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Abstract CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel waspNasonia vitripennis. However, current methods involving embryonic microinjection of CRISPR reagents are challenging. Delivery of Cas9 ribonucleoprotein into female ovaries is an alternative that has only been explored in a small handful of insects, such as mosquitoes, whiteflies and beetles. Here, we developed a simple protocol for germline gene editing by injecting Cas9 ribonucleoprotein in adultN. vitripennisfemales using either ReMOT control (Receptor‐Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules) as ovary delivery methods. For ReMOT Control we used theDrosophila melanogaster‐derived peptide ‘P2C’ fused to EGFP to visualize the ovary delivery, and fused to Cas9 protein for gene editing of thecinnabargene using saponin as an endosomal escape reagent. For BAPC we optimized the concentrations of protein, sgRNA and the transfection reagent. We demonstrate delivery of protein cargo such as EGFP and Cas9 into developing oocytes via P2C peptide and BAPC. Additionally, somatic and germline gene editing were demonstrated. This approach will greatly facilitate CRISPR‐applied genetic manipulation in this and other rising model organisms.
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In Vivo Editing of Macrophages through Systemic Delivery of CRISPR‐Cas9‐Ribonucleoprotein‐Nanoparticle Nanoassemblies
Abstract Macrophages are key effectors of host defense and metabolism, making them promising targets for transient genetic therapy. Gene editing through the delivery of Cas9‐ribonucleoprotein (RNP) provides multiple advantages over gene delivery–based strategies for introducing CRISPR machinery to the cell. There are, however, significant physiological, cellular, and intracellular barriers to the effective delivery of the Cas9 protein and guide RNA (sgRNA) that have to date, restricted in vivo Cas9 protein–based approaches to local/topical delivery applications. Described herein is a new nanoassembled platform featuring coengineered nanoparticles and Cas9 protein that has been developed to provide efficient Cas9‐sgRNA delivery and concomitant CRISPR editing through systemic tail‐vein injection into mice, achieving >8% gene editing efficiency in macrophages of the liver and spleen.
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- Award ID(s):
- 1808199
- PAR ID:
- 10459783
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Therapeutics
- Volume:
- 2
- Issue:
- 10
- ISSN:
- 2366-3987
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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