Expanding the Potential of Mammalian Genome Engineering via Targeted DNA Integration

Inserting custom designed DNA sequences into the mammalian genome plays an essential role in synthetic biology. In particular, the ability to introduce foreign DNA in a site-specific manner offers numerous advantages over random DNA integration. In this review, we focus on two mechanistically distinct systems that have been widely adopted for targeted DNA insertion in mammalian cells, the CRISPR/Cas9 system and site-specific recombinases. The CRISPR/Cas9 system has revolutionized the genome engineering field thanks to its high programmability and ease of use. However, due to its dependence on linearized DNA donor and endogenous cellular pathways to repair the induced double-strand break, CRISPR/Cas9-mediated DNA insertion still faces limitations such as small insert size, and undesired editing outcomes via error-prone repair pathways. In contrast, site-specific recombinases, in particular the Serine integrases, demonstrate large-cargo capability and no dependence on cellular repair pathways for DNA integration. Here we first describe recent advances in improving the overall efficacy of CRISPR/Cas9-based methods for DNA insertion. Moreover, we highlight the advantages of site-specific recombinases over CRISPR/Cas9 in the context of targeted DNA integration, with a special focus on the recent development of programmable recombinases. We conclude by discussing the importance of protein engineering to further expand the current toolkit for targeted DNA insertion in mammalian cells.