Abstract
The bacterial CRISPR/Cas9 system has a proven to be an efficient tool for genetic manipulation in various organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random creation of indels. Many studies focused on improving the efficiency of HDR using double sgRNA, cell synchronization cycle and the delivery of ssODN with a rational design. In the present study, we tested and compared the combination of these three methods to improve HDR efficiency. To our tests, we chosen the TNFα gene (NM_000594) for its crucial role in a variety of biological processes and diseases. Our results showed a dramatically increases of HDR efficiency from undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome targeting. Furthermore, we showed that TNFα gene could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.
Systematic Analysis of microRNA Targeting Impacted by Small Insertions and Deletions in Human Genome
