Figure 2

CRISPR/Cas9 interacting with target DNA. The CRISPR/Cas9 complex of Streptococcus pyogenes consists of the Cas9 protein (in gray) and a guide RNA that is a chimera of natural crRNA and tracRNA (in orange). The targeting sequence at the 5′end of the guide RNA base-pairs with complementary sequences on the target DNA (in blue); the targeting sequence is 20 nucleotides long, but may be shortened to increase specificity [88] (the addition of 1 to 2 unpaired nucleotides at the 5′ end is also tolerated [51, 88]). The presence of a PAM (protospacer adjacent motif, NGG for Streptococcus pyogenes), located immediately downstream of the 20-nucleotide sequence targeted by the guide RNA, is also essential for target recognition and cleavage. The PAM sequence does not have a counterpart on the guide RNA. Following recognition of the PAM and base-pairing between the guide RNA and the target, Cas9 cleaves each of the target DNA strands a few nucleotides upstream of the PAM (red arrowheads). Each strand is cleaved by a different nuclease domain present in Cas9 (HNH and RuvC domains). These domains have been mutated independently to generate Cas9 nickases [82, 87].