Bacterial Adaptive Immunity with CRISPR/Cas9

CRISPR systems evolved as a means of bacterial adaptive immunity. Understanding their behavior in vivo is essential to harnessing their power in vitro.


CRISPR, also known as Clustered Regularly Interspaced Short Palindromic Repeats, are systems that mediate bacterial adaptive immune responses. CRISPR systems are made up of arrays and clusters of CRISPR-associated, or Cas, genes.

Type 2 CRISPR systems incorporate approximately 20 base pair repeats of DNA from foreign viruses or plasmids amidst a series of short repeats to confer immunity. The CRISPR repeat array is transcribed into a primary transcript, which contains repeats of conserved sequences that are complimentary to part of a transactivating CRISPR RNA called tracrRNA, which is also encoded in the CRISPR locus.

TracrRNA base pairs with the primary transcript and is then processed into individual crRNAs. Each of these crRNAs contain a single 20 base pair sequence, complimentary to foreign DNA. The crRNA tracer RNA complex interacts with the Cas9 protein to form an active RNA-guided nuclease. The active Cas9 ribonucleoprotein complex interacts with the protospacer-associated motif, also known as the PAM sequence, in the foreign DNA.

Next, the Cas9 complex attempts to bind more tightly to sequence complimentary to the crRNA. If both PAM binding and complimentarity occur, the Cas9 nuclease cleaves the target DNA.

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