I know I'm a bit late, but thanks for a great video! It perfectly answered all the questions I had and was easy to understand (for a person working in a closely related field)
Hi, thank you for the great video. But I have some question: Based on my limited understand in transfection experiment, the antibiotic selection might keep the plasmid DNA being replicated or even random inserted to gDNA, which may disturb gene expression. So that is the reason why we need the exactly knock-in and harbor position. If I perform the transfection, why do I still need the CRISPR knock-in? Or is there any method to remove the unexpected insertion in animal model?
I was wondering why you included the PGK-Puro within the AAVS homology arms? If the CMV promoter is constitutive then can't we just select based on red flourescence?
Also because this way our knock-in only contains the GOI. My understanding is that this is necessary because we wouldn't want to transfect other selectable markers if we were using this for something commercial, like in agriculture.
Hello! Thank you for your comment! In the research setting, sometimes a selection marker is more desirable. For example, when you need a polyclonal overexpression cell line, a drug screen will be more applicable than a fluorescent marker screen. It could be a totally different scenario when it is commercial agriculture. Thank you for raising an interesting POV!
Great video and many thanks Let me ask, if there is no successful knock in, and we perform a pcr as you describe, isnt it possible for 1 primer to bind on puromycin gene and the second primer to one homology arm, both being on the original plasmid that we transfected? (And not on our desirable genomic loci)
Hi tavladwr, Yes, this may happen right after you transfect your plasmid, but after passaging your cells about 10 times your plasmid should be diluted out as it does not have a replication origin.
Shouldn't the gene with the homology arms flanking from both sides be linear for the homolygous recombination to occur? And how do you make the RFP gene get integrated and not the ampR gene
How can I be sure that the gene inserted is on the correct locus? I mean what kind of experiment can I perform to confirm the location of the inserted gene in the genome?
Typically, screening for correct integration can be done by PCR and sequencing. You can find more information about these techniques in our Knowledge Base, located here: info.abmgood.com/crispr-cas9-screening-validation.
hi, thanks for the video. I'm slightly confused, if I want to perform a gene knock in, do I administer the vector containing the Cas9 and gRNA separately from the donor DNA, or can the donor DNA be inserted into the host in the same vector? thanks again. Also, if I wanted to tag my vector with HA in order to follow the fate of my transduction, where would I add this? thanks in advance
@@abmgood sir, is there any info suitable stem cell therapy for pkd /adpkd diseases polycystic kidney disease sacs of fluid in both kidnies and high blood pressure. Which PKD1, PKD2 genes can be turn off from DNA, kidnies, liver,. Kindly provide me suitable info thanks
Hi! I had a dumb question. But if you are using the Puro plasmid to confer Puromycin resistance to select for only cells that took both plasmids, wouldn't it be possible that there are cells that only took the Puro/Ampicilin plasmid, and didn't intake the sgRNA/Cas9 vector plasmid? Like a false positive. Also i didnt understand what CMV and u6 promoters are. Thanks
Hello Obluda, CRISPR utilizes a sgRNA that directs the Cas9 enzyme to create breaks at specific locations in the genome. Genome stability will prompt repair of the breaks and thus a donor template containing homologous sequences upstream and downstream of the intended editing site is taken up by the cell. If the sgRNA/Cas9 plasmid is not introduced, this means that breaks will not created in the genome, and therefore the repair mechanism is not initiated to take up the donor template. The CMV promoter in the pCAs9-Guide-AAVS1 plasmid is used to drive expression of the Cas9, and the U6 promoter is used to drive expression of the sgRNA.
Thank you for great video! what if i would like to knockin reporter to the 3' end of my gene of interest, in pluoripotent cells so that i can track the particular lineage of cells. Now in this case i need to ignore the safe harbor region and just should design guide RNA which would be at 3' end of my gene of interest, and that can be on any chromosome. right?
Hi Arshad! Thanks for leaving a comment. Yes, you are correct! Keep in mind that if you wish for a fusion reporter, you will need to remove the gene of interest's stop codon. In this case, you will need to design the gRNA so it targets close to the 3' end of your gene of interest, and design the repair template so that in the process of recombination the stop codon is removed. Confirm the removal of the stop codon and the correct reading frame of the reporter by sequencing analysis. Hope this helps!
How all of those orange bases disappear at 2:15 ? i'm trying to understand crispr but everybody explain it differently . Why just 1 hole at this place could lead to the disappearance of all those orange bases ? If the orange bases weren't there on the bottom strand, and that the cut was made just between the blue and the pink part , i would understand that, with HDR , the upper strand orange part would've been copied to the bottom part with reparation system. But now i'm confused.
