Zinc Finger Nucleases (ZFNs)

okay so in this video we're going to talk about zinc finger nucleases and that is something we can use to edit the genome by cleaving sections of the DNA so first we'll talk about these restriction endonucleases and those are basically an enzyme that can cut DNA at a specific nucleotide sequence so in the e.coli organism we have a restriction endonuclease called eco r1 and that is targeting a specific section of DNA or a recognition site and in this case it's two amino acids or six base pairs of g-a-a-t-t-c and e-coli does this to prevent random cleavages in its DNA that could otherwise be lethal by removing this site so like eco r1 our zinc finger nucleases can target the specific recognition sites however the only difference is these sites we can now customize to a specific nucleotide sequence that the user desires so what exactly is a zinc finger and here I've outlined them in these different colored ovals so these are proteins that are held together by a zinc ion hence the name zinc finger and they usually work together so one zinc finger here if we look at the red one is targeting one amino acid so here it is ATG and then the blue one is targeting a TC and the purple G a G and then these Xing fingers usually are they usually pair or work together in groups of two to three also bind to an enzyme called Fock one and this you can imagine as like a pair of scissors it's a DNA cleavage domain and so if we have two groups of this sequence here then we're able to make a cut right here by heterodimer izing these two cleavage enzymes and so to remove an entire sequence like the one I've outlined in red we need to make a cut here and another cut here and that is why it's required that we have four of these groups of sync fingers and thought one to do that and that would be a minimum of eight sync fingers or usually a maximum of 12 like I've done here and so we can bring these sing fingers into the cell by transfection which is deliberately introducing nucleic acids or electroporation which is using a pulse of electricity to open the cell membrane and then once they bind we can make these cuts and remove this section of DNA so by removing that that would be a deletion mutation and then we could insert a new section of DNA with homologous pairs which would be an insertion mutation now there is an error because a certain percentage of cells can be Mis repaired and then result in deletion mutation again which is part of the risks of zinc fingers however we don't usually have frameshift mutations from Ayers and zinc fingers because we are working with the amino acids themselves and not the individual base pairs so for example here if we go to remove these two proteins here from the sequence like that we do not affect these proteins here now if we insert a new section in as well as long as that is inserted in groups of three bases or codons themselves then there is not a frameshift mutation now what's good about this is that these mutations made by Singh finger nucleases are permanent and they're heritable and we can do that through a single transfection and then they can also knockout genes that are not amendable to RNA interference now there are some risks and that is an off target effect what happens if our zinc fingers here bind to the wrong section of DNA then we will create a deletion mutation that is not intended by the user now the best known therapy of zinc finger nucleases an HIV treatment so the HIV virus will infect cells through this Co receptor called ccr5 and then it can proceed to disable the immune system by destroying cd4 Plus and T cells so by using these zinc finger nucleases we can target the ccr5 gene and we can remove it and many people can live a normal life without the ccr5 gene I think it's something like 20% of the population doesn't have it naturally but when zinc finger nucleases from entering the cells and it has been shown in some preliminary experiments that this has effectively repressed the expression of ccr5 now the only thing left is our ethical and legal implications of using a gene editing technology and in this case zinc finger nucleases so like any other medical treatment it would be the consent of the patient that's required to do something like this and if the patient is an embryo then the issue of consent is no longer there really so what is the best interest of the patient and that must be what is kept in mind so using this or other gene editing technologies to make these of gene edited super babies is definitely a real concern and then there's also people that are concerned about just preserving the natural development of human beings and not messing with our DNA or our blueprints so that's it for the zinc finger nucleases I hope you enjoyed this video

Channel: Will Assad

Views: 2,310

Rating: 4.9215684 out of 5

Keywords: zinc fingers, zinc finger nuclease, ZFN, zinc finger nuclease hiv treatment, restriction endonucleases

Id: TvySiie0KEA

Length: 6min 54sec (414 seconds)

Published: Wed Apr 01 2020