In birds, males are the homogametic sex (ZZ) and females the heterogametic sex (ZW). Primary sex determination is thought to depend on a sex chromosome gene dosage mechanism, and the most likely sex determinant is the Z chromosome gene Doublesex and Mab-3–Related Transcription factor 1 (DMRT1). To clarify this issue, we used a CRISPR-Cas9–based monoallelic targeting approach and sterile surrogate hosts to generate birds with targeted mutations in the DMRT1 gene. The resulting chromosomally male (ZZ) chicken with a single functional copy of DMRT1 developed ovaries in place of testes, demonstrating the avian sex-determining mechanism is based on DMRT1 dosage. These ZZ ovaries expressed typical female markers and showed clear evidence of follicular development. However, these ZZ adult birds with an ovary in place of testes were indistinguishable in appearance to wild-type adult males, supporting the concept of cell-autonomous sex identity (CASI) in birds. In experiments where estrogen synthesis was blocked in control ZW embryos, the resulting gonads developed as testes. In contrast, if estrogen synthesis was blocked in ZW embryos that lacked DMRT1, the gonads invariably adopted an ovarian fate. Our analysis shows that DMRT1 is the key sex determination switch in birds and that it is essential for testis development, but that production of estrogen is also a key factor in primary sex determination in chickens, and that this production is linked to DMRT1 expression.

Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that the gene currently designated as avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV relies solely on chicken ANP32A to support its replication in chicken cells. Amino acids 129I and 130N, accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resilient genome edited chickens.

Primordial germ cells (PGCs), the embryonic precursors of the sperm and egg, are used for the introduction of genetic modifications into avian genome. Introduction of small defined sequences using genome editing has not been demonstrated in bird species. Here, we compared oligonucleotide-mediated HDR using wild type SpCas9 (SpCas9-WT) and high fidelity SpCas9-HF1 in PGCs and show that many loci in chicken PGCs can be precise edited using donors containing CRISPR/Cas9-blocking mutations positioned in the protospacer adjacent motif (PAM). However, targeting was more efficient using SpCas9-HF1 when mutations were introduced only into the gRNA target sequence. We subsequently employed an eGFP-to-BFP conversion assay, to directly compare HDR mediated by SpCas9-WT and SpCas9-HF1 and discovered that SpCas9-HF1 increases HDR while reducing INDEL formation. Furthermore, SpCas9-HF1 increases the frequency of single allele editing in comparison to SpCas9-WT. We used SpCas9-HF1 to demonstrate the introduction of monoallelic and biallelic point mutations into the FGF20 gene and generate clonal populations of edited PGCs with defined homozygous and heterozygous genotypes. Our results demonstrate the use of oligonucleotide donors and high fidelity CRISPR/Cas9 variants to perform precise genome editing with high efficiency in PGCs.

The application of gene editing (GE) technology to create precise changes to the genome of bird species will provide new and exciting opportunities for the biomedical, agricultural and biotechnology industries, as well as providing new approaches for producing research models. Recent advances in modifying both the somatic and germ cell lineages in chicken indicate that this species, and conceivably soon other avian species, has joined a growing number of model organisms in the gene editing revolution.

In birds, males are the homogametic sex (ZZ) and females the heterogametic sex (ZW), and primary sex determination is thought to depend on a sex chromosome gene dosage mechanism. Previous studies have suggested that the most likely sex-determinant is the Z chromosome gene DMRT1 (Doublesex and Mab-3 Related Transcription factor 1). To clarify this issue, we used a CRISPR-Cas9 based mono-allelic targeting approach and sterile surrogate hosts to generate birds with targeted mutations in the DMRT1 gene. The resulting chromosomally male (ZZ) chicken with a single functional copy of DMRT1 developed ovaries in place of testes, demonstrating the avian sex determining mechanism is based on DMRT1 dosage. These ZZ ovaries expressed typical female markers and showed clear evidence of follicular development. However, these ZZ adult birds with an ovary in place of testes were indistinguishable in appearance to wild type adult males, supporting the concept of cell-autonomous sex identity (CASI) in birds. In experiments where oestrogen synthesis was blocked in control ZW embryos, the resulting gonads developed as testes. In contrast, if oestrogen synthesis was blocked in ZW embryos that lacked DMRT1, the gonads invariably adopted an ovarian fate. Our analysis shows that DMRT1 is the key sex determination switch in birds and that it is essential for testis development, but that production of oestrogen is also a key factor in primary sex determination in chickens, and that this production is linked to DMRT1 expression.

words) 12Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission 13 and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. 14 Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human 15 ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but 16 do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here 17 that avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does 18 not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV does not 19 replicate in chicken cells that lack ANP32A. Amino acid differences in LRR5 domain accounted for the 20 inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV 21 polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design 22 of influenza virus resistant genome edited chickens. 23 Keywords 24Influenza, polymerase, genome editing, disease, ANP32A, ANP32B, host pathogen interaction 25

Genome editing technology facilitates the creation of specific and precise genetic changes to unravel gene function and rapidly transfer unique alleles between chicken breeds in contrast to lengthy traditional crossbreeding methods for the study of poultry genetics. Innovations in genome sequencing technology have made it possible to map polymorphisms associated with both monogenic and multigenic traits in livestock species. We, and many others, have demonstrated the use of genome editing to introduce specific monogenic traits in chicken through targeting of cultured primordial germ cells. In this chapter, we describe materials and protocols for performing heritable genome editing in the chicken through targeting of in vitro propagated chicken primordial germ cells. Materials2.1 Avian KnockOut DMEM (KO-DMEM): a no-calcium, low osmolarity DMEM for culturing embryonic cells.1. Avian KO-DMEM (Life Technologies 041-96570M) or 2. Alternatively, prepare ~50 ml of avian KO-DMEM replacement as follows:

No abstract