Parallel Cloning by Site Specific Recombination

Kevin Deitz, Ph.D. is a Postdoctoral Research Scientist, Department of Biological Sciences, Columbia University & Visiting Research Collaborator at the Lewis-Sigler Institute for Integrative Genomics, Department of Ecology and Evolutionary Biology, Princeton University where he is dissecting the causative genes and mutations underlying complex traits, and tracing the stepwise processes by which the identified causative alleles arose and spread through an isolated population to generate a fixed morphological phenotype. MORE ABOUT THE AUTHOR

Blanco-Redondo and Langenhan (2018) report recently in G3 valuable improvements in the  φC31 system that enable the simultaneous targeted integration of two transgenes into distinct landing sites using a single source of  φC31 recombinase.

The development of modern genetic engineering techniques that leverage homologous recombination has allowed the site-specific integration of exogenous DNA into insect genomes. Engineering attP phage attachment sites into insect genomes has facilitated the efficient and reproducible transgenesis of loci through the use of φC31 site-specific integration.

PhiC 31 integration involving attP and attB sites. Image credit: https://www.biocat.com/genomics/gene-integration/phic31-integrase-vector-system-one-step-gene-addition-technology

φC31 is a favorite phage integration system for Drosophila because it allows the integration of DNA fragments over 10kb. First discovered in bacteria (Thorpe and Smith, 1988), this system uses φC31 recombinase to facilitate recombination between a donor plasmid harboring an attB site and a genomic attP site. With the establishment of an insect line with endogenous expression of φC31 recombinase and an attP site of interest, researchers can repeatedly target the site to generate allelic variants in a high-throughput fashion.

One draw-back of the φC31 system is that the specificity of the attB/attP pair has generally limited researchers to one integration site per strain. If multiple attP sites are engineered into a genome, each is equally receptive to different attB-harboring donor plasmids. While this problem can be overcome through crossing of strains with unlinked φC31 integrations, the targeting of linked sites (e.g. multiple genes in a tandem array) with the φC31 system has been impractical.

Figure 1

This is a figure from Blanco-Redondo and Langenham (2018) showing the critical modifications in attP and attB sites needed in order to permit parallel integrations using a single source of phiC 31 recombinase

Blanco-Redondo and Langenhan (2018) have overcome this obstacle by manipulating the φC31 system to integrate at a novel landing site. The cleavage of attB and attP sites by φC31 integrase occurs at a crossover dinucleotide where a matching two base pair 5’-TT overhang occurs in both (Fig. 1A). The two base pair overhangs must be reverse complimentary between attB and attP to facilitate recombination, however, their sequences are flexible (Colloms et al. 2014). Blanco-Redondo and Langenhan (2018) have capitalized on this property to engineer attB/attP pairs with crossover dinucleotides comprised of two cytosines, which they call attBCC/PCC, in contrast to the standard attBTT/PTT thymine dinucleotide pairs (Fig. 1B). This simple site modification allowed the authors to modify existing φC31 plasmids to encode orthogonal attBCC/PCC pairs and accomplish targeted transgenesis at two linked loci.

Related image

The Streptomyces phage phiC31 is in the family Siphoviridae and was the original source of the site specific recombination system now widely used for transgene integration. Image Credit: from DOI: 10.1002/9780470015902.a0020000.pub2

The authors targeted the closely-linked, homologous adhesion GPCR genes CG11318 and CG15556 in Drosophila and demonstrated that this technique can be used to efficiently and specifically engineer these loci both sequentially and simultaneously. They further discuss the possible extension of this modified protocol to target not only attB/attP sites carrying TT/AA and CC/GG sequences, but also other asymmetric central overlap dinucleotides (eg. GT/CA, CT/GA, TC/AG, CA/GT).

Thus, the φC31 system can likely be modified to target up to six genomic locations in parallel through the manipulation of the attP landing pads and their attB integration vectors. As our understanding of the complex molecular interactions within gene regulatory networks increases, this expanded capability of the φC31 system will help researchers test how specific combinations of alleles contribute to phenotypic variance among treatments, lines, populations, and species.

Beatriz Blanco-Redondo and Tobias Langenhan (2018) Parallel Genomic Engineering of Two Drosophila Genes Using Orthogonal attB/attP Sites. G3: Genes, Genomes, Genetics September 1, 2018 vol. 8 no. 9 3109-3118; https://doi.org/10.1534/g3.118.200565

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