Creating resistance to avian influenza infection through genome editing of the ANP32 gene family

Chickens genetically resistant to avian influenza could prevent future outbreaks. In chickens, influenza A virus (IAV) relies on host protein ANP32A. Here we use CRISPR/Cas9 to generate homozygous gene edited (GE) chickens containing two ANP32A amino acid substitutions that prevent viral polymerase interaction. After IAV challenge, 9/10 edited chickens remain uninfected. Challenge with a higher dose, however, led to breakthrough infections. Breakthrough IAV virus contained IAV polymerase gene mutations that conferred adaptation to the edited chicken ANP32A. Unexpectedly, this virus also replicated in chicken embryos edited to remove the entire ANP32A gene and instead co-opted alternative ANP32 protein family members, chicken ANP32B and ANP32E. Additional genome editing for removal of ANP32B and ANP32E eliminated all viral growth in chicken cells. Our data illustrate a first proof of concept step to generate IAV-resistant chickens and show that multiple genetic modifications will be required to curtail viral escape.

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Life sciences
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. We used the minimum amount of animals allowed for the generation of statistically significant conclusions. We performed a power calculation to determine the group sizes that we required to see a biologically relevant difference in viral shedding (area under the curve) between the WT and GE ANP32a chickens. From our previous work we have information about the average area under the curve of virus shedding using the low pathogenicity H9N2 influenza virus in chickens with a WT ANP32a protein and have a measure of the variability seen (Area under the curve =5.471 log10 plaque forming units (pfu) with a standard deviation of 0.24). From the literature we can see multiple examples of experimental analysis of the infectious dose 50 (ID50) of avian influenza virus for chickens, the median being approximately 103 plaque forming units (1-3). A reduction of viral shedding to this level (the area under the curve estimated as 3.506 log10 pfu) therefore would correspond to reduce the ability of the virus to replicate in the chicken host and also onwards transmission. Therefore we used a t-test power analysis to determine the group size required to give a significance level of 0.05 and a power of 85%. The group size was determine as 8 experimental units per group and therefore if each chicken is an experimental unit 8 chickens as directly inoculated and 8 as in-contact recipients were required. We require 4 additional animals in both directly inoculated and in-contact groups to sacrifice at day 3 post inoculation and exposure to determine viral dissemination profile in the chickens. Mouse anti-!-tubulin antibody (Sigma-Aldrich TS6557) was validated by the manufacturer for western blot detection of chicken !tubulin. IRDye 800CW goat anti-mouse secondary antibody (LI-COR) was validated by LI-COR for detection of mouse immunoglobulins in western blot assays. IRDye 680RD goat anti-rabbit secondary antibody (LI-COR) was validated by LI-COR for detection of rabbit immunoglobulins in western blot assays.
Chicken primordial germ cells (PGCs) were derived from fertile eggs obtained from commercial Hy-line layer flocks bred at the National Avian Research Facility, Midlothian, United Kingdom. PGC cell lines consistently tested negative for Mycoplama. Fibroblast-like cells were derived from these PGCs. ANP32A-ANP32B-double-knockout human EHAP1 cells were supplied by Horizon Discovery. Madin-Darby canine kidney cells (ATCC) were obtained commercially. Human airway epithelial cells were purchased from Epithelix Sarl, Inc.
All cell lines were commonly identified by microscopy. Chicken PGCs, PGC-derived fibroblast-like cells and human EHAP1 cells were validated by Sanger sequencing and western blot analysis of ANP32A.
PGC lines were regulaly tested for mycoplasma. Human airway epithelial cells were tested by the company for mycoplasma.
No commonly misindentified cell lines were used in this study.