Mechanisms of target and off-target DNA recognition and cleavage of CRISPR-Cas effectors. (A) Top: Schematic diagram of the structure and full domain of the CRISPR-Cas9 protein. In the figure, each inset represents the interaction between the Cas9 domain and PAM distal region (left inset) and the PAM proximal region (right inset) within the guide RNA-DNA heteroduplex. Bottom: Schematic diagram of the interaction between guide RNA-target DNA heteroduplex and CRISPR-Cas9 protein. (B) Top: Schematic diagram of the structure and full domain of the CRISPR-Cas12a protein. In the figure, each inset represents the interaction between the Cas12a domain and PAM proximal region (left inset) and PAM distal region (right inset) within the crRNA-DNA heteroduplex. Bottom: Schematic diagram of the interaction between the guide RNA-target DNA heteroduplex and the CRISPR-Cas12a protein. NTS: non-target strand, TS: target strand, REC: Recognition domain, RuvC: RuvC domain, PI: PAM interaction domain, WED: Wedge domain, Nuc: Nuclease domain. The structures (A, B) are obtained from Protein Data Bank (PDB) and molecular images of SpCas9 (A, PDB: 7QQS) and FnCas12a (B, PDB: 6I1K) were illustrated using PyMOL software (The PyMOL Molecular Graphics System, Version 2.5.4 Schrödinger, LLC.). (C) Schematic diagram of the cleavage mechanism of CRISPR-Cas9 (Top) and Cas12a (Bottom) effectors for on-target and off-target DNA. Red arrowhead: cleavage of DNA. Red triangles: Mismatched sequence between guide RNA and target DNA. Images were created with BioRender.com.
