What is RNA in CRISPR
How CRISPR-Cas9 works
First, with CRISPR-Cas9, the CRISPR and “spacer” sequences are translated into CRISPR RNA (crRNA). Before this can then show the Cas9 protein the point of intersection on the DNA, it has to be brought into its final form by cutting proteins and parts of it have to be removed. One such enzyme is RNase III. Together with the tracrRNA, it converts the original form of the crRNA into a mature and functional molecule.
The finished crRNA contains a copy of the CRISPR sequence and the foreign DNA. In this way, it provides Cas9 with the recognition sequence at which the cutting molecule is supposed to cut through the DNA. The crRNA combines with the tracrRNA, because only the two together can show the Cas9 intersection.
However, the recognition sequence that matches the crRNA alone is not enough for Cas9 to bind to DNA: It also requires a so-called “proto-spacer adjacent motif”, or PAM for short. Cas9 can only bind to the DNA thread if such a three-letter section - two guanines and any base - is next to the recognition sequence.
Now the two strands of DNA unwind and the crRNA / tracrRNA molecule - or an artificial “guide RNA” - can attach itself. The enzyme then cuts both strands at the same point. So Cas9 needs both the recognition sequence and a PAM so that it can cut DNA. Since the genetic material of the bacterium itself does not have any PAMs, it is protected from being destroyed by its own defense system.
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