Stable knock-in of duck RIG-I into DF-1 chicken cells via CRISPR/Cas9 homology directed repair

Abstract

Chickens and ducks differ in their susceptibility to avian influenza virus infection. Ducks are the primordial hosts and reservoir of avian influenza A viruses. Mallard ducks can asymptomatically harbour high-pathogenicity avian influenza (HPAI) and low-pathogenicity avian influenza (LPAI). Chickens, however, succumb to HPAI infection and die within 1-2 days. Ducks possess the innate immune RNA sensor retinoic acid-inducible gene I (RIG-I), whereas chickens do not. The lack of this key receptor is likely a major factor in the pronounced susceptibility of chickens to influenza A viruses. Here we insert the duck RIG-I gene into the chicken genome, using a CRISPR/Cas 9 approach in the chicken fibroblast cell line, DF-1. For a knock-in locus we chose a region on the chicken Z chromosome, homologous to where RIG-I is found in ducks. To achieve the desired knock-in we used a two-plasmid system. Firstly, a commercial cas9 expression plasmid was modified for use in avian cells. A homology directed repair plasmid was constructed to supply a knock-in template, consisting of duck RIG-I under the control of its own promoter and GFP under the control of the hCMV promoter. DF-1 cells were co-transfected with both plasmids, and successful transfectants were sorted via FACS. Sorted cells then underwent a negative selection process utilizing the human HSV thymidine kinase/ganciclovir inducible suicide gene system to kill cells with off-target knock-ins expressing HSV thymidine kinase. Genomic DNA was harvested from successfully selected cells and each resultant cell line was characterized by genomic PCR, rtPCR, and qPCR for the expression of duck RIG-I. All cell lines recovered showed transgenic duck RIG-I and HSV thymidine kinase in genomic DNA suggesting a knock-in had occurred. However, no expression of duck RIG-I or HSV thymidine kinase was observed by rtPCR or qPCR, and expression of GFP was silenced after ~7 days post transfection. We therefore cannot conclude whether the knock-in of duck RIG-I was on target. Despite the lack of transgene expression our work is a proof of the principle that large fragments of foreign DNA comprised of multiple protein coding genes each with a different promoter can be transfected into DF-1 cells. The production of a transgenic chicken cell line expressing duck RIG-I has the potential to be a vital tool in researching innate immunity in birds.