title: CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment.
abstract: The CRISPR/Cas9 complex, a bacterial immune response system, has been widely adopted for RNA-guided genome editing and transcription regulation in applications such as targeted genome modification and site-directed mutagenesis. However, the physical basis for its target specificity is not fully understood. In this study, based on a statistical mechanical analysis for the whole ensemble of sgRNA-target complex conformations, we identify a strong correlation between Cas9 cleavage efficiency and the stability of the DNA-RNA (R-loop) complex structures, with a Pearson correlation coefficient ranging from 0.775 to 0.886 for the tested systems. The finding leads to quantitative insights into important experimental results, such as the greater Cas9 tolerance to single-base mismatches in PAM-distal region than to PAM-proximal region and the high specificity and efficiency for shorter protospacers. Moreover, the results from the genome-wide off-target assessments, compared with other off-target scoring tools, indicate that the statistical mechanics-based approach provides more reliable off-target analyses and sgRNA design. To facilitate the genome engineering applications, a new web-based tool for genome-wide off-target assessment is established.
authors.affiliation: Department of Physics, Department of Biochemistry, and Informatics Institute, University of Missouri, Columbia, MO, USA.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Xu
authors.affiliation: Department of Molecular Microbiology and Immunology, Department of Neurology, School of Medicine; Department of Biomedical Sciences, College of Veterinary Medicine; and Department of Bioengineering, University of Missouri, Columbia, MO, USA.
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Duan
authors.affiliation: Department of Physics, Department of Biochemistry, and Informatics Institute, University of Missouri, Columbia, MO, USA. chenshi@missouri.edu.
authors.forename: Shi-Jie
authors.initials: SJ
authors.lastname: Chen
mesh_terms: D064113:CRISPR-Cas Systems; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D000072669:Gene Editing; D008958:Models, Molecular; D015233:Models, Statistical; D009154:Mutation; D009690:Nucleic Acid Conformation; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA; D004720:Endonucleases