What is the PAM? - A CRISPR Whiteboard Lesson

[@조건희-u9c]

The most excellent video I ever see. No one explains what is PAM and why it is important exactly until now.

[@eugeniedeschamps4530]

Couldn't find this explanation anywhere else, super clear and answered all my questions, great video really

[@shubhenduhazra4620]

Great...the best crispr video in youtube

[@pramodkc2309]

indeed

[@jirimuller8945]

Amazing video. Thank you for sharing.

[@zqxzqxzqx1]

I'm a simple housewife/grandma, but have been fascinated by CRISPR Cas9 since its first mentions in the news. Since then, I've striven to learn all I can, and have read Code Breaker (which was much more dramatic than I expected!) I'm truly happy to see it being used now to correct sickle cell and blindness, and have great hopes for the future. Thanks for this video, and for shedding a little more light into my understanding of the subject.

[@sundorenaesir2327]

Amazing video. Thats how a brilliant human explains science... Brilliant drawings.

[@maisunn]

Wow, that was extremely helpful! I don't normally comment on vids but I had to thank you for making such a fantastic video.

[@acidithiobacillusferrooxid3687]

your explanation is so clear but yet so thorough, salute from a non-bio phd trying to understand what my colleagues are doing...

[@innovativegenomicsinstitute]

Thank you! A lot of work went into this one. :)

[@HowardBPerer]

I have studied numerous sources to explain how PAM works and none came close to the clarity of this video....great work, thank you

[@franciscoagus]

Congrats on the video. You explained what none of the all the most viewed and most liked videos didn't.

[@aquamarine3699]

Had to slow this down to 0.75 to take clear notes for myself lol Thanks for the clear explanation!

[@peepdi]

same , I kept pausing throughout and watched the video twice

[@autumnalgal]

Wow! Thank you for this video, it makes CRISPR Cas-9 so much easier to understand! Great vid

[@jpoppenheim1460]

This video just goes to show that you don't need any fancy modeling animations. This white board explanation is better than anything else out there. 5-Stars.

[@moritzberger2483]

Needed this Video, because my professor rushed through the PAM part way too quickly. Thankfully, you've explained the details and even the consequences for the "genome editing tool"

[@milicaplavsic39]

Thank you so much for creating this video! Finally I have a clear picture of how PAM works and why it is so useful!

[@abdulrahmanalsadiq6656]

This video exactly answered my questions about PAM and even with further important and useful details. Thank you so much!

[@rogercastellanosfernandez1349]

This is an example of what well-explained means. Thanks a lot

[@Jonathan544200]

You have just answered my whole questions. Thank you. Amazing video.

[@sirapobpotjanapimol5148]

honestly, you deserve 10M like bro. Thank you

[@gabriellerizzo8278]

Thank you so so much for this video! It really helped me understand the detailed mechanisms of CRISPR Cas9 for my human developmental bio midterm presentation! Also going to be doing CRISPR undergraduate research this summer, so this is SUPER helpful! Cheers!

[@innovativegenomicsinstitute]

So happy we could help! Hope you enjoy your research experience. :)

[@steveholmes161]

A wonderful and thorough explanation of the Pam sequence. Thank you so much

[@lindseycarlson5852]

So helpful! The only video I could find to clearly understand the concepts for my exam!

[@innovativegenomicsinstitute]

Glad you enjoyed the video! Which school was teaching CRISPR in one of their courses? Also, we will be making more whiteboard videos and would love some suggestions about which CRISPR topics to explain.

[@lindseycarlson5852]

RPI! We had a lecture on CRISPR/Cas9 and it's current uses in editing!

[@julialipowicz6670]

the BEST explanation of CRISPR, hands down

[@mariociencia12]

Amazing! Incredible! Fantastic! Astounding! Mighty! Wonder! Super!

[@mariociencia12]

I watch the video a second time and is still incredible!

[@julia3983able]

This is super helpful The pictures and explanation really helped me wrap my head around CRISPR Thank you 😊 💓

[@KiwiiStyle]

This is a great video explaining the PAM, we only learned about the NGG PAM site. But now you explained that cas1 and cas2 look for different PAM site's it makes way more sense! Thank you, this will help me with my exam!

