The ideal method for genetic engineering would meld efficiency with precision. Judging from work published in Genome Research, Maresca et al. may have found the sweet spot between these competing goals with a method christened ObLiGaRe (Obligate Ligation-Gated Recombination). The new strategy arose from a chance observation the authors made while employing zinc finger nucleases (ZFNs) for gene targeting in mouse zygotes. They observed that if the donor plasmid contained the same ZFN recognition sequence as the site of the ZFN-induced double strand break, the vector could end up being inserted into the genome. Because this insertion occurs in the absence of sequence homology between the targeted sequence and the donor vector, the authors speculated that it could offer a more convenient and efficient path to gene targeting than the traditional homologous recombination-dependent approach. A prime worry would of course be precision, and Maresca et al. quickly established that the ZFN recognition sites must be designed such that they do not re-form the recognition sequence after genomic insertion. With this condition met, the authors found that co-transfecting an ObLiGaRe donor plasmid containing a selectable marker with a ZFN-expressing plasmid gave clones which, in most cases, had precise end-joining products. Although there was variation in the proportion of aberrant ligations, ObLiGaRe worked in a variety of murine and human cell types; tests with primary nonreplicating cells and mouse and zebrafish embryos are under way. The technique is not limited to ZFNs, as the authors also succeeded in using a TALEN (transcription activator–like effector nuclease) for gene targeting. The article shows successful integration of a 15-kb sequence by ObLiGaRe, allowing one-step insertion of an inducible transgenic expression cassette, a feat that would have required two steps using traditional ZFN-based methods. When tested in parallel with homologous recombination, ObLiGaRe gave a frequency of successful marker gene insertion that was several-fold greater than the usual method. This work shows that non-homologous end joining can generate precisely joined knock-ins under the right conditions, and provides a welcome addition to the armamentarium of gene modification strategies.
Maresca et al. 2012. Obligate Ligation-Gated Recombination (ObLiGaRe): Custom designed nucleases mediated targeted integration through non-homologous end joining. Genome Res. [Epub ahead of print, November 14, 2012; doi:10.1101/gr.145441.112].Catch and release
A seaside vacation may not seem like the best way to make progress at the bench, but it could be the secret behind a new microfluidic strategy for isolating rare cells. Writing in Proceedings of the National Academy of Sciences, Zhao et al. describe how jellyfish tentacles inspired their DNA-based cell capture methodology. Just as dangling tentacles enable Cnidaria to pluck food from the ocean surging around them, long aptamer-containing DNA strands might capture target cells by entangling cell-surface proteins recognized by the nucleic acid sequence. To test their miniature tentacle idea, the authors used rolling circle amplification to prepare long, surface-immobilized DNA molecules containing hundreds of copies of an aptamer to the leukemia marker PTK7. Single-cell force experiments showed the RCA product to cling more tenaciously to PTK7-positive cells than did a singleton aptamer, and to capture cells at a much greater distance from the immobilization surface. In the context of a microfluidic cell, the RCA reaction creates 3D clusters of DNA strands that extend tens of micrometers into the solution—a 1000-fold longer reach than traditional monovalent capture systems. In microfluidic analysis of whole blood spiked with a PTK7-expressing T cell line, cell capture by the RCA-aptamer product was two-fold greater than a surface-immobilized PTK7 antibody at a low flow rate of 60 µL/h. At higher throughput (600 µL/h), the performance of the antibody-based capture method dropped to negligible levels, while the cell capture efficiency of the tentacle-like DNA strands remained a respectable 50%. The purity of the cells captured the RCA-aptamer chip also outperformed the antibody-coated chip by a factor of five. In other tests, the authors show that the majority of captured cells could be liberated by DNase treatment, a much gentler way of releasing isolated cells than available in traditional affinity-capture cell isolation chips. Zhao et al. propose that more sophisticated cell release would also be feasible if restriction enzyme recognition sites were included in the RCA template, and a single chip containing distinct aptamer–restriction site RCA products could even enable staggered release of different target cells by sequential application of restriction enzymes. Time will tell if this bioinspired chip will capture the market, but the results presented by Zhao et al. should snare the attention of anyone seeking to enrich rare cells for diagnostic or characterization purposes.
Zhao et al. 2012. Bioinspired multivalent DNA network for capture and release of cells. Proc Natl Acad Sci U S A. 109(48):19626-19631.