The CRISPR/Cas family of proteins comprises DNA- or RNA-cleaving enzymes that can be programmed to cut specific sequences.

CRISPR RNA (crRNA) protospacer, typically, 20mer, offers target-specificity, as it programs Cas9 to bind and cleave DNA at sites that are identical (or highly similar) to the crRNA protospacer sequence. In addition, Cas9 requires the protospacer-adjacent motif (PAM - sequence NGG).

The PAM is immediately next to the 3’ end of the target and defines the boundaries of the protospacer sequence. Candidate crRNAs can be designed by programs such as Chopchop and ordered through manufacturers such as IDT.

When Cas9 is loaded with sequence-specific crRNA protospacer, together with trans-activating CRISPR RNA (tracrRNA - a structural RNA required for catalytic activity), it forms a ribonucleoprotein complex (RNP).

This complex of RNA and protein searches the genomic DNA for the target region using the crRNA protospacer sequence. The complex attempts to seed the crRNA by melting duplex DNA, and, if the crRNA protospacer matches and base-pairs with the target sequence, Cas9 cuts both strands of the target sequence 3 bp upstream of the PAM.

More information on the mechanism of CRISPR/Cas9 cleavage and crRNA design can be found in the Targeted, amplification-free DNA sequencing using CRISPR/Cas9 info sheet.