[@innovativegenomicsinstitute]

Glad you enjoyed the video and found it helpful! Most people are familiar with the NGG PAM because that is what SpyCas9 recognizes and SpyCas9 is the most common Cas9 protein used for genome editing. Other species of Cas9 have different PAM sequences. In our video at 3:10, we show that Cas9 works with the Cas1-Cas2 complex to find the PAM sequence and then Cas1-Cas2 removes the piece of viral DNA. In this instance Cas1-Cas2 is not recognizing the PAM sequence, but is working with Cas9. This is still and active area of research with many exciting questions to answer! What course or institution is teaching and testing these concepts in class?

[@danielholley2495]

Thank you for this OUTSTANDING video!

[@nisrinthuff1604]

Just answered my whole question about CRISPR :") really helpful thank you soo much!!

[@chouwachan]

this was the best video ive ever seen in my life

[@fional7649]

this video saved my life!! twice! hhhhh. It explained so well and the drawing makes it even more clear! Thank you so much for making this video!!!

[@flouflou99]

thanks for your input and great work! I will always remember PAM now :)

[@neal84243]

So many of the very long videos entirely left aside either or both the evolutionary and recombinant highjacking significance of the PAM. This very short video gave necessary and sufficient description of both.

[@beththompson4296]

Such a good & clear explanation! The drawings really help the understanding. Thank you!

[@laurawheeler2749]

Excellent video...great job explaining PAM. Thank you!

[@rixu2671]

Love this video! Great help! Thank you!

[@mahimapandey6003]

This help in my interview 👍 thanks a million!!

[@JenniferReif-pu5qr]

Thank you so much, this literally answered every question I possibly had.

[@lola7993]

Great video!!! It helped me a lot preparing for my exams!

[@innovativegenomicsinstitute]

We are curious to learn which school or course is teaching and testing these concepts?

[@jaredwainwright4621]

My undergrad molecular genetics course at the University of Florida teaches a section of CRISPR-CAS9. Awesome video hope to see more

[@innovativegenomicsinstitute]

Awesome!

[@erossi98]

Really excellent video, thank you IGI

[@peepdi]

Please make more such content. You explained really well. I am doing a project on CRISPR so it'd be helpful.

[@innovativegenomicsinstitute]

Thank you! We're always interested in learning what topics people want to learn about, feel free to share your thoughts! And check out our website for lots of other CRISPR resources: innovativegenomics.org/

[@peepdi]

@@innovativegenomicsinstitute Sure I'll definitely check that out. :)

[@citrus_aves]

This was so helpful! Thank you for creating this

[@allyroses928]

Amazing explanation! Thank you so much

[@JasleenKaur-lu5or]

Awesome video! Super helpful. So glad I was able to find this.

[@SpeciesPlantarum]

Crystal clear explanation! Thank you!

[@lordbebech]

Thank you so much! I had a lecture about CRISPR and this PAM sequence was on many different slides but nowhere was explained it purpose nor what exactly actually this is :)

[@lisaaa7130]

Helping so much to understand the complex consent

[@rosscwilson]

Great work, Eugene!

[@inshaishrat4371]

Thank you so much. It helped

[@Kill29-tf7ki]

Seriously I love the way you question to every point and answering them nice 👌👌 Motived for my video 👌👌

[@williamsluman5298]

Thank you! Just thank you!

[@ٹیڑھا]

Ah amazing. Thank you so much. You are a live saver.

[@cinnabarina]

Wow, I tried to understend that part in wikipedia. But it was impossible. Here everything seems so simple. Thanks

[@anhmai9859]

I love your video! Thanks a bunch.

[@cryptocurrency.information]

thankyou! doing a SHE task on CRISPR and know i understand the topic

[@Kurchack]

Great explanation, and you're great too.

[@locosquad1049]

OMG bro i searched for hours to understand this but google just gives definition which explain nothing. In 5 minutes i Checked everything. Thx you bro i liked and subsribed

[@innovativegenomicsinstitute]

Glad you liked the video! We are always looking for new topics to explain, so please tell us if you would like to see another CRISPR concept animated.

[@locosquad1049]

@@innovativegenomicsinstitute i dont know what you could put new on the new vid this here had nearly every Thing:) i Need to Finish a presentation till wednesday this Video helped a lot to understand. I dont ''need" more Infos but a 2nd One would still be interesting!

