Constructing Plasmids for Cas9/gRNA Expression and Knock Out Experiments
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- เผยแพร่เมื่อ 28 ส.ค. 2019
- Overview of techniques used to modify a Cas9 expression plasmids with a new gRNA sequence that directs Cas9 to cut/mutate a target gene in the genome.
- แนวปฏิบัติและการใช้ชีวิต
I just finished taking a full semester University course with one of the leading CRISPR scientists and after watching this 20min video, I finally get it. Thanks!
AT 5 ' end of the oligo it should be CTCC overhang which is complementary to GAGG.
I guess the entire overhang is not shown.
I noticed that too. The CACC would not properly pair with the GAGG as there are two adenosines and no thymines
Excellent video. Just subscribed to your channel. I see you have not uploaded in a year. Hope you start putting more videos on youtube. You sir, are amazing.
After 20 minutes of this, I wont be intimidated by my colleagues' knowledge anymore.
😍😘 Tank you so much for this.
This is a very underated video
The best description of the CRISPR/Cas9 experiment I have ever had!
Thank you so much, you save my life when I try to understand this without any instruction.
God bless you, this video is absolute heaven for beginners. THANK YOUUUUU AHHHHH
Your videos are so helpful! Thanks, Jacob ✅✅✅
This was super clear and easy to follow, THANK YOU!!
thanks jacob for such a great service. plz keep making such informative videos
Amazing explanation!
Wonderful!! This was really helpful and easy to understand.
Outstanding! I browsed the whole TH-cam for such a video. Thanks God I found you eventually! Thanks a lot!
Amazingly explained. Thanks a lot 🙏
So great. So helpful.Thank you.
awesome...........i understood every concept related with cas9...
Finally I understood! Thank youuuu, It was amazing👏👏👏
One of the most clear and helpful videos out there on the topic ...Big thankyou..👏
thankiew so much for ur video I’m learning this for my internship
Wonderful! Helps me a lot. many thanks!
very good at explanation, thank you!
Wish to see more synbio related video tutorials from you! Thanks
Very helpful! Thanks!
It’s really helpful! I want to learn more about the cre- protein that make conditional mices
thanks! helping with my cell biology final!
very helpful! Thank you
Thank you so much 🙌🏽
Thanka a lot, You are amazing
Very clear, a great help. Thank you so much. Just one question: Is it only one restriction enzyme?
Perfect 😊
Why you didn't add "G" after 5' CACC and "C" 3' end of the antisence? Also, Thank you for your videos. It really helped me a lot
This is great, DO you have the protocol to know the concentration of the reagents to do the ligation ,etc? Thanks
But on the sticky ends, the 5-prime GAGG on the sense strand should complement with CTCC on the anti-sense strand. What am I missing?
Great video! is there any reaction mix to setup oligo duplex?
Great tutorial. I have a question:
Do you mean that in the same tube, we mix guide1 and guide 2 and load the cells with both guides at the same time?
Many thanks.
Thank you for such a nice description of the CRISPR system. Can CRISPR be used to induce mutation on a plasmid or Linear DNA?
why would you want to do that ? cloning would be more suitable
Thank you so much. But if you could answer one thing about the scRNA? is the sequence already present in the RNA or do we have to put that there as well?
Thank you. So Plasmids already available commercially so you have just to add you guide RNA?
could you explain how you get those sticky ends? I can't figure it out if I look to the BbsI restriction site.
Thank you for this video. After watching this video a question come to my mind. Why we use the Bbsl restriction enzyme for cutting the vector? Can't we use another restriction enzyme?
Thank you for answer.
Using Type IIs restriction enzymes that cut away from their binding site (e.g., BbsI) allows for Golden Gate cloning, which is much easier and potentially reliable than traditional cloning. All you need to do is mix the annealed oligos with the plasmid, add BbsI and T4 DNA Ligase, then cycle between 37C and 16C for several cycles to obtain nearly 100% the desired plasmid. No agarose gels needed!
@@jakelmer1985 Thanks a lot
How do u do a crispr knockin ?
6:28 This is the part where I start getting really lost... the point between theory and practice. OK, so... I have my target organism and its genome. I find the sequence of the gene whose expression I want to disable. I picked my target site for mutation within the gene, I have the 20 bp long sequence for the guide RNA - what then? In both your approaches, I have to pay some company to synthesize and deliver actual physical RNA, and Cas9. You say the second approach is cheaper, but wouldn't it also require doing the above + paying for the rest of the plasmid (the DNA sequences required for the amp. resistance, GFP, etc) and therefore be more expensive?
Also, if I'm trying to integrate other DNA mid-way into a gene I'm trying to knock out, aren't I by definition using HDR as an integration mechanism rather than NHEJ? And if so, don't I need 2 strands of guide RNA and a third sequence of the DNA I'm trying to incorporate in between them?
As a concrete project... Let's say I have a living culture of a fungi. I want to disable expression of a specific gene, and simultaneously insert a different gene (that would convey resistance to gentamicin for eventual identification and isolation of mutants, as well as ease of culture maintenance) into the break-point. I want to make this a permanent integration into the host organisms DNA, so I can maintain a live culture of modified organisms in long-term storage under the usual conditions for them. How exactly do I go from knowing all that and having all the relevant guide RNA sequences, to actually having all the physical DNA, reagents and equipment necessary to execute the experiment?
Plz do crispr knock in video
How does cas9 migrate into the nucleus?
How do you design the guide RNA's oligos using the software?
Is this just for the eukaryotic system?
Can I get this presentation please?
Hi,
Please, how I can contact with you? I want to check about my cas9 and my grand that I did, it is first time for me to do it, and I want to make sure that everything going right.
Is it sticky end compliment CACC or CTCC in Oligos design?
CTCC is what it should be! "CACC" is a typo in the slide deck.
I have a question.Maybe it is silly but please help me out. Why do we have two bands in uncut plasmid and one band in cut pKO plasmid? Please help
Uncut plasmid may still be nicked by shear stress or other damage. Therefore, a pure uncut plasmid sample may have some supercooled DNA (a band that travels farther through the gel) and some relaxed circular DNA that travels slower through a gel
@@jakelmer1985 Thank you so much, Sir. Learnt a lot from your videos. Keep posting such wonderful videos.
19:00 You say no PAM should be in a structure of gRNA sequence. But you have NGG there several times.. I am lost, could you, please, explain that.
Good question - let me clarify. You can certainly have NGG motifs within a gRNA, but you should not include the specific PAM that Cas9 cuts at the end of the gRNA.
For example, if the target site was:
AAAAAAAAGGAAAAAAAAAAACGG (where CGG is the PAM bound by Cas9)
Your gRNA would be:
AAAAAAAAGGAAAAAAAAAAA
Notice that the PAM bound by Cas9 (CGG) is omitted, but the internal AGG site is not removed.
@@jakelmer1985 Thank you!
Subtitle to spanish please
i am using two plasmid ...one for guide RNA and another is for cas 9 .......but how we make together to work ...how we make cassete that they will work together ...i mean expression cassette ...please do reply... further for more queries i want your email ID ....