Constructing Plasmids for Cas9/gRNA Expression and Knock Out Experiments

[@benbishop7775]

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!

[@muhammadwaqasluqman2977]

After 20 minutes of this, I wont be intimidated by my colleagues' knowledge anymore. 😍😘 Tank you so much for this.

[@KhaNguyen-os3uo]

Thank you so much, you save my life when I try to understand this without any instruction.

[@김찬희-h8d]

The best description of the CRISPR/Cas9 experiment I have ever had!

[@rehabalsaleh166]

Outstanding! I browsed the whole KZbin for such a video. Thanks God I found you eventually! Thanks a lot!

[@farahhammoud6457]

God bless you, this video is absolute heaven for beginners. THANK YOUUUUU AHHHHH

[@kajalyadav1020]

One of the most clear and helpful videos out there on the topic ...Big thankyou..👏

[@Justin-dv7ul]

This is a very underated video

[@moizkhan1501]

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.

[@yourlifeonpower]

An absolute gem! Thank you so much!

[@FreeFall73]

Amazingly explained. Thanks a lot 🙏

[@starrani5264]

awesome...........i understood every concept related with cas9...

[@silvartarabian1408]

Amazing explanation!

[@tapasbhattacharyya9956]

AT 5 ' end of the oligo it should be CTCC overhang which is complementary to GAGG.

[@LSC124377322]

I guess the entire overhang is not shown.

[@bobstewart8269]

I noticed that too. The CACC would not properly pair with the GAGG as there are two adenosines and no thymines

[@isabelasoares1168]

Finally I understood! Thank youuuu, It was amazing👏👏👏

[@kashifhameed2650]

thanks jacob for such a great service. plz keep making such informative videos

[@YunusPolat-h7x]

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

[@喵-v4y]

thankiew so much for ur video I’m learning this for my internship

[@tessalania5802]

This was super clear and easy to follow, THANK YOU!!

[@jingjingwang7585]

So great. So helpful.Thank you.

[@gregsmith6756]

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?

[@kigaruantony5632]

Thank you. So Plasmids already available commercially so you have just to add you guide RNA?

[@damialaoalao7137]

Wonderful!! This was really helpful and easy to understand.

[@tylerbradley8124]

thanks! helping with my cell biology final!

[@poloikary]

This is great, DO you have the protocol to know the concentration of the reagents to do the ligation ,etc? Thanks

[@puppyanddestiel]

this is really helpful!!!

[@adwoabiotech]

Your videos are so helpful! Thanks, Jacob ✅✅✅

[@dan-ni3772]

It’s really helpful! I want to learn more about the cre- protein that make conditional mices

[@tiamheydari7729]

Wish to see more synbio related video tutorials from you! Thanks

[@yqyu9843]

Very helpful! Thanks!

[@naveedshahzad8589]

Thank you for such a nice description of the CRISPR system. Can CRISPR be used to induce mutation on a plasmid or Linear DNA?

[@zakweed4322]

why would you want to do that ? cloning would be more suitable

[@cacevedo07]

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.

[@ٹیڑھا]

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?

[@zhixinli5861]

very helpful! Thank you

[@amonoracheal9118]

very good at explanation, thank you!

[@shadenalthakafy7766]

Could you tell us about your reference?

[@Tennyhu]

Thank you so much 🙌🏽

[@aishwaryakarn7437]

Is this just for the eukaryotic system?

[@elhamrastegar]

Very clear, a great help. Thank you so much. Just one question: Is it only one restriction enzyme?

[@weldutesfagabr6453]

Wonderful! Helps me a lot. many thanks!

[@sushreesangitapriyadarshin6441]

Thanka a lot, You are amazing

[@kaorumugen991]

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?

[@karunakalichamy8539]

Great video! is there any reaction mix to setup oligo duplex?

[@jodscience3741]

How does cas9 migrate into the nucleus?

[@phshorooqqaz6541]

Perfect 😊

[@berilboran1469]

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.

[@jakelmer1985]

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!

[@berilboran1469]

@@jakelmer1985 Thanks a lot

[@pasinduniroshan5273]

Can I get this presentation please?

[@1ALJawhara]

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.

[@anjalipatni2580]

How do you design the guide RNA's oligos using the software?

[@oleksandraskorobohatko2125]

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.

[@jakelmer1985]

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.

[@oleksandraskorobohatko1522]

@@jakelmer1985 Thank you!

[@terraslof2978]

could you explain how you get those sticky ends? I can't figure it out if I look to the BbsI restriction site.

[@Diogenesthedog0]

Wow! Wow...

[@manishadas7142]

Plz do crispr knock in video

[@indianexpoin]

How do u do a crispr knockin ?

[@skullcrusher6227]

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

[@jakelmer1985]

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

[@skullcrusher6227]

@@jakelmer1985 Thank you so much, Sir. Learnt a lot from your videos. Keep posting such wonderful videos.

[@uzoechisamuel]

Is it sticky end compliment CACC or CTCC in Oligos design?

[@jakelmer1985]

CTCC is what it should be! "CACC" is a typo in the slide deck.

[@cesartopper1545]

Subtitle to spanish please

[@ankitasaroya319]

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 ....