[@AlphenLi-h5g]

love your introduction, it is so useful to me, much thanks!

[@gregorysagegreene]

So Cas9 has specificity to seeing only the unique PAM of the virus (bacteriophage), while the cRNA piece attached to it has specificity to that corresponding part of viral RNA to be cut ... excellent description ! Need more detail on TracRNA, it's pairing with the cRNA array, and how the trimming occurs before hooking up with CAS9 ???

[@innovativegenomicsinstitute]

This is a pretty good illustration of how the tracr and crRNAs are processed: www.biorxiv.org/content/biorxiv/early/2020/05/21/2020.05.21.102756/F9.large.jpg (you can ignore the part about "short" and "long" tracrRNAs). At least in Streptococcus thermophilius, RNase III trims both the crRNA and tracrRNA where they're base-paired, and an unknown nuclease trims the single-stranded 5' end of the crRNA.

[@umuthaskok1465]

Great job, ı havent seen any video about pam sequence before

[@akshayavidhya9325]

I needed this video .. thank you so much

[@youtubechannel5429]

Thanks for a great video!

[@matthewanderson1916]

Incredible video, sooo helpful!

[@rehabalsaleh166]

Amazing, thank you!

[@Champion9255]

Excellent video

[@mrphysh]

CRISPR; The repeats are palindromic. the target sites for restriction endonucleases are palindromic. RNA stem loops are made from palindromes, but the palindrome includes extra bases for the loop. Do the CRISPR repeats include a spacer, allowing for a stem loop? (I assume that they do) (Your explanation of PAM was great)

[@innovativegenomicsinstitute]

Yes! That's exactly right.

[@aakashprasad8956]

Incredible video! Helped me so much

[@aimanjalmukhambetova5021]

thank you! amazing video, it helped me a lot

[@LAinLA86]

Great video

[@charpunk]

Muchas gracias por la explicación!, ahora la función de PAM me queda mas claro :)

[@eliz1222]

Thank you x 100!

[@Jacobin777]

I wish there were KZbin videos like this when I was in college. 🙁

[@AnitaRadmanic]

Great video, i just wish that some references were attached in the description 😅 would make my essay writing so much easier hahaha

[@najibawar4861]

thank you so much

[@lukidurer28]

Awesome video, thank you!

[@Phloneme]

But what happens if the Cas1-Cas2 complex finds a PAM in the bacteria's own genome?

[@innovativegenomicsinstitute]

This can and does happen! Self-targeting spacers will kill the cell, removing it from the population. Thus, we mostly just see the "successful" bacteria with foreign DNA-targeting spacers in their CRISPR arrays. Luckily for microbes, the CRISPR acquisition machinery is known to "prefer" foreign DNA, making acquisition of lethal self-targeting spacers less common than capture of invasive DNA. There are several reasons for this. In 2015, an awesome paper from the Sorek group began to explain this preference: www.ncbi.nlm.nih.gov/pmc/articles/PMC4561520/. Basically, the machinery is more likely to get new spacers from DNA that is being actively replicated and/or repaired. Invasive plasmids or phage genomes must replicate to generate lots of copies, so Cas1-Cas2 (and accessory proteins) go for these abundant DNA targets. A lot of acquisition is also triggered by the failure of old spacers. For example, if the target site in a phage's DNA gets mutated and the CRISPR effector complex is no longer able to find and cut it efficiently, this can stimulate the acquisition machinery to capture new, better spacers from the invasive DNA. This process is called "priming." Here's a recent review on the process that describes both of these processes much more thoroughly: www.ncbi.nlm.nih.gov/pubmed/26949040

[@Phloneme]

@@innovativegenomicsinstitute Wow, thank you so much for the detailed reply! This does clear up my questions. I was guessing there might be a micro-evolutionary effect involved in this, very much like you described, but I hadn't heard of that sort of preference. Thanks!

[@julianacarvalho2156]

This is amazing!

[@shalinicDNA]

One correction...the Cas9 enzyme does not recognise other PAMs such as TTTA. There are different Cas enzymes eg., Cas12a recognises TTTA.

