Abstract
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.
Highlights
Over the last decade, the novel CRISPR-associated endonuclease Cas9 protein has been used for therapeutic and analytical approaches in a vast spectrum of cell types and animal model [1, 2]
Intending to improve the efficiency of homologous direct recombination repair (HDR), we tested and further combined three strategies already reported in the literature able to increase the HDR efficiency but never used together
We wondered if these protocols could work in synergy to improve the HDR efficiency
Summary
The novel CRISPR-associated endonuclease Cas protein has been used for therapeutic and analytical approaches in a vast spectrum of cell types and animal model [1, 2]. Thanks this method, the genes editing has become as simple, fast, and economical as never before. DSB activates the cell’s repair mechanisms, including non-homologous end joining (NHEJ) and homologous direct recombination repair (HDR) to repair the DNA strands.
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