In vivo cleavage of transgene donors promotes nuclease‐mediated targeted integration

Abstract

Targeted DNA integration is commonly used to eliminate position effects on transgene expression. Integration can be targeted to specific sites in the genome via both homology-based and homology-independent processes. Both pathways start the integration process with a site-specific break in the chromosome, typically from a zinc-finger nuclease (ZFN). We previously described an efficient homology-independent targeted integration technique that captures short (<100 bp) pieces of DNA at chromosomal breaks created by ZFNs. We show here that inclusion of a nuclease target site on the donor plasmid followed by in vivo nuclease cleavage of both the donor and the chromosome results in efficient integration of large, transgene-sized DNA molecules into the chromosomal double-strand break. Successful targeted integration via in vivo donor linearization is demonstrated at five distinct loci in two mammalian cell types, highlighting the generality of the approach. Finally, we show that CHO cells, a cell type recalcitrant to homology-based integration, are proficient at capture of in vivo-linearized transgene donors. Moreover, we demonstrate knockout of the hamster FUT8 gene via the simultaneous ZFN- or TALE nuclease-mediated integration of an antibody cassette. Our results enable efficient targeted transgene addition to cells and organisms that fare poorly with traditional homology-driven approaches.

Obligate Ligation-Gated Recombination (ObLiGaRe): Custom-designed nuclease-mediated targeted integration through nonhomologous end joining

“…In fact no integration of a surrogate reporter plasmid bringing a ZFN site identical to the one in the genome was observed in a recent study (Kim et al 2011). While our manuscript was under revision, Cristea et al (2012) reported that introducing a ZFN site in a donor plasmid could promote its integration into the ZFN targeting site in the genome. Because the same ZFN sites were used in donor plasmids, only a minority of genomic insertions had precise 59 and 39 junctions, presumably due to repetitive digestions by ZFNs (Cristea et al 2012).…”

“…While our manuscript was under revision, Cristea et al (2012) reported that introducing a ZFN site in a donor plasmid could promote its integration into the ZFN targeting site in the genome. Because the same ZFN sites were used in donor plasmids, only a minority of genomic insertions had precise 59 and 39 junctions, presumably due to repetitive digestions by ZFNs (Cristea et al 2012). Interestingly, they found that inefficient donor integration could also happen when the ZFNs used for donor linearization and chromosomal cleavage were not identical.…”

Targeted chromosomal deletions and inversions in zebrafish

“…Thus, it should be possible to capture two different ssODNs simultaneously at different positions on the same chromosome (e.g., for the introduction of two loxP sites flanking a set of exons) depending on the efficiency of this process. Furthermore, combination of the segmental deletions with the application of ssODNs (Chen et al 2011;Bedell et al 2012) or the introduction of nuclease-stimulated ligation of an exogenous donor DNA (Cristea et al 2013;Maresca et al 2013) could facilitate the targeted replacement of a desired genomic segment with a specific sequence variant for a more complete deconvolution of function.…”

Magic wands of CRISPR—lots of choices for gene knock-in

“…Since artificial nuclease systems such as ZFNs produce the sticky ends, these overhangs were used as the tabs for sticking, by simply adding the overhangs to the knock-in cassette or the target site of ZFNs to the donor plasmid (Orlando et al 2010;Cristea et al 2013). This system was subsequently refined as the ObLiGaRe method, which enabled obligate ligation by switching the left and right target sites of ZFNs and TALENs added in the plasmid donor and utilizing the obligate heterodimeric nucleases (Maresca et al 2013).…”