Incomplete activation of developmentally required genes Alyref1 and Gabpb1 leads to preimplantation arrest in cloned mouse embryos

Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes, and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Single embryo RNA-seq revealed that Alyref is needed for the formation of inner cell mass. The supplement of Alyref and Gabpb1 by mRNA injection supported efficient preimplantation development of cloned embryos. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.