where both strands of the double helix are split. So cells really need to repair these, and in fact there's two mechanisms they have of doing so:

    a) Non-homologous end joining - where they just take the two broken bits and splice them together - this isn't precise and can often lead to insertions/deletions with a possible frameshift mutation.

    b) Homologous recombinantion - this is when the cell uses the homologous chromosome as a template to fix the DSB. The details of how it works aren't important but just understand that the cell looks for homologous sequences upstream and downstream of the DSB (5' and 3' homology arms respectively) and matches that to the homology arms in the template DNA and copies over everything between them in to fill in the gap and repair the DSB.

    Name:  1D4B6650-FEA3-4427-8B2B-EAFBA872F8BB-9442-00000846449DD4C1.png
Views: 24
Size:  243.9 KB

    What this means then, is that we can exploit this system to insert genes simply by putting in a fake template DNA for the broken DNA to use as a repair copy. All we need are 5' and 3' homology arms but with whatever gene we want between them. This known as gene targeting:

    Attachment 700014700018

    If you have a selection marker (e.g. Antibiotic resistance), we can select for cells that have taken up the gene in that spot. But really the system as it is, relies on waiting for DSBs to happen by chance.

    This is where the CRISPR/Cas9 system comes in. As you say, it is basically a programmable pair molecular of molecular scissors that can make a DSB anywhere. This is simply done by making a guide RNA that is complementary to the locus where you want to make the cut. So overall, if you inject the guide RNA with the cas9 enzyme - you'll get a DSB where you want. You also need to inject a homologous recombination repair template with homology arms to insert in your gene when the DSB tries to repair itself.


    Incidentally, you can also use the CRISPR/Cas9 system in a knockout mode instead of a gene insertion mode. Here you do the same steps but don't have a repair template. Instead you want repair to occur through non-homologous end joining to create indel, and thus frameshift, mutations. This will create a premature stop codon and the mRNA product will be degraded so you've knocked that gene out.

    Attachment 700014700018700000
    Attached Images
      
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    (Original post by Asklepios)
    It can be used to insert genetic sequences into a locus of choice. To understand how you really need to understand gene targeting by homologous recombination, and to understand that, you need to understand homologous recombination. You can find material to cover this in more depth online but I'll try explain at the most simple level.

    DNA damage is pretty bad for cells, especially double-strand breaks (DSBs) where both strands of the double helix are split. So cells really need to repair these, and in fact there's two mechanisms they have of doing so:

    a) Non-homologous end joining - where they just take the two broken bits and splice them together - this isn't precise and can often lead to insertions/deletions with a possible frameshift mutation.

    b) Homologous recombinantion - this is when the cell uses the homologous chromosome as a template to fix the DSB. The details of how it works aren't important but just understand that the cell looks for homologous sequences upstream and downstream of the DSB (5' and 3' homology arms respectively) and matches that to the homology arms in the template DNA and copies over everything between them in to fill in the gap and repair the DSB.

    Name:  1D4B6650-FEA3-4427-8B2B-EAFBA872F8BB-9442-00000846449DD4C1.png
Views: 24
Size:  243.9 KB

    What this means then, is that we can exploit this system to insert genes simply by putting in a fake template DNA for the broken DNA to use as a repair copy. All we need are 5' and 3' homology arms but with whatever gene we want between them. This known as gene targeting:

    Attachment 700014700018

    If you have a selection marker (e.g. Antibiotic resistance), we can select for cells that have taken up the gene in that spot. But really the system as it is, relies on waiting for DSBs to happen by chance.

    This is where the CRISPR/Cas9 system comes in. As you say, it is basically a programmable pair molecular of molecular scissors that can make a DSB anywhere. This is simply done by making a guide RNA that is complementary to the locus where you want to make the cut. So overall, if you inject the guide RNA with the cas9 enzyme - you'll get a DSB where you want. You also need to inject a homologous recombination repair template with homology arms to insert in your gene when the DSB tries to repair itself.


