Experimental Study to Increase the Viability Rate of Cas9 Knock in Chicken Embryos and Examination of Differentially Expressed Genes in Early Chicken Embryos
BACKGROUND: CRISPR-Cas9 can be used to silence undesirable traits in chickens. However, due to the challenges associated with gene editing in chickens, including the need to window the egg shell, chicken gene editing requires lengthy, precise protocols to produce gene edited chickens. Due to the challenges involved, several factors were addressed here to increase viability of SaCas9 edited chicken embryos. It was also important to examine gene expression patterns of the genes that regulate angiogenesis and vascularization and osmosis transport, namely HIF1α and AQP8, during chicken embryonic development. METHODS: Viability was assessed on fertilized chicken embryos using disinfection techniques in combination with a 1 cm or 0.5 cm window, either microinjected with a SaCas9 lentiviral vector, or uninjected. RNA sequencing and bioinformatics were used to examine HIF1α and AQP8 gene expression patterns in 24- and 48-hour old shell-less and intact chicken embryos. RESULTS: Intact controls had a 90% viability rate to Hamburger Hamilton Stage 46. The 1 cm windowed embryos had a 15% viability rate to Stage 36. The 0.5 cm windowed embryos had a 5% viability rate to Stage 46. The 0.5 cm windowed embryos microinjected with SaCas9 had a 30% viability rate to Stage 43. The most efficient protocol combined disinfection techniques with a 0.5 cm window. RNA sequencing and bioinformatics showed that HIF1 α was upregulated in 24-hour old intact embryos with an RPKM of 27.45. In 48-hour old intact and 24- and 48-hour old shell-less embryos, RPKM was 17.46, 20.76 and 15.91, respectively. The RT-qPCR expression pattern of HIF1α was similar to the bioinformatics analysis in 24-hour old intact and shell-less embryos. AQP8 was down-regulated in intact 24-hour old embryos, at 1.46 RPKM. In 24-hour old shell-less and 48-hour old intact and shell-less embryos, AQP8 was upregulated at 7.15, 43.97 and 130.02 RPKM, respectively. CONCLUSIONS: More work needs to be done to produce SaCas9 knock in chickens. Abnormal gene expression patterns of HIF1α and AQP8 may be responsible for lethality in early developing chicken embryos. Since normoxia/hyperoxia and dehydration occur simultaneously within the same embryo, those processes could create a significantly damaging effect to developing chicken embryos.
Mary Shannon Byers,
"Experimental Study to Increase the Viability Rate of Cas9 Knock in Chicken Embryos and Examination of Differentially Expressed Genes in Early Chicken Embryos"
ETD Collection for Tennessee State University.