CRISPR/Cas9 genome editing to create nonhuman primate models for studying stem cell therapies for HIV infection

Abstract

Nonhuman primates (NHPs) are well-established basic and translational research models for human immunodeficiency virus (HIV) infections and pathophysiology, hematopoietic stem cell (HSC) transplantation, and assisted reproductive technologies. Recent advances in CRISPR/Cas9 gene editing technologies present opportunities to refine NHP HIV models for investigating genetic factors that affect HIV replication and designing cellular therapies that exploit genetic barriers to HIV infections, including engineering mutations into CCR5 and conferring resistance to HIV/simian immunodeficiency virus (SIV) infections. In this report, we provide an overview of recent advances and challenges in gene editing NHP embryos and discuss the value of genetically engineered animal models for developing novel stem cell-based therapies for curing HIV.

Keywords: CCR5; CRISPR/Cas9; HIV; HSC transplantation; Nonhuman primates; Pluripotent stem cells; SIV.

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.

Fig. 2

Potential on- and off-target editing outcomes. CRISPR/Cas9 editing can result in both desired on-target editing events and the potential for introducing unexpected gene modifications. Editing errors that may be incurred include off-target edits, whole or segmental chromosome losses and translocations, large-scale insertions and/or deletions (INDELS), and loss of heterozygosity due to a loss of one parental allele and homologous recombination of the retained allele. Editing anomalies can occur at the on-target site as well as at an off-target site(s) that shares homology to the gRNA sequence