    Incidentally, you can also use the CRISPR/Cas9 system in a knockout mode instead of a gene insertion mode. Here you do the same steps but don't have a repair template. Instead you want repair to occur through non-homologous end joining to create indel, and thus frameshift, mutations. This will create a premature stop codon and the mRNA product will be degraded so you've knocked that gene out.

    Attachment 700014700018700000
    Thank you for your response . How does the CRISPR system work in bacteriophages when viruses attack?And what does the term CRISPR refer to?
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    (Original post by Asklepios)
    It can be used to insert genetic sequences into a locus of choice. To understand how you really need to understand gene targeting by homologous recombination, and to understand that, you need to understand homologous recombination. You can find material to cover this in more depth online but I'll try explain at the most simple level.

    DNA damage is pretty bad for cells, especially double-strand breaks (DSBs) where both strands of the double helix are split. So cells really need to repair these, and in fact there's two mechanisms they have of doing so:

    a) Non-homologous end joining - where they just take the two broken bits and splice them together - this isn't precise and can often lead to insertions/deletions with a possible frameshift mutation.

    b) Homologous recombinantion - this is when the cell uses the homologous chromosome as a template to fix the DSB. The details of how it works aren't important but just understand that the cell looks for homologous sequences upstream and downstream of the DSB (5' and 3' homology arms respectively) and matches that to the homology arms in the template DNA and copies over everything between them in to fill in the gap and repair the DSB.

    Name:  1D4B6650-FEA3-4427-8B2B-EAFBA872F8BB-9442-00000846449DD4C1.png
Views: 24
Size:  243.9 KB

    What this means then, is that we can exploit this system to insert genes simply by putting in a fake template DNA for the broken DNA to use as a repair copy. All we need are 5' and 3' homology arms but with whatever gene we want between them. This known as gene targeting:

    Attachment 700014700018

    If you have a selection marker (e.g. Antibiotic resistance), we can select for cells that have taken up the gene in that spot. But really the system as it is, relies on waiting for DSBs to happen by chance.

    This is where the CRISPR/Cas9 system comes in. As you say, it is basically a programmable pair molecular of molecular scissors that can make a DSB anywhere. This is simply done by making a guide RNA that is complementary to the locus where you want to make the cut. So overall, if you inject the guide RNA with the cas9 enzyme - you'll get a DSB where you want. You also need to inject a homologous recombination repair template with homology arms to insert in your gene when the DSB tries to repair itself.


    Incidentally, you can also use the CRISPR/Cas9 system in a knockout mode instead of a gene insertion mode. Here you do the same steps but don't have a repair template. Instead you want repair to occur through non-homologous end joining to create indel, and thus frameshift, mutations. This will create a premature stop codon and the mRNA product will be degraded so you've knocked that gene out.

    Attachment 700014700018700000
    Could you please explain what the term homologous and non homologous means and how the dbs is repaired using homologous chromosomes again but in a more straightforward way?
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    (Original post by Anonymous1502)
    Could you please explain what the term homologous and non homologous means and how the dbs is repaired using homologous chromosomes again but in a more straightforward way?
    The key thing to remember is that you have two copies of each chromosome in an animal cell. These have the same genes (albeit can be different alleles) so are termed "homologous chromosomes."

    This means that if you damage one of the chromosomes, the cell can copy the homologous chromosome to fix the broken one. If the cell uses this method, it's called homologous recombination. The other method to repair a DSB is simply to locate the two broken ends together - this is called non-homologous end joining.

    Maybe an easier way to think of it is imagine you have two identical pencils and snap one in half. 'Non-homologous end joining' would be to try and glue the two broken bits together. Homologous recombination would be to use the intact pencil as a guide how to fix the broken one, since you know they were identical to begin with. As you can imagine, the latter repair mechanism will be a lot more precise than the former!
 
 
 
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