Designer Nuclease-Assisted Targeting to Engineer Mammalian Genomes

Designer nucleases have greatly simplified small genome modifications in many genomes. They can precisely target a specific DNA sequence within a genome and make a double stranded break (DSB). DNA repair mechanisms of the DSB lead to gene mutations or gene modification by homologous directed repair (HDR) if a repair template is exogenously supplied. Thus, small, site directed mutations are easily and quickly achieved. However, strategies that utilize designer nucleases for more complex tasks are emerging and require optimization. To optimize CRISPR/Cas9 assisted targeting, an HPRT rescue assay was utilized to measure the relationship between targeting frequency and homology arm length in targeting constructs in mouse embryonic stem cells. The results show that different gene engineering exercises had different homology requirements.