[@shilpisehgal5613]

nice simple and straighforward

[@indujagannathan1163]

I would like clarification on the additional role of the PAM sequence mention at "3:35". You say helps with the search process of Cas9 to find the match sequence. From the first part of the video I understood that the PAM sequence is only present in the virus next to the protospacer. Is there a PAM sequence in bacteria too?

[@innovativegenomicsinstitute]

Hi Indu, thanks for your question! Because PAM sequences are very short, they will end up being present quite often throughout the bacterium's own genome, and in multiple places in the viral genome. The key is that there will only be one region that has BOTH a PAM AND a complementary protospacer next to it, and that will only be in the viral genome. Thus, instead of Cas9 unwinding every single bit of DNA in the entire cell, searching for a complementary match, it just looks for a PAM first, and then checks to see if there's a match next to it. This cuts down on the overall search time. Does that help?

[@emilyanklesaria2624]

fantastic video thank you

[@lianahaig7298]

Great video but I wish you explained how the bacteria makes the TracrRNA that bind to the repeat RNA

[@chrispybelles]

Making sure I understand. There is a PAM after a protospacer in the virus, and a PAM after the spacer in the CRISPR array? But the PAM in the virus and the PAM in the CRISPR array are different. The PAM in the viral DNA makes it a target for Cas9, the PAM in the CRISPR array accelerates the search process for Cas9 in the bacterium. Please (anyone) correct me if I'm wrong. Great video!

[@innovativegenomicsinstitute]

Hi Chrisabelle! The PAM for SpyCas9 (NGG) sits next to the protospacer in the viral genome, but this same PAM sequence does NOT sit next to the spacer in the CRISPR array. Instead of NGG there is a GTT next to the spacer sequence. GTT is not the PAM sequence. This means that SpyCas9 will not cut the CRISPR array. When SpyCas9 is searching around a genome it is looking for NGG. To alleviate any confusion, just remember that PAM stands for "protospacer adjacent motif" so it refers to the sequence next to the protospacer, which is NGG in the case of SpyCas9.

[@arsh-qn8bw]

@@innovativegenomicsinstitute how does cas9 know to look for NGG?

[@irishairachel]

Hello. Can you make a video like this about cas12 or cas13? Thanks

[@innovativegenomicsinstitute]

You're in luck-we did! :) kzbin.info/www/bejne/m2i2l4qem7CZb9U

[@w1nniedapooh]

This was great. Tysm!

[@artr0x93]

Really nice video! But I wonder why viruses don't evolve to not have the PAM sequence next to important genes, seems like a vulnerability. I guess its so short it's impossible to avoid practically?

[@innovativegenomicsinstitute]

Viruses DO often mutate and escape CRISPR targeting, either in the spacer region or in the PAM. There might be another PAM elsewhere, but the CRISPR proteins can only use that PAM if the adjacent sequence matches their guide RNA. Since this is an ancient evolutionary battle, bacteria have figured out a way around this. There is a process called "priming," where the CRISPR system can sometimes sense a slight mismatch in a viral target and trigger the acquisition machinery to find and add a new spacer. So even if a virus mutates, the system has a chance to detect the mutation and start with a fresh spacer.

[@bakerd773]

So it needs to have the GG after in order to attach

[@l.j.a.]

This is super helpful than you so much!!! :D

[@zuzanakubiszova7169]

Thanks!

[@thanganhvu4557]

It is really helpful, thanks a lot!

[@maryellard-ivey9753]

Outstanding!

[@alexandrao.2814]

You just saved me from a headache

[@WhirlwindHeatAndFlash]

Okay but if the "PAM area" next to the spacer on the bacterial genome reads GTT (GUU) then how the h. does the executive complex magically become able to recognize nGG in Viral DNA considering crRNA is SPACER + spacer adjacent leading strand (palindrome)?

[@mrphysh]

The CAS9 searches the viral DNA for a GG and identifies that region as the target for destruction. The "GG" is specific for one CAS protein, but it could be...even ...almost anything . Am I understanding this correctly?

[@innovativegenomicsinstitute]

Yes! Each Cas effector protein has its own PAM, and for the most popular Cas9, it's NGG. For others, it's something else. It's usually pretty short (like 2-6 nucleotides long), but otherwise it can be almost anything. Just depends on how the particular system happened to evolve.

