The Science Behind CRISPR
Hi all,
Looking back at the topic synopsis, I realized that the picture provided of how CRISPR-Cas9 works could be confusing. Even I didn't really understand how it worked just by looking at that. But as a molecular and cell biology major, my curiousity was peaked, so I wanted to better understand that for myself and share it with you. Please note: this is in no way required for committee, just trying to help those who were curious like myself find resources that do a good job of explaining how the technology works. Many sources online either over-simplify and focus on wild speculations, or favor jargon too heavily, to the exclusion of novices. So I've compiled a list of videos and a few articles that explain CRISPR to a satisfying extent(at least for me), but know that there's a lot more out there and an expanding body of academic work on CRISPR that might be unaccessible without a very sold foundation in biology. Also note: the sources I have focus only on CRISPR, so if you haven't taken an introductory biology class before they might still be a bit fast. But there are many good sources online such as a range of Crash Course biology videos if you don't have any background in biology but want to so that you can understand how the technology works.
To start, here's the nitty-gritty of CRISPR-Cas9:
CRISPR stands for Clustered Regularly Interspaces Short Palindromic Repeats. They're sections in the DNA of bacteria that have viral DNA from a previous bacteria in them. Basically, if a bacteria is infected with a virus but manages to survive, it takes in part of the viral DNA and puts it into it's own DNA. A protein(Cas9) that can cut DNA is then combined with an RNA template that mirrors the viral DNA the bacteria took in, and the protein complex then scans through the DNA regularly, looking for this viral DNA segment in the bacteria's DNA. If it finds it, the protein cuts the viral DNA to inhibit the virus from infecting and killing the bacteria. This relies on a couple of key aspects of biology: 1) viruses infect cells by putting their DNA inside other organisms so that the infected cell(bacteria in our case) makes more of the virus, so bacteria putting viral DNA in their DNA mirrors what happened during an infection, so CRISPR-Cas9 knows what to look for, and 2) RNA can match with DNA because of a universal genetic code. Normally DNA makes an RNA that mirrors it to go out into the cell to preform a function, but CRISPR-Cas9 uses this important mirror property in the reverse order- finding the DNA section that would have made this RNA segment.
I've organized the videos from least to most complicated, so you can watch them in order up to a level you feel comfortable with or that you feel gives you as much of the science as you want to know.
Looking back at the topic synopsis, I realized that the picture provided of how CRISPR-Cas9 works could be confusing. Even I didn't really understand how it worked just by looking at that. But as a molecular and cell biology major, my curiousity was peaked, so I wanted to better understand that for myself and share it with you. Please note: this is in no way required for committee, just trying to help those who were curious like myself find resources that do a good job of explaining how the technology works. Many sources online either over-simplify and focus on wild speculations, or favor jargon too heavily, to the exclusion of novices. So I've compiled a list of videos and a few articles that explain CRISPR to a satisfying extent(at least for me), but know that there's a lot more out there and an expanding body of academic work on CRISPR that might be unaccessible without a very sold foundation in biology. Also note: the sources I have focus only on CRISPR, so if you haven't taken an introductory biology class before they might still be a bit fast. But there are many good sources online such as a range of Crash Course biology videos if you don't have any background in biology but want to so that you can understand how the technology works.
To start, here's the nitty-gritty of CRISPR-Cas9:
CRISPR stands for Clustered Regularly Interspaces Short Palindromic Repeats. They're sections in the DNA of bacteria that have viral DNA from a previous bacteria in them. Basically, if a bacteria is infected with a virus but manages to survive, it takes in part of the viral DNA and puts it into it's own DNA. A protein(Cas9) that can cut DNA is then combined with an RNA template that mirrors the viral DNA the bacteria took in, and the protein complex then scans through the DNA regularly, looking for this viral DNA segment in the bacteria's DNA. If it finds it, the protein cuts the viral DNA to inhibit the virus from infecting and killing the bacteria. This relies on a couple of key aspects of biology: 1) viruses infect cells by putting their DNA inside other organisms so that the infected cell(bacteria in our case) makes more of the virus, so bacteria putting viral DNA in their DNA mirrors what happened during an infection, so CRISPR-Cas9 knows what to look for, and 2) RNA can match with DNA because of a universal genetic code. Normally DNA makes an RNA that mirrors it to go out into the cell to preform a function, but CRISPR-Cas9 uses this important mirror property in the reverse order- finding the DNA section that would have made this RNA segment.