The orange bases did not disappear. The orange bases were included in the image to illustrate the location of the gene following insertion. We apologize if the animation was confusing. To clarify the steps: (1) Cas9 and sgRNA are used to produce a double-stranded break; (2) the repair template, shown in the video as "Donor Plasmid", is used by the cell to repair the break using homologous recombination; and (3) the gene of interest, which is included between the homology arms on the repair template, is integrated into the genome.
As part of knock-in, sgRNA sequences are designed to target the AAVS1 site specifically. As well, HDR templates are designed such that they included flanking homology arms with homology to the AAVS1 integration site. As for verification, whole genome sequencing can be employed to check for the presence/absence of off-target effects in the edited cell line.
@@abmgood very nice vid. I am confused in 2:11 to 2:22. If there is 1 cut by sgRNA, so on vid seems that orange GoI replaces no homology DNA part on the genome, despite of correct HDR left and right. Is it correct illustration? I am wondering, can you use two sgRNAs designed to target two genomic sites of AAVS1near HR arm left and rigth? I hope my question is clear.
Ok, this is all well and great, but when are we going to start using this technology to help people with medical conditions? Seriously, seems like all fluff when you never see real world administration. Do we have to wait until the 22nd century for this, or within our lifetime to cure diseases, or make people impervious to cancer
I finally understand how my grandparents feel using an ATM machine. Thank you!
Haha, I hope this video helped and wasn't too overwhelming!
as an outsider to the field, watching this really reminded me of that episode in Rick and Morty where they were explaining how the Plumbus is made.
This is an excellent introduction. Brilliant for students early revision as well.
Thanks for watching and for the nice comment!
I know I'm a bit late, but thanks for a great video! It perfectly answered all the questions I had and was easy to understand (for a person working in a closely related field)
This is a really well made video. Great job
Hi Goretast, thanks for watching! Let us know if you have any questions!
Great explanation ❤
fantastic video
Nice video, do you guys offer any in-vivo CRISPR knock-ins and knock-outs?
Thanks for watching our video! Currently, we only offer in vitro CRISPR tools and services.
@@abmgood how would it differ in methods whether in vitro or in vivo?
Hi, thank you for the great video.
But I have some question:
Based on my limited understand in transfection experiment, the antibiotic selection might keep the plasmid DNA being replicated or even random inserted to gDNA, which may disturb gene expression. So that is the reason why we need the exactly knock-in and harbor position.
If I perform the transfection, why do I still need the CRISPR knock-in?
Or is there any method to remove the unexpected insertion in animal model?
I was wondering why you included the PGK-Puro within the AAVS homology arms? If the CMV promoter is constitutive then can't we just select based on red flourescence?
Also because this way our knock-in only contains the GOI. My understanding is that this is necessary because we wouldn't want to transfect other selectable markers if we were using this for something commercial, like in agriculture.
Hello! Thank you for your comment! In the research setting, sometimes a selection marker is more desirable. For example, when you need a polyclonal overexpression cell line, a drug screen will be more applicable than a fluorescent marker screen. It could be a totally different scenario when it is commercial agriculture. Thank you for raising an interesting POV!
What is the use of ColE1? does that the origin of replication for the replication of plasmid inside the mammalian cells?
Great video and many thanks
Let me ask, if there is no successful knock in, and we perform a pcr as you describe, isnt it possible for 1 primer to bind on puromycin gene and the second primer to one homology arm, both being on the original plasmid that we transfected? (And not on our desirable genomic loci)
Hi tavladwr,
Yes, this may happen right after you transfect your plasmid, but after passaging your cells about 10 times your plasmid should be diluted out as it does not have a replication origin.
ah yes, now i see
thanks!
Shouldn't the gene with the homology arms flanking from both sides be linear for the homolygous recombination to occur?
And how do you make the RFP gene get integrated and not the ampR gene
How can I be sure that the gene inserted is on the correct locus? I mean what kind of experiment can I perform to confirm the location of the inserted gene in the genome?
Typically, screening for correct integration can be done by PCR and sequencing. You can find more information about these techniques in our Knowledge Base, located here: info.abmgood.com/crispr-cas9-screening-validation.
hi, thanks for the video. I'm slightly confused, if I want to perform a gene knock in, do I administer the vector containing the Cas9 and gRNA separately from the donor DNA, or can the donor DNA be inserted into the host in the same vector? thanks again. Also, if I wanted to tag my vector with HA in order to follow the fate of my transduction, where would I add this? thanks in advance
Hello Aine,
The donor DNA is typically supplied as (1) a single-stranded oligonucleotide (for small insertion sequences,
@@abmgood sir, is there any info suitable stem cell therapy for pkd /adpkd diseases polycystic kidney disease sacs of fluid in both kidnies and high blood pressure. Which PKD1, PKD2 genes can be turn off from DNA, kidnies, liver,. Kindly provide me suitable info thanks
Awesome video,Keep it up!
