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

Schmidt, Jenna Kropp; Reynolds, Matthew R.; Golos, Thaddeus G.; Slukvin, Igor I.

doi:10.1186/s12977-022-00604-5

Cited by 6 publications

(5 citation statements)

References 97 publications

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“…The incidence of chromothripsis was not assessed in the present study given the limitations in interpreting WGA artifacts in light of an incomplete reference genome, in addition to the difficulty in discerning whether chromosome loss was due to CRISPR-Cas9 targeting or naturally-occurring cellular fragmentation events. As CRISPR-Cas9 editing errors can result in chromosomal disruption and/or elimination ( Leibowitz et al, 2021 ; Papathanasiou et al, 2021 ) it is plausible that the embryo development may be negatively impacted and could explain the poor embryo transfer rate observed in our previous study ( Schmidt et al, 2020 ) and in general the low live birth rates of CRISPR-Cas9 targeted NHPs ( Schmidt et al, 2022a ; Schmidt et al, 2022b ).…”

Section: Discussionmentioning

confidence: 89%

“…Transplantation of hematopoietic stem cells (HSCs) containing the CCR5 -Δ32 mutation to human HIV patients led to the cure of HIV infection in some but not all cases ( Hütter et al, 2009 ; Allers et al, 2011 ; Henrich et al, 2014 ; Cummins et al, 2017 ). Generating NHPs with CCR5 -deletions would aid in determining the mechanisms of HIV elimination following transplantation of allogeneic HSCs with CCR5 mutations and the development of clinical protocols for reproducible HIV cure ( Schmidt et al, 2022b ).…”

Section: Introductionmentioning

confidence: 99%

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Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits

et al. 2023

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Introduction: Genome editing by CRISPR-Cas9 approaches offers promise for introducing or correcting disease-associated mutations for research and clinical applications. Nonhuman primates are physiologically closer to humans than other laboratory animal models, providing ideal candidates for introducing human disease-associated mutations to develop models of human disease. The incidence of large chromosomal anomalies in CRISPR-Cas9-edited human embryos and cells warrants comprehensive genotypic investigation of editing outcomes in primate embryos. Our objective was to evaluate on- and off-target editing outcomes in CCR5 CRISPR-Cas9-targeted Mauritian cynomolgus macaque embryos.Methods: DNA isolated from individual blastomeres of two embryos, along with paternal and maternal DNA, was subjected to whole genome sequencing (WGS) analysis.Results: Large deletions were identified in macaque blastomeres at the on-target site that were not previously detected using PCR-based methods. De novo mutations were also identified at predicted CRISPR-Cas9 off-target sites.Discussion: This is the first report of WGS analysis of CRISPR-Cas9-targeted nonhuman primate embryonic cells, in which a high editing efficiency was coupled with the incidence of editing errors in cells from two embryos. These data demonstrate that comprehensive sequencing-based methods are warranted for evaluating editing outcomes in primate embryos, as well as any resultant offspring to ensure that the observed phenotype is due to the targeted edit and not due to unidentified off-target mutations.

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Section: Discussionmentioning

confidence: 89%

Section: Introductionmentioning

confidence: 99%

Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits

et al. 2023

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“…The incorporation of morphokinetic analysis enables the quantification of differences in timing of developmental events to assess the impact of experimental manipulation such as genome editing, gene knockdown, or experimental infection during this developmental time frame. Recent research has focused on developing nonhuman primate genetic models of human disease through genome editing of embryos [31, 32], yet the impact that CRISPR-Cas9 on- and off-target editing has on early embryo development remains uncertain. Time-lapse imaging could be used to deselect embryos for transfer that have had an atypical cleavage pattern, although no differences were observed in this study between blastocysts with normal or abnormal cleavage patterns in their ability to attach to an extracellular matrix and grow.…”

Section: Discussionmentioning

confidence: 99%

Atypical initial cleavage patterns minimally impact rhesus macaque in vitro embryo morphokinetics and embryo outgrowth development