[@mrphysh]

@@innovativegenomicsinstitute Thanks! New question: The guide RNA includes a long tail with two RNA stem loops. Do these have anything to do with the palindromic repeats? The loops must have been transcribed from somewhere!

[@innovativegenomicsinstitute]

@@mrphysh You got it! The single-stranded part of the guide RNA is the sequence that matches the invading virus (the 'spacer') and it's the section that scientists reprogram to match a desired DNA site for genome editing. The rest of the guide (the part with the loops, which are called 'stem-loops' or 'hairpins') comes from the repeat. Cas9 is a little bit different from other Cas targeting proteins, because there are actually two pieces of RNA involved. One piece, the 'CRISPR RNA' (crRNA) comes from the spacer and repeat, and the other, the 'tracrRNA' (pronounced "tracer") is a separate piece that's complementary to the repeat and contains a bunch of stem-loops. We have some diagrams in our glossary (visuals help!): innovativegenomics.org/glossary/

[@mrphysh]

@@innovativegenomicsinstitute Thanks, and thanks for the glossary. The tracker RNA and crRNA seem to be connected with ligase (rather than through complementary bases … is that correct?) I cannot visualize how the stem loops in the tracrRNA came from the palindromic repeated clusters. Also, do the repeated palindromic clusters include the bases that will allow the stem loop. Are the palindromic sections absolutely conserved. Maybe pass me a reference.

[@innovativegenomicsinstitute]

@@mrphysh In nature, they're two separately-transcribed pieces of RNA that come together and stay connected solely through base-pairing. Scientists have also developed a 'single-guide' RNA (also in the glossary!) that is connected with an RNA linker. This is an engineered version of the gRNA, where the entire thing is encoded as a single, long stretch in the DNA, and then transcribed into one long guide RNA that folds up into the final shape that Cas9 binds. The repeats are really only palindromic in systems other than the one that uses Cas9, so Cas9 is kind of a terrible example in that sense. In most cases, the repeat folds into a stem-loop once it's transcribed. Here's a diagram of how that works: journals.plos.org/plosone/article/figure/image?id=10.1371/journal.pone.0146422.g005&size=large. This also shows you that the repeats are not absolutely conserved. For Cas9, maybe this figure will help: www.researchgate.net/publication/326747405/figure/fig4/AS:654865487388679@1533143507232/crRNAtracrRNA-duplexes-A-The-consensus-structure-of-each-crRNAtracrRNA-duplex-is.png The crRNA is shown on the left and the tracrRNA is on the right. If this were showing the single-guide RNA instead, you'd see a little loop connecting the two ends labeled "upper stem" in this diagram.

[@guixien]

What if the target region never contains PAM, i.e. the NGG on the 3' side? Would that imply that CRISPR will fail at cutting out the sequence?

[@ithirstyforknowledge]

Awesome, thanks

[@zacherywhite156]

Hello, I was brought to this video after clicking on a question and answer on Google and my question is related to the answer I was reading where it says "This Is Where The Pam Comes In The Pan Which Stands For Photo Spacer Adjacent Motif " So What I Would Like To Know Is What Is The Pan Cause Thats What The Pam Is And What Does Come In The Pan Mean Also? Thanks 😊

[@joseantoniodavila2752]

So interesting. What I didn't understand is why Cas doesn't cut the bacterial chromosome. There must be many thousands of PAL sequences outside the CRISPR.

[@innovativegenomicsinstitute]

Good question! Yes, there are PAM-like sequences all over the genome, but they aren't paired with the protospacer sequence that matches the CRISPR RNA guide. It's like two-factor authentication: having one or the other isn't sufficient, you need both. This system prevents the accidental cutting of the genome at other PAM-like sites (PAM present but protospacer absent), and it prevents cutting the CRISPR array because it also contains the matching piece of viral DNA (protospacer present but PAM absent).

[@joseantoniodavila2752]

@@innovativegenomicsinstitute But about Cas1-Cas2? They just need a PAM and there are more PAM's in the bacterial genome than in the viral one. My English is too rusty.

[@AliAli-y6m2g]

Hello.. How can I get this lecture in the form of a PDF or PowerPoint file ?

[@sarahiortega6260]

Thank you so much 😮‍💨🩷

[@LeonEllisZ]

Oof, FINALLY, someone explained the PAM! Lol, thank you!