Applications of homologous recombination-mediated genome engineering

We recently demonstrated an improved method for homologous recombination (HR)-mediated genome editing using TALEN (Transcription activator-like effector nuclease) in zebrafish (Shin et al., 2014). In the study, we identified that a total of 3kb of homology in the targeting construct, 1kb of one arm and 2kb of the other arm, is sufficient to induce HR-mediated knock-in. Importantly, our results suggest that a specific targeting construct configuration (a double stranded break in the short homology arm) can enhance the efficiency of HR-mediated knock-in. I believe that the method is highly valuable due to its fundamental advantage, which is the ability to manipulate in vivo genome precisely. In this post, I discuss several potential applications using HR-dependent genome engineering.

Potential applications using HR-mediated genome engineering

1. Tagging fluorescent protein

HR-mediated gene targeting technology allows us to precisely deliver a relatively large DNA fragment encoding a fluorescent protein into a specific locus of the chromosome. For example, HR dependent GFP knock-in alleles will be useful to monitor the spatiotemporal localization of a target protein in vivo. Although there is a potential caveat such as altered protein functions due to tagging, this method allows visualization of tagged proteins in the physiologically relevant condition rather than overexpression context. Tagging a flouorescent protein along with biotinylation tag, such as avitag, will have an additional capacity to study protein-protein interactions. Because in vivo BirA-Avitag system (de Boer et al., 2003) can carry the advantage of mass spectrometry (MS) analysis, the resulting knock-in lines will be useful for tracing the tagged target gene product as well as screening for novel protein binding partners.

2. Mirroring gene expression

As shown in our paper, the expression of a target gene can be monitored by precisely inserted sequences encoding a fluorescent protein that is linked with ‘self-cleaving’ 2A peptides just in front of the a target gene’s stop codon. I believe this strategy will allow tracing of specific cells, which express both a target gene and a fluorescent reporter protein, without functional alterations of the gene products. In addition to this, the physical separation of a target gene product and a fluorescent reporter protein allows for independent protein behaviors, which is important for diversifying methodologies. For example, application of this method with Cre recombinase instead of a fluorescent reporter will result in Cre knock-in lines that can be used to generate tissue specific Cre driver lines for conditional knock-out mutants. Alternatively, specific cell types can be permanently labeled by crossing the ubi:Switch transgenic line (Mosimann et al., 2011) with Cre knock-in lines. Moreover, replacement of the bacterial biotin ligase BirA by a fluorescent reporter will serve as a biotinylation driver for tissue or cell specific gene expression profiling (Housley et al., 2014).

3. Generation of floxed alleles

To create conditional knock-out lines, a gene of interest must be modified by the insertion of two loxP sites to ecise the floxed exon(s) via Cre-mediated recombination. Although the insertion of a loxP site in the target locus can be accomplished by using loxP site containing single-stranded oligonucleotides (ssONs), insertion of two loxP sites can pose a challenge due to low efficiency of ssONs knock-in. Therefore, using a combination of two loxP site containing targeting constructs with TALEN is an useful and efficient strategy to obtain floxed alleles.

I believe that many other applications using HR-mediated genome engineering are already developed or will be invented for innovative research. Hence, our efficient HR-dependent knock-in method will allow many laboratories to generate multi-purpose knock-in lines and serve as a platform for development of more sophisticated genome manipulation methods.

Reference article:

de Boer, E., Rodriguez, P., Bonte, E., Krijgsveld, J., Katsantoni, E., Heck, A., Grosveld, F., & Strouboulis, J. (2003). Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice Proceedings of the National Academy of Sciences, 100 (13), 7480-7485 DOI: 10.1073/pnas.1332608100

Housley, M., Reischauer, S., Dieu, M., Raes, M., Stainier, D., & Vanhollebeke, B. (2014). Translational profiling through biotinylation of tagged ribosomes in zebrafish Development, 141 (20), 3988-3993 DOI: 10.1242/dev.111849

Mosimann, C., Kaufman, C., Li, P., Pugach, E., Tamplin, O., & Zon, L. (2010). Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish Development, 138 (1), 169-177 DOI: 10.1242/dev.059345

Shin, J., Chen, J., & Solnica-Krezel, L. (2014). Efficient homologous recombination-mediated genome engineering in zebrafish using TALE nucleases Development, 141 (19), 3807-3818 DOI: 10.1242/dev.108019

I thank Nanbing Li-Villarreal for comments.

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