🧵Intro to CRISPR
Here we will look at the discovery of CRISPR.
Here we will look at the discovery of CRISPR.
1/ What does CRISPR stand for? It stands for Clustered Regularly Interspaced Short Palindromic Repeats. This was discovered by scientists studying bacteria. Bacteria get infected by viruses called Bacteriophages. They attach to the bacteria and inject their viral DNA.
2/ Scientists noticed that the bacteria genome had a sequence of repeats. It showed a small sequence of DNA then a repeat followed by another small sequence and another repeat. They named this area of the bacteria genome the CRISPR region. This is the bacteria's immune system.
3/ It works through type 1 CRISPR enzymes like CAS1 and CAS2, chopping up the bacteria DNA. Then a small sequence of the viral DNA called the protospacer is inserted into the CRISPR region of the bacteria genome. What happens next is very awesome.
4/ The bacteria transcribes the entire CRISPR region into one long messenger RNA called the pre-transcript. This gets chopped up so each DNA sequence that matches the virus' protospacer becomes the CRISPR RNA (crRNA) and the repeat becomes the trans activating RNA (tracrRNA).
5/ The repeat segments become the tracrRNA that will get loaded into an enzyme called CAS9. CAS9 stands for CRISPR Associated Protein 9. The CAS9 will recognize the tracrRNA and bind to it.
6/ The crRNA will then bind to the tracrRNA to make a complete guide RNA. The two RNA segments bind together by complementary base pair binding by hydrogen bonding as shown in the picture above.
7/ Once the CAS9 is loaded with the complete guide RNA, it will go and find any DNA that has the matching protospacer for the guide. The guide is a complete complementary sequence of RNA that matches that protospacer in the Viral genome.
8/ The actual guide sequence is about 20 nucleotides long. Once it finds the matching viral DNA, the CAS9 enzyme will cut that DNA. This destroys the viral DNA.
9/ I know what you are thinking. If the DNA sequence copied into the CRISPR region of the Bacteria matches the viral DNA, how does the CAS9 not cut up that bacteria's DNA too?
10/ The answer to that is the PAM sequence of the CAS9 enzyme. This stands for Protospacer Adjacent Motif. This is a small segment of nucleotides in the DNA that is recognized by the CAS9 enzyme which is N-G-G.
11/ The N stands for any nucleotide while the G stands for Guanine. This basically means the CAS9 enzyme not only needs to match the guide RNA sequence to the protospacer, but it also needs to match its PAM to the 2 Guanines near the protospacer.
12/ If both don't exist, the CAS9 will not cut. As you can guess, the CRISPR region of the bacteria genome would not contain the PAM sequence so the CAS9 would be selective for targeting the viral DNA, but not the bacteria's DNA.
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