Tell me bout genomic technique at least there will work...
What would happen if we send DNA into outer space ? I think it would show some connections between human genes and dark particle. What's your answer?
Hi! I had a dumb question. But if you are using the Puro plasmid to confer Puromycin resistance to select for only cells that took both plasmids, wouldn't it be possible that there are cells that only took the Puro/Ampicilin plasmid, and didn't intake the sgRNA/Cas9 vector plasmid? Like a false positive. Also i didnt understand what CMV and u6 promoters are. Thanks
Hello Obluda,
CRISPR utilizes a sgRNA that directs the Cas9 enzyme to create breaks at specific locations in the genome. Genome stability will prompt repair of the breaks and thus a donor template containing homologous sequences upstream and downstream of the intended editing site is taken up by the cell. If the sgRNA/Cas9 plasmid is not introduced, this means that breaks will not created in the genome, and therefore the repair mechanism is not initiated to take up the donor template. The CMV promoter in the pCAs9-Guide-AAVS1 plasmid is used to drive expression of the Cas9, and the U6 promoter is used to drive expression of the sgRNA.
Thank you for great video! what if i would like to knockin reporter to the 3' end of my gene of interest, in pluoripotent cells so that i can track the particular lineage of cells. Now in this case i need to ignore the safe harbor region and just should design guide RNA which would be at 3' end of my gene of interest, and that can be on any chromosome. right?
Hi Arshad! Thanks for leaving a comment. Yes, you are correct! Keep in mind that if you wish for a fusion reporter, you will need to remove the gene of interest's stop codon. In this case, you will need to design the gRNA so it targets close to the 3' end of your gene of interest, and design the repair template so that in the process of recombination the stop codon is removed. Confirm the removal of the stop codon and the correct reading frame of the reporter by sequencing analysis. Hope this helps!
the vocal fry is very real here
Can this technique be used to generate knock-in mice?
Thank you for your comment! I believe the protocol for a CRISPR Knock-in mouse model is different but it is achievable!
How all of those orange bases disappear at 2:15 ? i'm trying to understand crispr but everybody explain it differently . Why just 1 hole at this place could lead to the disappearance of all those orange bases ? If the orange bases weren't there on the bottom strand, and that the cut was made just between the blue and the pink part , i would understand that, with HDR , the upper strand orange part would've been copied to the bottom part with reparation system. But now i'm confused.
The orange bases did not disappear. The orange bases were included in the image to illustrate the location of the gene following insertion. We apologize if the animation was confusing.
To clarify the steps:
(1) Cas9 and sgRNA are used to produce a double-stranded break; (2) the repair template, shown in the video as "Donor Plasmid", is used by the cell to repair the break using homologous recombination; and (3) the gene of interest, which is included between the homology arms on the repair template, is integrated into the genome.
@@abmgood thank you :p
Nice explanation ..
How are you sure that knockin took place at AAVS1 locus and nowhere else?
As part of knock-in, sgRNA sequences are designed to target the AAVS1 site specifically. As well, HDR templates are designed such that they included flanking homology arms with homology to the AAVS1 integration site. As for verification, whole genome sequencing can be employed to check for the presence/absence of off-target effects in the edited cell line.
@@abmgood very nice vid. I am confused in 2:11 to 2:22. If there is 1 cut by sgRNA, so on vid seems that orange GoI replaces no homology DNA part on the genome, despite of correct HDR left and right. Is it correct illustration?
I am wondering, can you use two sgRNAs designed to target two genomic sites of AAVS1near HR arm left and rigth? I hope my question is clear.
I would be grateful , if you please answer me (asap).
Great！
Didn't know that guys had started with vocal fry too.
I found this a bit too complex of an explanation. I guess I need to learn more about the context.
Hello there,
We are sorry to hear that! Feel free to contact us at technical@abmgood.com if you have any questions.
So countries can use this for military purposes?
Ok, this is all well and great, but when are we going to start using this technology to help people with medical conditions? Seriously, seems like all fluff when you never see real world administration. Do we have to wait until the 22nd century for this, or within our lifetime to cure diseases, or make people impervious to cancer