Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats

Abstract

BAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human SIRPA transgene have previously been shown to support improved human cell hematopoiesis. Here, we have generated for the first time, human SIRPA BAC transgenic rats, for which the gene is faithfully expressed, functionally active, and germline transmissible. To do this, human SIRPA BAC was modified with elements to work in coordination with genome engineering technologies-piggyBac, CRISPR/Cas9 or TALEN. Our findings show that piggyBac transposition is a more efficient approach than the classical BAC transgenesis, resulting in complete BAC integration with predictable end sequences, thereby permitting precise assessment of the integration site. Neither CRISPR/Cas9 nor TALEN increased BAC transgenesis. Therefore, an efficient generation of human SIRPA transgenic rats using piggyBac opens opportunities for expansion of humanized transgenic rat models in the future to advance biomedical research and therapeutic applications.

Figure 1. Strategy for converting hSIRPA-BAC DNA into a piggyBac transposon.

(A) Diagram illustrating the strategy used for retrofitting hSIRPA-BAC DNA (RP11-887J4) with piggyBac TIR elements. 5′ TIR (green) and 3′ TIR (orange) elements were sub-cloned into pUC19 vector backbone with spectinomycin resistance gene (purple), and 50 bp homology arm sequences (red) used for replacing the chloramphenicol resistance gene in the BAC vector backbone via recombineering technology. The diagram also indicates that the genomic DNA insert in the RP11-887J4 BAC is 176,233 bps, covering the SIRPA genic region, on chromosome 20 between 1,842,086-2,018,318. (B) The green arrows indicate the primer pairs used to verify hSIRPA-BAC retrofitting after the recombineering process. (C) A schematic diagram describing the transpositioning strategy of hSIRPA-BAC retrofitted with TIR elements mediated by piggyBac transposase. Illustration (i) shows the retrofitted BAC DNA. Illustrations (ii) and (iii) show the process by which the piggyBac transposase proteins bind to the TIR sequences, initiating nicking of the DNA strands, allowing 3′ hydroxyl group at both ends of the transposon to hydrophilic attack the flanking TTAA sequence and freeing the BAC from the spectinomycin resistance gene by forming hairpin structure at the TIR ends. Once the BAC DNA is released from spectinomycin resistance gene, illustration (iv) shows repairing of the linearized BAC DNA by ligating into the complementary TTAA overhangs in the genomic DNA through the mediation of the piggyBac transposase proteins.

Figure 2. Analysis of piggyBac mediated hSIRPA-BAC-TIR transposition.

(A) Genotyping result showing PCR positive pups for a 201 bp regions in the human SIRPA gene. The diagram shows pup numbers 1.6, 4.2, 5.4, 5.5, 5.6, 5.7, 8.1, 9.2 and 9.4 to be positive for PCR. In the upper panel, BAC refers to RP11-887J4 used as a positive control, gDNA refers to human genomic DNA as a positive control, and Negative as a negative control. In the lower panel, 5.7 (Control) refers to genomic DNA from pup number 5.7 used as a positive control. (B and C) Show PCR result of rat zygotes injected with Cas9 mRNA transcript, instead of piggyBac transposase, as a negative control. BAC refers to RP11-887J4 used as a positive control. To determine transposition, splinkerette PCR was performed in order to sequence the region adjacent to the TIR ends. (D) Shows the sequencing result, indicating that six pups (1.6, 5.4, 5.7, 8.1, 9.2 and 9.4) carry transpositioned hSIRPA-BACs. The first column shows the pup identities, the second and third columns show the location of the transposition sites, the fourth column lists the TTAA site of transposition, and sequences ten base up and downstream. The last column indicates whether the transposition occurred in genic or intergenic region.

Figure 3. Integrity of transpositioned hSIRPA-BACs and copy number analysis.

(A) Graphic illustration showing the genomic DNA insert in RP11-887J4. The chromosomal locations of start and end points are indicated. Exon in the SIRPA gene are blocked in bold. Green arrows indicate the primer pairs (10) spanning along entire BAC at approximately 20 kb intervals, used to verify the presence of the full BAC in pups confirmed to have hSIRPA-BAC transpositioned. (B) PCR result showing that six pups (1.6, 5.4, 5.7, 8.1, 9.2 and 9.4) with transpositioned hSIRPA-BACs are confirmed to be positive for all ten primer pairs. Genomic DNAs from pups 2.3, 3.2 and 6.2 were used as negative controls (Fig. 2A). (C) PCR result two pups (4.2 and 5.5) that were found to be positive for the presence of the human SIRPA gene during the genotyping screening, but negative for piggyBac mediated transposition. The results show that pup 4.2 is positive only for primer pair 5, suggesting random insertion of a fragmented BAC DNA. Pup 5.5 is found to be positive for all ten primer pairs, suggesting random insertion of hSIRPA-BAC DNA. (D) Bar graph showing RT-qPCR result indicating the copy number of human SIRPA BAC in piggyBac transpositioned founders (1.6, 5.4, 5.7, 8.1, 9.2, 9.4), random insertion founder (5.5), two negative controls (#1, #2). RT-qPCR results were analyzed relative to the rat GAPDH, for which there are two copies in the genome.

Figure 4. Analysis of 1.6 F1 offspring for BAC transmission and hSIRPA protein expression.

(A) The PCR analysis shows human SIRPA BAC detection in 9 out of 13 offspring from the founder 1.6. Genomic DNA from founder 5.7 was used as a positive control. (B) Animals 1.6 F1.3 and 1.6 F1.4 are F1 offspring animals representative of PCR SIRPA-BAC positive or negative animals, respectively. Peripheral blood mononuclear cells (PBMCs) were first gated by morphology (dot plot SSC-FSC), then CD11b+ cells (mostly expressed by monocytes) were labelled with a rat anti-CD11b antibody (dot plot CD11b-FSC) and finally, cells were stained with combinations of human and rat anti-SIRPα antibodies (left dot-plots) or human anti-SIRPA antibodies and human CD47-Fc (right dot-plots). The large majority of rat monocytes from BAC-SIRPA-TIR⁺ transgenic rats expressed human SIRPα, as detected using both anti-human SIRPα antibodies and human CD47-Fc whereas all monocytes from BAC-SIRPA-TIR⁻ transgenic rats were negative for both labels.