CRISPR Cas9 : How CRISPR can be performed in the lab ?

in 21st century one of the best way to manipulate and change DNA is CRISPR cast 9 and this is really revolutionized molecular biology itself in this video we'll look at a little bit about the use of the CRISPR and how CRISPR can be achieved in the laboratory so CRISPR casts nine systems can be used to generate knock in point mutations and knock out lines so all sort of genetic manipulation at DNA level is possible using CRISPR not only DNA CRISPR has a brilliant way of targeting a genomic locus so a particular genomic loci can be highlighted and can be imaged using CRISPR mediated fish if you add a fluorophore with the kazna enzyme and allow the guide RNA to target a particular genomic loci so the genomic loci would be highlighted and that too in a very specific manner then at the RNA level or at RNA production level you can also control you can perform transcriptional activation and transcriptional repression paradigm using CRISPR so in this paradigm you use the guide RNA to target this machinery and this particular cache line instead of cleaving just stay there near the promoter now depending upon which sequences they are binding them either allow or facilitate the binding of the RNA polymerase or they might hinder the binding of RNA polymerase as a result transcriptional activation or repression could be achieved not only that the futuristic gene therapy could be designed or the custom build medicines could be designed using this CRISPR strategy yet we are far away from it but it's not so far there are possibilities now let's talk about that how CRISPR can be achieved using cell culture method or how it is performed in the laboratory how the scientist can perform it in the lab so it is possible now to manipulate human cells human stem cells using CRISPR cast line technology in this video we are going to look at that so stay tuned till the end of this video exactly it would give you a flavor how things are done in the lab so here are some cells which are human embryonic stem cell lines in a plate now what we are going to perform is to perform several Jimmy genetic manipulations in these stem cells definitely the gene that we want to manipulate we are going to use a guide ER and against it and the crystal machinery would cut it and inside the cell there would be repair machinery such as an h ej or homologous directed repair mechanism mechanism and when this repair mechanism would happen we would incorporate our changes so you are to perform that we have to add a mixture of the cells several plasmids about which I'm going to talk about and a plasmid which would help you to scream in this case it's a transient GFP expression plasmid it would only tell you that your transfection reaction has worked properly the next step would be electroporation so electroporation is one of the efficient way of transferring gene inside the cells using electric current use from small pores on the cell and the DNA that means in this case the plasmids would get inside the cell after that you take the transfected cells or electroporated cells and you played them on our fresh plate and allow them to grow for a little while now since we have a expression plasmid for GFP so the transfected colonies would be highlighted in green if you see under a microscope now there is a chance that the colonies which would be green or which would be gfp positive they also have other plasmids which is required for the CRISPR so most probably some of these colonies would be positive for this genetic so the next step would be sorting out all the GFP positive cells from these transfected cell pool after the fact sorting when you really sort GFP positive cells and collect them separately you put those gfp positive cells in a new plate and allow the cells to grow for a while after the cells grow you would see in a plate there are colonies and these colonies are achieved from one single cell which was checked be positive that means which was transfected at least we know the transfection has worked on those cells but we yet don't know all the plasmids are present in all of these colonies or not in one colony it is possible that all the three plasmids are present in one colony it is also possible let's say two plasmids are present the third one is not there so what we really don't know in which colonies all of the plasmid are present and all the genetic manipulation has taken place so what we have to do under the microscope we have to put collagenous and take out all of these colonies and distribute these single colonies into 96-well plates or a 24 well plate in each well you put one colony and this one colony is derived from one single clone from this you do something which is very similar to replica plating when the split is called fluent you split them into two different plates such that one plate is exact replica of the other from the material presenting one plate you extract the DNA and used PCR based screening or sequencing based screening and let's say after screening you figure out that these three wells are actually positive for whatever you want whatever genetic manipulation you wanted to do so you track back to the root plate and take out the cells from those plates and expand them and keep them for future uses and these this is your cell line you revalidate with sequencing as well so let's look at what are the plasmids here the plasmids are depending upon what type of manipulation we are doing in the first case that I'm going to share with you we are going to do a knock in experiment that means we are adding some component inside the genomic DNA which was not present many of the cases scientists attach a GFP or a fluorophore coding gene to mark many proteins endogenously in this case the donor plasmid has that sequence another plasmid has the sequence forecast line and also had the guide RNA which is shown here in the plaque the third plasmid is a transient GFP expressing plasmid which would tell us the transfection has worked or not so the guide RNA would target the cache line system to a particular genomic locus which it should be targeted and then the donor plasmid have sequence homology with the strand which is targeted and based on homology it would be actually incorporated in the place of the double strand break and a homology dependent repair would take place using this strand the the donor stand strand as a template as a result the donor strand should be incorporated into the genome and using specific PCR primers you can amplify the particular region because you know this particular GFP containing region is not present in this cell in the cells normally right so the wild-type would not show this band but I'm at the but the mutant or the knockin line would show a band which would amplify a portion of the GFP and the portion of the normal genomic DNA so using that screening method you can have an rough idea which cells cell lines are positive then definitely you'd send it for sequencing to understand the incorporation of the sequence later on you can also use the same principle to target the guide RNA and instead of having a donor plasmid you put a repair all equal and this repeal illegal is basically having a point mutation inside of me now when this repair oligo is replacing in the double strand break it incorporates that mutation inside the genome and you have several ways to understand it many of the cases a new restriction site would appear there which was not previously there so you can cut it with a different restriction enzyme it would give you a particular restriction balance but the ultimate way to understand the point mutation is to perform sequencing that should tell you exactly the sequence change has happened or not though CRISPR looks highly precise high-throughput but you need to screen as many as hundreds of colonies to get specific mutants so this is also a tedious process as well in the laboratory but this process has a lot of promise and it is really really targeted so with that I would say goodbye and if you liked this video give it a thumbs up don't forget to Like share and subscribe thank you

Channel: Animated biology With arpan

Views: 27,316

Rating: 4.9101124 out of 5

Keywords: CRISPR Cas9 protocol, how crispr is performed in the lab?, crispr on embryonic stemcell, crispr, crispr medicine, use of crispr, csir ugc net, GATE, IIT JAM

Id: pNseb_U6gu0

Length: 10min 0sec (600 seconds)

Published: Thu Jan 30 2020