Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

Mosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector 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 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.

Efficient Targeted Transgenesis of Large Donor DNA Into Multiple Mouse Genetic Backgrounds Using Bacteriophage Bxb1 Integrase

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene “landing pad” composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies >40% with up to ~43kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development combined with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.

Murine SEC24D can substitute functionally for SEC24C during embryonic development

The COPII component SEC24 mediates the recruitment of transmembrane cargos or cargo adaptors into newly forming COPII vesicles on the ER membrane. Mammalian genomes encode four Sec24 paralogs (Sec24a-d), with two subfamilies based on sequence homology (SEC24A/B and C/D), though little is known about their comparative functions and cargo-specificities. Complete deficiency for Sec24d results in very early embryonic lethality in mice (before the 8 cell stage), with later embryonic lethality (E7.5) observed in Sec24c null mice. To test the potential overlap in function between SEC24C/D, we employed dual recombinase mediated cassette exchange to generate a Sec24cc-d allele, in which the C-terminal 90% of SEC24C has been replaced by SEC24D coding sequence. In contrast to the embryonic lethality at E7.5 of SEC24C-deficiency, Sec24cc-d/c-d pups survive to term, though dying shortly after birth. Sec24cc-d/c-d pups are smaller in size, but exhibit no other obvious developmental abnormality by pathologic evaluation. These results suggest that tissue-specific and/or stage-specific expression of the Sec24c/d genes rather than differences in cargo export function explain the early embryonic requirements for SEC24C and SEC24D.

Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase

Efficient, targeted integration of large DNA constructs represent a significant hurdle in genetic engineering for the development of mouse models of human disease and synthetic biology research. To address this, we developed a system for efficient and precise, targeted single-copy integration of large transgenes directly into the zygote using multiple mouse genetic backgrounds. Conventional approaches, such as random transgenesis, CRISPR/Cas9-mediated homology-directed repair (HDR), lentivirus-based insertion, or DNA transposases all have significant limitations. Our strategy uses in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) to efficiently generate precise single-copy integrations of transgenes. This is achieved using a transgene landing pad composed of dual heterologous Bxb1 attachment (att) sites in cis, pre-positioned in the Gt(ROSA)26Sor safe harbor locus. Successful RMCE is achieved in att carrier zygotes using donor DNA carrying cognate attachment sites flanking the desired donor transgene microinjected along with Bxb1-integrase mRNA. This approach routinely achieves perfect vector-free integration of donor constructs at efficiencies as high as 43% and has generated transgenic animals containing inserts up to ~43kb. Furthermore, when coupled with a nanopore-based Cas9-targeted sequencing (nCATS) approach, complete verification of the precise insertion sequence can be achieved. As a proof-of-concept we describe the creation and characterization of C57BL/6J and NSG Krt18-ACE2 transgenic mouse models for SARS-CoV2 research with verified heterozygous N1 animals available for experimental use in ~4 months. In addition, we created a diverse series of mouse backgrounds carrying a single att site version of the landing pad allele in C57BL/6J, NSG, B6(Cg)-Tyrc-2J/J, FVB/NJ, PWK/PhJ, 129S1/SvImJ, A/J, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, and DBA/2J for rapid transgene insertion. Combined, this system enables predictable, rapid creation of precisely targeted transgenic animals across multiple genetic backgrounds, simplifying characterization, speeding expansion and use.

Identification of the safe harbor locus, AAVS1, from porcine genome and site-specific integration of recombinase-mediated cassette exchange system in porcine fibroblasts using CRISPR/Cas9

Gene integration at site-specific loci , such as safe harbor regions for s table expression via transgenesis , is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor site for human and mouse studies. In the present study, we found an AAVS1-like sequence in the porcine genome using the UCSC Genome Browser and designed TALEN and CRISPR/Cas9 to target AAVS1. The efficiency of CRISPR/Cas9 for targeting the AAVS1 locus in porcine cells was superior to that of TALEN. An AAVS1-targeting donor vector containing GFP was designed and cloned. We added a loxP-lox2272 cassette sequence to the donor vector for further exchange of various transgenes in the AAVS1-targeted cell line. The donor vector and CRISPR/Cas9 components targeting AAVS1 were transfected into a porcine fibroblast cell line. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in at the AAVS1 locus was confirmed by PCR analysis. To induce recombinase-mediated cassette exchange (RMCE), another donor vector containing the loxP-lox2272 cassette and inducible (Tet-on) Cre recombinase was cloned. The Cre-donor vector was transfected into the AAVS1-targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR analysis. In conclusion, gene targeting at the AAVS1 locus and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.

Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans

Therapeutic antibodies are decorated with complex-type N-glycans that significantly affect their biodistribution and bioactivity. The N-glycan structures on antibodies are incompletely processed in wild-type CHO cells due to their limited glycosylation capacity. To improve N-glycan processing, glycosyltransferase genes have been traditionally overexpressed in CHO cells to engineer the cellular N-glycosylation pathway by using random integration, which is often associated with large clonal variations in gene expression levels. In order to minimize the clonal variations, we used recombinase-mediated-cassette-exchange (RMCE) technology to overexpress a panel of 42 human glycosyltransferase genes to screen their impact on antibody N-linked glycosylation. The bottlenecks in the N-glycosylation pathway were identified and then released by overexpressing single or multiple critical genes. Overexpressing B4GalT1 gene alone in the CHO cells produced antibodies with more than 80% galactosylated bi-antennary N-glycans. Combinatorial overexpression of B4GalT1 and ST6Gal1 produced antibodies containing more than 70% sialylated bi-antennary N-glycans. In addition, antibodies with various tri-antennary N-glycans were obtained for the first time by overexpressing MGAT5 alone or in combination with B4GalT1 and ST6Gal1. The various N-glycan structures and the method for producing them in this work provide opportunities to study the glycan structure-and-function and develop novel recombinant antibodies for addressing different therapeutic applications.

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.