Targeted Genomic Integration by Engineered Transposon and CRISPR/Cas9 Components

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An ideal gene transfer tool would enable chromosomal integration of therapeutic nucleic acids in an efficient and site-specific manner without introducing unwanted alterations of the target genome, which might lead to potentially severe side effects. None of the currently available technologies fulfill this requirement. Designer nucleases, including the CRISPR/Cas9 system, are highly specific in their action, but rely on introducing a double-strand break (DSB) into the genome to promote transgene integration at the desired target. This is one of the most dangerous forms of DNA damage the genome can experience. In addition, unintended off-target introduction of DSBs into the genome represents a significant safety concern. On the other hand, integrating gene delivery vectors, including viruses and transposons, have the advantage of inserting their genetic cargo without breakage of the target genome. However, integration occurs in a semi-random fashion, thereby presenting a risk of insertional activation or inactivation of cellular genes. In this project we aim at enhancing genome engineering tools to achieve highly efficient and precise integration of therapeutic transgenes in a DSB-free manner. We will incorporate components of CRISPR/Cas9 into the framework of non-viral, Sleeping Beauty (SB) transposon-mediated gene integration to increase its specificity. We will evaluate the performance of fusion proteins consisting of catalytically inactive Cas9 and the SB transposase with respect to their potency of targeting SB transposition events into multi-copy targets and single-copy chromosomal targets that satisfy the criteria of genomic safe harbors, specified by guide RNAs (gRNAs). We will explore a novel SB transposase mutant that displays a significant gain in specificity, in conjunction with gRNA-mediated targeting to pre-selected genomic sites. Finally, we will engineer the SB transposase to lower its intrinsic integration activity, thereby shifting the balance towards targeted insertions in the presence of CRISPR/Cas9 components. We will engineer fusion proteins composed of DNA repair factors that promote transgene integration through homologous recombination and Cas9-derived nickases that cut only one strand of the target DNA and therefore would alleviate the need for a DSB. Finally, we will evaluate the performance of the most promising experimental system in therapeutically relevant human T lymphocytes. Technologies for efficient, site-directed transgene integration into safe regions in the human genome would significantly contribute to an overall improvement of the safety profile of gene transfer in human applications.