phiC31 integrase for recombination mediated single copy insertion and genome manipulation in C. elegans

C. elegans benefits from a large set of tools for genome manipulation. Yet, the precise single-copy insertion of very large DNA constructs (>10 kb) and the generation of inversions are still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that serves as a landing pad for integration of transgenes by recombination mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations were obtained for insert sizes up to ∼33.4 kb. Taking advantage of this integration method we establish a dual color fluorescent operon reporter system able to study post-transcriptional regulation of mRNA. Last, we show that large chromosomal segments can be inverted using phiC31 integrase. Thus, the phiC31 integrase system should be a useful addition to the C. elegans toolkit.

Extrachromosomal markers for bacteriophage phiC31 recombinase actions on C. elegans genome.

We report on the experimental implementation of the transgenic phiC31 recombinase C. elegans intron-split system. The three-component plasmid-based phiC31 recombinase system consists of i) two intron-split segments of the C. briggsae-unc-119 gene, and ii) the plasmid that provides phiC31 recombinase activity. Described results constitute the proof-of-concept assay for the implementation of bacteriophage phiC31 integrase in C. elegans.

Generation of Drosophila attP containing cell lines using CRISPR-Cas9

The generation of Drosophila stable cell lines have become invaluable for complementing in vivo experiments and as tools for genetic screens. Recent advances utilizing attP/PhiC31 integrase system has permitted the creation of Drosophila cells in which recombination mediated cassette exchange (RMCE) can be utilized to generate stably integrated transgenic cell lines that contain a single copy of the transgene at the desired locus. Current techniques, besides being laborious and introducing extraneous elements, are limited to a handful of cell lines of embryonic origin. Nonetheless, with well over 100 Drosophila cell lines available, including an ever-increasing number CRISPR/Cas9 modified cell lines, a more universal methodology is needed to generate a stably integrated transgenic line from any one of the available Drosophila melanogaster cell lines. Here we describe a toolkit and procedure that combines CRISPR/Cas9 and the PhiC31 integrase system. We have generated and isolated single cell clones containing an Actin5C::dsRed cassette flanked by attP sites into the genome of Kc167 and S2R+ cell lines that mimic the in vivo attP sites located at 25C6 and 99F8 of the Drosophila genome. Furthermore, we tested the functionality of the attP docking sites utilizing two independent GFP expressing constructs flanked by attB sites that permit RMCE and therefore the insertion of any DNA of interest. Lastly, to demonstrate the universality of our methodology and existing constructs, we have successfully integrated the Actin5C::dsRed cassette flanked by attP sites into two different CNS cell lines, ML-DmBG2-c2 and ML-DmBG3-c2. Overall, the reagents and methodology reported here permit the efficient generation of stable transgenic cassettes with minimal change in the cellular genomes in existing D. melanogaster cell lines.

Generation of Drosophila attP containing cell lines using CRISPR-Cas9

The generation of Drosophila stable cell lines have become invaluable for complementing in vivo experiments and as tools for genetic screens. Recent advances utilizing attP/PhiC31 integrase system has permitted the creation of Drosophila cells in which recombination mediated cassette exchange (RMCE) can be utilized to generate stably integrated transgenic cell lines that contain a single copy of the transgene at the desired locus. Current techniques, besides being laborious and introducing extraneous elements, are limited to a handful of cell lines of embryonic origin. Nonetheless, with well over 100 Drosophila cell lines available, including an ever-increasing number CRISPR/Cas9 modified cell lines, a more universal methodology is needed to generate a stably integrated transgenic line from any one of the available Drosophila melanogaster cell lines. Here we describe a toolkit and procedure that combines CRISPR/Cas9 and the PhiC31 integrase system. We have generated and isolated single cell clones containing an Actin5C::dsRed cassette flanked by attP sites into the genome of Kc167 and S2R+ cell lines that mimic the in vivo attP sites located at 25C6 and 99F8 of the Drosophila genome. Furthermore, we tested the functionality of the attP docking sites utilizing two independent GFP expressing constructs flanked by attB sites that permit RMCE and therefore the insertion of any DNA of interest. Lastly, to demonstrate the universality of our methodology and existing constructs, we have successfully integrated the Actin5C::dsRed cassette flanked by attP sites into two different CNS cell lines, ML-DmBG2-c2 and ML-DmBG3-c2. Overall, the reagents and methodology reported here permit the efficient generation of stable transgenic cassettes with minimal change in the cellular genomes in existing D. melanogaster cell lines.

Efficient Production of Human Hyaluronidase PH20 in HEK293T Cells Using Site Specific Integration

PH20 is hyaluronidase that hydrolyze the glycosidic bond of hyaluronic acid as a major proteoglycan found in extracellular matrices. PH20 is used in the subcutaneous space to increase the dispersion and absorption of co-administered drugs. PH20 is also injected against solid tumors for to better penetration of anticancer agents into the tumor tissue and inhibiting the tumor cell growth. In the present study, we have developed HEK293T stable cell lines secreting His-tagged human recombinant PH20 (rhPH20) in the culture supernatant through the PhiC31 integrase system. The produced rhPH20 was quantify using ELISA and turbidimetric assay and its activity was assessed through treatment of mouse cumulus-oocyte-complex (COCs). Furthermore, we have characterized genomic integration of PH20-containing vectors in one of the isolated clone with the highest levels of rhPH20 production. Our results demonstrated that the secreted rhPH20 in the culture supernatant contained a specific activity of approximately 3.5 IU/ml and it was properly able to denote all mouse oocytes. Consequently, it was revealed that PH20-expressing vectors integrated site-specifically in the PhiC31 pseudo attP sites in the host genome. Taken together, these results confirmed successful application of PhiC31 integrase as a robust approach for production of soluble, active rhPH20 in HEK293T cells.

Scarless engineering of the Drosophila genome near any site-specific integration site

We describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This two-step method combines phiC31 integrase-mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.

phiC31 integrase for recombination mediated single copy insertion and genome manipulation in C. elegans

C. elegans benefits from a large set of tools for genome manipulation. Yet, the insertion of large DNA constructs and the generation of inversions is still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that also serves as a landing pad for integration of transgenes by recombination mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations are relatively easy to obtain for insert sizes up to ~15 kb. Taking advantage of this integration method we establish a dual color fluorescent operon reporter system to study post-transcriptional regulation of mRNA. Last we show that large chromosomal segments can be inverted using phiC31 integrase. Thus the phiC31 integrase system should be a useful addition to the C. elegans toolkit.

Scarless engineering of the Drosophila genome near any site-specific integration site

We describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This 2-step method combines phiC31 integrase mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.