| Abstract [eng] |
The recent development of the Cas9 technology revolutionized the genome editing field. The simple modular organization of Cas9 complex, where specificity for the DNA target is encoded by a small crRNA and the cleavage reaction is executed by the Cas9 endonuclease, provides a versatile platform for the engineering of universal RNA-directed DNA endonucleases. In this work, we identified all molecular components required for an assembly of the functional Cas9 complex of Streptococcus thermophilus CRISPR3-Cas system and demonstrated targeted DNA cleavage by the reconstituted Cas9 complex in vitro and in vivo. More specifically, we showed for the first time that Cas9 complex can be used as a tool for DNA cloning and targeted genome editing by chemical transfection of the in vitro assembled Cas9 ribonucleoprotein complex into the cells. Aiming to understand the molecular mechanism governing DNA recognition and cleavage by the Cas9 complex, we analyzed mechanism of R-loop formation and provided first direct evidence for directional R-loop formation, starting from PAM recognition and expanding toward the distal protospacer end. Realizing the importance of PAM sequence for Cas9 function, we developed an assay for rapid PAM identification for newly identified Cas9 proteins and characterized Type II-C Cas9 protein of Brevibacillus laterosporus, expanding Cas9 toolbox for genome editing applications. |
