Cas9-Protein Bioconjugation via Click Chemistry to Enhance HDR

Abstract

The CRISPR/Cas9 system is a newly-discovered method in gene therapy, showing great potential. However, after the introduction of a double strand break by the Cas9 protein to the genomic DNA, the error-prone non-homologous end-joining DNA repair pathway predominates over the precise homology-directed repair (HDR) one. For the CRISPR/Cas9 system to be more efficient in accurate genome editing and thus be utilized for the correction of disease-causing mutations, the efficiency of the HDR response should be increased. In this study, an effort has been made to conjugate the Cas9 protein with other protein domains, namely the dominant-negative domain of 53BP1, the human phosphatase inhibitor-2 and the N-terminal region of the human geminin, in order to enhance the frequency of the HDR response either directly or in a time-controlled manner by taking advantage of the characteristics of those three protein domains. An unnatural amino acid was incorporated in the Cas9 sequence, for the purpose of the bioconjugation, which was done utilizing copper-free click chemistry. Our results show that the incorporation of the unnatural amino acid is possible in different positions and can be used for conjugation of the protein with molecules, such as PEG or fluorescent dye. Based on the conjugation efficiency and the relative activity that the SpCas9 variants demonstrated, a preferred position for the amino acid substition was highlighted. The corresponding SpCas9 variant is highly active, even when conjugated with the fluorescent dye. The results of the protein-protein bioconjugation remain inconclusive.