Components of the CRISPR-Cas9 system.

<p>Individual components in both panels are not drawn to scale. <b>A)</b> RNA guided nuclease Cas9 specifically cleaves a genomic target sequence. The protospacer and scaffold sections of the sgRNA are represented in light green and green, respectively. The protospacer basepairs with the complementary strand of the target sequence to form a D-loop. Note that efficient cleavage depends on the presence of the three bp PAM sequence in the target sequence, which is located directly downstream of the region invaded by the protospacer. Blunt-end cleavage occurs between bases located 3–5 bp upstream of the PAM, as indicated by scissors <b>B)</b> Liberation of the sgRNA, represented in light green/green, from a polymerase II transcript by intrinsic hammerhead (HH) ribozyme and hepatitis delta virus ribozymes (HDV) ribozyme represented in red/blue and orange respectively. Cleavage points are indicated by scissors. The blue part of HH basepairs with the protospacer for efficient cleavage. <b>C)</b> A fungal AMA1 based vector harboring Cas9 and sgRNA encoding genes are transformed into a fungus. The Cas9/sgRNA riboprotein induces a site specific DNA DSB. In the absence of a homologous template for HR repair (left lane), the DNA DSB is repaired by NHEJ; and error/prone repair by NHEJ results in mutations as indicated by yellow base pairs. In the presence of a linear or circular gene targeting substrate (right lane), specific Cas9 induced DNA DSBs may be repaired by HR resulting in a gene targeting event illustrated as insertion of an orange marker gene in this example.