The Rise of CRISPR/Cas for Genome Editing in Stem Cells

Figure 1

A proposed model for Cas9 endonuclease to trigger DNA cleavage. A conformational change is induced once the Cas9 protein binds to gRNA, allowing it to search for the DNA substrate. The REC lobe of Cas9 scans for the PAM in the genome. PAM recognition helps local unwinding of dsDNA 5′ to the PAM region. The unwound DNA is transiently stabilized by protein/ssDNA interaction. Successful base pairing between the ssDNA portion and the gRNA further extends the ssDNA loop. A critical loop size may trigger the enzymatic activity of Cas9 to make the double-stranded cut. Afterwards, Cas9 remains bound to the DNA substrate. If the base pairing between ssDNA and gRNA is blocked by mismatches, the ssDNA loop collapses to release the Cas9 protein.