Transgene integration at "safe harbor" (AAVS1) site

    Exogenous expression of mammalian genes has been widely used to study gene functions. Most transgene expression is based on either a simple transformation with plasmid vectors or transduction with viral particles. The main drawback of these methods is random integration of transgenes in the host genome, which cannot be tightly controlled and usually result in large variation among different experiments. On the other hand , targeted integration using genome editing technologies enables researchers to express their gene of interest (GOI) at a safe location, such as the proven "safe harbor" site or AAVS1 site on the human chromosome 19.  Integration at the "safe harbor" site eliminate the uncertainty and variation caused by random integration, such as gene copy number variation, position effect, transgene silencing, and unwanted disruption of endogenous genes, etc.  


    To facilitate targeted integration at "safe harbor" site, CIB scientists have screened and identified a CRISPR/Cas9 that targeted the AAVS1 site with high on-site cutting efficiency and no detectable off-target cutting. In the meantime, we have optimized the process of making donors and improved the efficiency of homologous-driven recombination (HDR). These improvements enable us to integrate gene of interest in pluripotent stem cells with high efficiency and predictability.

 

Example (CRISPR/Cas9):

BACE-1 Alzheimer Disease-related Gene

                        A


                        B

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                        C

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A: Donor plasmid structure; B: Junction PCR to verify integration site (b-1: left arm length 1489bp; b-2: three positive clones with expected inserts); and C: Sequences at the junction sites (partial). 


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