The genome editing protein Cas9 faces engineering challenges in improving off–target DNA cleavage and low editing efficiency. In this study, we aimed to engineer Cas9 to be able to slide along DNA, which might facilitate genome editing and reduce off-target cleavage. We used two approaches to achieve this: reducing the sliding friction along DNA by removing the interactions of Cas9 residues with DNA and facilitating sliding by introducing the sliding-promoting tail of Nhp6A. Seven engineered mutants of Cas9 were prepared, and their performance was tested using single-molecule fluorescence microscopy.
Fig: dCas9 exhibits sliding and paused modes on DNA in the absence of sgRNA. (A)Single-molecule tracking of dCas9 mutants labeled with ATTO488 in the flow cell. DNA (pink line) is tethered at one end and stretched by buffer flow. The labelled dCas9 mutant (green circle) is illuminated by HILO (blue sheet). The movements of dCas9 mutants bound to DNA were recorded. (B) Typical kymographs of dCas9. White lines or curves denote the traces of single molecules. Yellow arrows demonstrate sliding molecules. (C) MSD plots of all tracked molecules of dCas9 (N = 232). The errors denote standard errors. A best-fit linear line is shown (R2 = 0.80). (D) Displacement distribution of dCas9 at a time interval of 176 ms. The black curve shows the best-fit curve based on the sum of two Gaussian functions. The dashed curves represent the distributions of each mode.
Comparison of the mutations enabled the identification of key residues of Cas9 to enhance the sliding along DNA in the presence and absence of single guide RNA (sgRNA). The attachment of the tail to Cas9 mutants enhanced sliding along DNA, particularly in the presence of sgRNA. Together, using the proposed approaches, the sliding ability of Cas9 was improved up to eightfold in the presence of sgRNA.. A sliding model of Cas9 and its engineering action are discussed herein.
Banerjee, T., Takahashi, H., Subekti, D.R.G. et al. Engineering of the genome editing protein Cas9 to slide along DNA. Sci Rep 11, 14165 (2021). https://doi.org/10.1038/s41598-021-93685-9