What is the PAM? - A CRISPR Whiteboard Lesson
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- Опубликовано: 16 июн 2024
- CRISPR-Cas9 searches and chops up invading viral DNA to defend bacterium against destruction. What prevents Cas9 from cutting the bacteria's own genome? The answer is the PAM sequence. Watch this whiteboard video to learn more about this important stretch of DNA. Video recorded and produced by Eugene Lo. Learn more in CRISPRpedia: innovativegenomics.org/crispr...
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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.
Great...the best crispr video in youtube
indeed
Amazing video. Thank you for sharing.
The most excellent video I ever see. No one explains what is PAM and why it is important exactly until now.
Couldn't find this explanation anywhere else, super clear and answered all my questions, great video really
Amazing video. Thats how a brilliant human explains science... Brilliant drawings.
You have just answered my whole questions. Thank you. Amazing video.
Thank you for this OUTSTANDING video!
Wow, that was extremely helpful! I don't normally comment on vids but I had to thank you for making such a fantastic video.
Such a good & clear explanation! The drawings really help the understanding. Thank you!
Had to slow this down to 0.75 to take clear notes for myself lol Thanks for the clear explanation!
same , I kept pausing throughout and watched the video twice
A wonderful and thorough explanation of the Pam sequence. Thank you so much
Thank you so much for creating this video! Finally I have a clear picture of how PAM works and why it is so useful!
Wow! Thank you for this video, it makes CRISPR Cas-9 so much easier to understand! Great vid
Great work, Eugene!
This is an example of what well-explained means. Thanks a lot
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"
Congrats on the video.
You explained what none of the all the most viewed and most liked videos didn't.
This is super helpful
The pictures and explanation really helped me wrap my head around CRISPR
Thank you 😊 💓
Just answered my whole question about CRISPR :") really helpful thank you soo much!!
Amazing! Incredible! Fantastic! Astounding! Mighty! Wonder! Super!
I have studied numerous sources to explain how PAM works and none came close to the clarity of this video....great work, thank you
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.
This was so helpful! Thank you for creating this
I watch the video a second time and is still incredible!
Excellent video...great job explaining PAM. Thank you!
Love this video! Great help! Thank you!
Crystal clear explanation! Thank you!
Helping so much to understand the complex consent
Awesome video! Super helpful. So glad I was able to find this.
honestly, you deserve 10M like bro. Thank you
Thank you so much, this literally answered every question I possibly had.
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!
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?
thanks for your input and great work! I will always remember PAM now :)
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!
So happy we could help! Hope you enjoy your research experience. :)
Amazing explanation! Thank you so much
your explanation is so clear but yet so thorough, salute from a non-bio phd trying to understand what my colleagues are doing...
Thank you! A lot of work went into this one. :)
the BEST explanation of CRISPR, hands down
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!!!
So helpful! The only video I could find to clearly understand the concepts for my exam!
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.
RPI! We had a lecture on CRISPR/Cas9 and it's current uses in editing!
Ah amazing. Thank you so much. You are a live saver.
This help in my interview 👍 thanks a million!!
Thank you! Just thank you!
Thanks for a great video!
Amazing, thank you!
this was the best video ive ever seen in my life
I needed this video .. thank you so much
Incredible video! Helped me so much
I love your video! Thanks a bunch.
Incredible video, sooo helpful!
This was great. Tysm!
fantastic video thank you
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.
Really excellent video, thank you IGI
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 :)
love your introduction, it is so useful to me, much thanks!
thank you! amazing video, it helped me a lot
This is amazing!
Muchas gracias por la explicación!, ahora la función de PAM me queda mas claro :)
Thank you so much. It helped
Seriously I love the way you question to every point and answering them nice 👌👌
Motived for my video 👌👌
Great explanation, and you're great too.
It is really helpful, thanks a lot!
thank you so much
Excellent video
Great video!!! It helped me a lot preparing for my exams!
We are curious to learn which school or course is teaching and testing these concepts?
My undergrad molecular genetics course at the University of Florida teaches a section of CRISPR-CAS9. Awesome video hope to see more
Awesome!
Please make more such content. You explained really well. I am doing a project on CRISPR so it'd be helpful.
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/
@@innovativegenomicsinstitute Sure I'll definitely check that out. :)
thankyou! doing a SHE task on CRISPR and know i understand the topic
Awesome, thanks
Wow, I tried to understend that part in wikipedia. But it was impossible. Here everything seems so simple. Thanks
Thank you x 100!
AWESOME!!!!!!!
excellent, thank you
Great job, ı havent seen any video about pam sequence before
Great video
Thanks!
Outstanding!
cannot thank you enough.
nice simple and straighforward
Brilliant!
This is super helpful than you so much!!! :D
Great video, i just wish that some references were attached in the description 😅 would make my essay writing so much easier hahaha
I wish there were RUclips videos like this when I was in college. 🙁
incredibly explained
i love this video so much
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 ???
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.
perfect !
Excelent! i love it
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)
Yes! That's exactly right.
Thank yuuuuu
Brilliant~
Thanks
Great video but I wish you explained how the bacteria makes the TracrRNA that bind to the repeat RNA
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
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.
@@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!
I Love You.
You just saved me from a headache
Danke Bruder
amazing
So it needs to have the GG after in order to attach
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?
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?
Thank you very much for making this video, really helped me understand the PAM! I'm currently writing a monograph on CRISPR-cas9 so this definetly really helped. I do however still have one question, can this PAM sequence be on the 5' side of the DNA sequence or is it always on the 3' side?
Great question! The PAM sequence can be closer to the 5' end of the gRNA sequence for certain CRISPR systems. In the case of Cas9 it will be near the 3' end of the guide. For Type I (Cascade) and Cpf1 (Cas12) complexes, it is near the 5' end of the crRNA guide, opposite of Cas9. Check out this figure: goo.gl/d7W4k7 to compare Cas9 + Cascade; check out this one to compare Cas9 + Cas12: goo.gl/87WqoZ Hope this helps!
Thanks, definetly helped a lot! I'll make sure to mention this on my monograph!
Hi, as I'm writing my monograph, I now have a new question, how are the CRISPR system and the needed tools delivered to the target cells?
We have a great resource on our website called "Ask a Scientist" where we can connect you will a CRISPR researchers to answer questions, such as delivery and cell targeting. innovativegenomics.org/resources/educational-materials/ask-a-scientist/
this is very late but if you're still curious it is standard DNA plasmid electroporation nucleofection if you're working with cell cultures. The Cas9 and guide RNA sequence of interest are encoded into a DNA plasmid construct which is then introduced into the cells and expressed in the nucleus where it becomes a mature and active CrisprCas9 against the desired target sequence.
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?
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.