RNA-Guided Gene Targeting in Human Cells

Project Summary/Abstract The goal of the proposed Phase I research is to develop a set of commercial kits for footprint-free genome engineering. The core technologies that serve as the foundation of these kits are based on the CRISPR/cas system, which wasoriginally identified in bacteria and recently shown to also function in mammalian cells. The main advantage of the proposed gene targeting system is that the specificity of the target DNA sequence is dictated by a guide RNA, which can base-pair with onestrand of double-stranded DNA by forming an R-loop . Currently, zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) provide possibilities to specifically and permanently modify the human genome. There are commercial reagents and services available for these two technologies. However, these two technologies are based on protein-DNA recognition, which requires designing and creating new enzymes every time a unique DNA sequence is to be targeted. The assembly of new ZFNs or