I've organized the videos from least to most complicated, so you can watch them in order up to a level you feel comfortable with or that you feel gives you as much of the science as you want to know.
- https://www.youtube.com/watch?v=2pp17E4E-O8
- https://www.youtube.com/watch?v=MnYppmstxIs
- https://www.youtube.com/watch?v=bXnWIk8FgKc
- Please note that in the beginning of this video she refers to the nucleus of a bacteria, but bacteria don't have nucleuses. They have regions where DNA tends to be, called the nucleoid region, but they don't actually have a "nucleus". The rest of the video is accurate and i'm sure she just did it for simplicity, but as a biology major this annoyed me and I don't want others to wrongly think that bacteria have nucleuses.
- https://www.youtube.com/watch?v=TdBAHexVYzc
- I particularly enjoyed this video; it's a TED talk from Jennifer Doudna herself, touches upon implications of the technology, and is generally entertaining.
These sources are articles that I think explain key elements not fully explained in the videos, but that appear in the picture of how CRISPR works in the topic synopsis.
- https://www.wired.com/story/what-is-crispr-gene-editing/
- This source explains what the PAM sequence is and is another good overall explanation of what CRISPR-Cas9 is.
- https://www.horizondiscovery.com/gene-editing/crispr-cas9
- This sources has a good diagram near the end that explains how CRISPR-Cas9 can be used for genetic engineering to add a desired sequence into DNA rather than just "knockout" a gene.
- https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology
- And lastly I'll give you this source. It's by far the most complicated and jargon intensive source i've provided, but really explains the molecular biology of CRISPR-Cas9. Please don't feel frustrated if you're having trouble with this source, it really is complicated.
Hopefully this gives a pretty good overall picture of how CRISPR-Cas9 works. I'd encourage those interested to find more academic sources online, but academic articles in the sciences are notoriously hard to engage with if you're not also an expert, so be aware that some sources just won't make sense to you(or me) and that's perfectly fine. If you have any questions about information in the sources, post it as a comment and I'll do my best to reply and try to answer it.
How did you find the sources? Were they useful?
Do you think this technology is going to be more helpful or harmful?
Good luck, and see you at conference!
How does the Cas9 complex target the right gene? Unless the guideRNA is incredibly long, won't there be an identical section of DNA that is not the intended target?
ReplyDeleteAlso, what is the point of the tracerRNA other than creating the hairpin shape that grips onto the Cas9 protein?
Thanks for all the great resources!
Delegation of the United Kingdom
Hi, thanks for commenting!
DeleteFirst, the Cas9 complex likely doesn't have to be incredibly long. I acknowledge that it could potentially happen that if the segment is too short, an incorrect gene could be targeted. However, viral proteins are different enough from other organisms that the exact same RNA segment of a viral protein shouldn't be appearing in the host's DNA unless the host is infected. I know this sounds a bit vague, but the proteins are different. This difference is also how your immune system is able to react specifically to viruses in our bodies; their proteins are unique enough to appear as foreign. And the protein is coded for by a unique RNA, so the DNA template of that RNA also stands out as different from the host cell's DNA.
And to the best of my knowledge tracerRNA is only used to help guide the Cas9 endonuclease, which splits the DNA. However, I'm not an expert. There could be other uses, but all the results I could find about tracerRNA connected it to CRISPR, so I doubt these other uses are common or very important.
Thanks again for commenting. I hope my response was helpful.
Thanks for the quick response! It was indeed helpful.
DeleteI was actually referring to the use of the Cas9 complex combined with RNA created by scientists to target specific genetic mutations that are causing diseases rather than viral proteins. However, I suppose that if the guide RNA matches to the DNA, then that section of DNA would have produced the undesired protein regardless if that was the section of DNA that was meant to be targeted.