Schmidt,

Block,

Jones

et al. 2023

Biology of Reproduction

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Embryo morphokinetic analysis through time-lapse embryo imaging is envisioned as a method to improve selection of developmentally competent embryos. Morphokinetic analysis could be utilized to evaluate the effects of experimental manipulation on pre-implantation embryo development. The objectives of this study were to establish a normative morphokinetic database for in vitro fertilized rhesus macaque embryos and to assess the impact of atypical initial cleavage patterns on subsequent embryo development and formation of embryo outgrowths. The cleavage pattern and the timing of embryo developmental events were annotated retrospectively for unmanipulated in vitro fertilized rhesus macaque blastocysts produced over four breeding seasons. Approximately 50% of the blastocysts analyzed had an abnormal early cleavage event. The time to the initiation of embryo compaction and the time to completion of hatching was significantly delayed in blastocysts with an abnormal early cleavage event compared to blastocysts that had cleaved normally. Embryo hatching, attachment to an extracellular matrix, and growth during the implantation stage in vitro was not impacted by the initial cleavage pattern. These data establish normative morphokinetic parameters for in vitro fertilized rhesus macaque embryos and suggest that cleavage anomalies may not impact embryo implantation rates following embryo transfer.

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“…In addition, nanoparticle‐mediated CRISPR/Cas9‐encoding mRNA delivery is the most frequently used gene‐editing technology (Figure 3). Several studies have shown that in non‐human primate models, CRISPR/Cas9‐mediated CCR5 disruption is used to give resistance to HIV/SIV infection 144 . Therefore, mRNA‐based genome editing will be a promising modality for gene therapy, and several of them are undergoing clinical studies.…”

Section: Mrna For Therapeutic Applicationsmentioning

confidence: 99%

“…Several studies have shown that in non‐human primate models, CRISPR/Cas9‐mediated CCR5 disruption is used to give resistance to HIV/SIV infection. 144 Therefore, mRNA‐based genome editing will be a promising modality for gene therapy, and several of them are undergoing clinical studies.…”

Section: Mrna For Therapeutic Applicationsmentioning

confidence: 99%

Nanomaterials for mRNA‐based therapeutics: Challenges and opportunities

Liu

Yang

et al. 2023

Bioengineering & Transla Med

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Messenger RNA (mRNA) holds great potential in developing immunotherapy, protein replacement, and genome editing. In general, mRNA does not have the risk of being incorporated into the host genome and does not need to enter the nucleus for transfection, and it can be expressed even in nondividing cells. Therefore, mRNA‐based therapeutics provide a promising strategy for clinical treatment. However, the efficient and safe delivery of mRNA remains a crucial constraint for the clinical application of mRNA therapeutics. Although the stability and tolerability of mRNA can be enhanced by directly retouching the mRNA structure, there is still an urgent need to improve the delivery of mRNA. Recently, significant progress has been made in nanobiotechnology, providing tools for developing mRNA nanocarriers. Nano‐drug delivery system is directly used for loading, protecting, and releasing mRNA in the biological microenvironment and can be used to stimulate the translation of mRNA to develop effective intervention strategies. In the present review, we summarized the concept of emerging nanomaterials for mRNA delivery and the latest progress in enhancing the function of mRNA, primarily focusing on the role of exosomes in mRNA delivery. Moreover, we outlined its clinical applications so far. Finally, the key obstacles of mRNA nanocarriers are emphasized, and promising strategies to overcome these obstacles are proposed. Collectively, nano‐design materials exert functions for specific mRNA applications, provide new perception for next‐generation nanomaterials, and thus revolution of mRNA technology.

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

Cited by 6 publications

References 97 publications

Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits

Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits

Atypical initial cleavage patterns minimally impact rhesus macaque in vitro embryo morphokinetics and embryo outgrowth development

Nanomaterials for mRNA‐based therapeutics: Challenges and opportunities

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