The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods' synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.
