Fig. 1

Cell and embryo based genome editing approaches. To introduce a mutation associated with human disease (orange nucleotide pair) into monkey iPSCs and embryos, a Cas9-gRNA ribonucleoprotein complex (RNP) with or without a single-strand oligodeoxynucleotide (ssODN) template containing the desired mutation may be delivered via cell electroporation or microinjection into one-cell embryos. The double-stranded DNA break incurred upon Cas9 cleavage may be repaired preferentially by non-homologous end joining (NHEJ) or alternatively, by homology directed repair (HDR). Repair by canonical NHEJ or an alternative NHEJ pathway via microhomology-mediated end joining (MMEJ) are the cellular default repair mechanisms which often introduce insertions or deletions resulting in gene disruption due to frameshift, nonsense or missense mutations. When provided an ssODN template, repair may occur by HDR to create more precise edits by utilizing the provided template to introduce the desired mutation. Of note, despite co-delivery of an ssODN template, repair by NHEJ will predominate. Upon introducing edits, iPSCs can be differentiated into immune cells and subjected to experimental infection to assess phenotypic and functional responses to validate gene editing strategy and targeted embryos may be transferred to a surrogate to produce edited offspring containing the desired mutation. Abbreviations: iPSC induced pluripotent stem cells, WT wild-type.