Dissection of Splicing Regulation at an Endogenous Locus by Zinc-Finger Nuclease-Mediated Gene Editing

Sequences governing RNA splicing are difficult to study in situ due to the great difficulty of traditional targeted mutagenesis. Zinc-finger nuclease (ZFN) technology allows for the rapid and efficient introduction of site-specific mutations into mammalian chromosomes. Using a ZFN pair along with a donor plasmid to manipulate the outcomes of DNA repair, we introduced several discrete, targeted mutations into the fourth intron of the endogenous BAX gene in Chinese hamster ovary cells. Putative lariat branch points, the polypyrimidine tract, and the splice acceptor site were targeted. We recovered numerous otherwise isogenic clones carrying the intended mutations and analyzed the effect of each on BAX pre-mRNA splicing. Mutation of one of three possible branch points, the polypyrimidine tract, and the splice acceptor site all caused exclusion of exon five from BAX mRNA. Interestingly, these exon-skipping mutations allowed usage of cryptic splice acceptor sites within intron four. These data demonstrate that ZFN-mediated gene editing is a highly effective tool for dissection of pre-mRNA splicing regulatory sequences in their endogenous context

Targeting the absence: Homozygous DNA deletions as immutable signposts for cancer therapy

Many cancers harbor homozygous DNA deletions (HDs). In contrast to other attributes of cancer cells, their HDs are immutable features that cannot change during tumor progression or therapy. I describe an approach, termed deletion-specific targeting (DST), that employs HDs (not their effects on RNA/protein circuits, but deletions themselves) as the targets of cancer therapy. The DST strategy brings together both existing and new methodologies, including the ubiquitin fusion technique, the split-ubiquitin assay, zinc-finger DNA-recognizing proteins and split restriction nucleases. The DST strategy also employs a feedback mechanism that receives input from a circuit operating as a Boolean OR gate and involves the activation of split nucleases, which destroy DST vector in normal (nontarget) cells. The logic of DST makes possible an incremental and essentially unlimited increase in the selectivity of therapy. If DST strategy can be implemented in a clinical setting, it may prove to be curative and substantially free of side effects

Mammalian interspecies substitution of immune modulatory alleles by genome editing

We describe a fundamentally novel feat of animal genetic engineering: the precise and efficient substitution of an agronomic haplotype into a domesticated species. Zinc finger nuclease in-embryo editing of the RELA locus generated live born domestic pigs with the warthog RELA orthologue, associated with resilience to African Swine Fever. The ability to efficiently achieve interspecies allele introgression in one generation opens unprecedented opportunities for agriculture and basic research

Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification

Non-invasive epigenome editing is a promising strategy for engineering gene expression programs, yet potency, specificity, and persistence remain challenging. Here we show that effective epigenome editing is gated at single-base precision via 'keyhole' sites in endogenous regulatory DNA. Synthetic repressors targeting promoter keyholes can ablate gene expression in up to 99% of primary cells with single-gene specificity and can seamlessly repress multiple genes in combination. Transient exposure of primary T cells to keyhole repressors confers mitotically heritable silencing that persists to the limit of primary cultures in vitro and for at least 4 weeks in vivo, enabling manufacturing of cell products with enhanced therapeutic efficacy. DNA recognition and effector domains can be encoded as separate proteins that reassemble at keyhole sites and function with the same efficiency as single chain effectors, enabling gated control and rapid screening for novel functional domains that modulate endogenous gene expression patterns. Our results provide a powerful and exponentially flexible system for programming gene expression and therapeutic cell products

1003. Targeted Site-Specific Integration in Human Cells Using Designed Zinc Finger Nucleases

We have shown previously that human genome editing can be performed at high efficiency using designed zinc finger nucleases (ZFNs). ZFNs can be engineered to generate a double-strand break at a specific chromosomal location both in transformed and primary human cells. These breaks are repaired by the cell's own DNA repair machinery, and when a homologous extrachromosomal donor molecule is provided, a homology-directed repair process leads to highly efficient transfer of single-base-pair changes into the chromosome (Urnov et al. Nature 435: 646). ZFNs can be engineered to target virtually any chromosomal location (Pabo et al., Ann. Rev. Biochem. 70: 313), and function robustly in human hematopoietic stem cells, hence such localized gene modification may potentially be useful in the correction and treatment of certain monogenic diseases. Here we show that ZFNs can also be used for the preciseinsertion of a novel sequence into a pre-determined location in the human genome. Remarkably, this novel sequence can constitute a short patch sequence or several kilobases of one or more transgenes. We have observed ZFN-driven targeted integration into endogenous chromosomal loci in human cells of entire open reading frames and promoter-transcription units at a frequency of 5% in the absence of any selection. We also show the use of this process to disrupt with high efficiency an endogenous chromosomal locus with a selectable marker ORF. Finally, we describe our work on using ZFN-driven integration to insert a therapeutically-relevant transgene into a |[ldquo]|safe-harbor|[rdquo]| locus in the human genome, potentially avoiding the problem of insertional mutagenesis. Hence, the homology-directed repair process invoked by the ZFNs can be used to carry out high-efficiency targeted integration to potentially improve the safety and efficacy of gene therapy