ReMOT control delivery technology holds promise for CRISPR-Cas9 activity independent of embryonic injection

Bill Reid, Ph.D. is a postdoctoral researcher in the laboratory of Dr. Alexander Franz in the Department of Veterinary Pathology at the University of Missouri, Columbia where is is studying mosquito/arbovirus interactions and the application of insect genetic technologies to the control or elimination of arbovirus transmission.More About Author

A new report in Nature Communications by Chaverra-Rodriguez et al. details a novel Cas9-delivery approach for insect genetic modification. In their work, Chaverra-Rodriguez et al introduce a technology they term “ReMOT control”, or Receptor-Mediated Ovary Transduction of Cargo.

In their study, Chaverra-Rodriguez et al. identified a 41 aa peptide sequence (termed P2C) of Drosophila yolk protein 1 that allowed for uptake of hemocoelic P2C-fused EGFP in six species of mosquitoes during vitellogenic egg provisioning (Figure 1). Further fusion of P2C to Cas9, (or EGFP-Cas9) demonstrated that sgRNA-programmed Cas9 could also be delivered to the developing eggs during vitellogenesis, providing a novel method of Cas9/sgRNA delivery. The identification of a functional peptide allowing for the endocytic uptake of Cas9 from the hemocoel, however, requires additional effort in order to allow for robust CRISPR-Cas9 activity.

Insect vitell0genin and yolk protein uptake into developing ovaries is a receptor mediated process.

During egg development, yolk protein precursors (YPP), are transported to the egg through receptor-mediated endocytosis, resulting in endosomal YPPs. Since the uptake of the P2C-Cas9 into the egg relies on the same mechanism, P2C-vectored Cas9 faces the same endosomal fate, meaning that the Cas9 will reach its DNA target only if it escapes from the endosome (Figure 2).

Through the testing of various carriers, Chaverra-Rodriguez et al. identified suitable endosomal escape reagents/concentrations that consistently allowed for P2C-Cas9 release from the endosome, resulting in CRISPR-Cas9 editing reaching up to an impressive 11% activity. In addition, ReMOT control delivered P2C-Cas9 resulted in heritable germline edits, which they demonstrated by targeting kynurenine 3-monoxygenase (kmo) using a validated sgRNA from Basu et al. (2015). By targeting a locus downstream of a kmo+/- mutant (with a different deletion locus), their assay allowed them to confirm P2C-Cas9 meditated germline editing, and to further confirm CRISPR-Cas9 editing for both maternal and paternal alleles.

To deliver proteins and nucleic acids to the cytoplasm or nucleus following endocytosis they must escape form the endosome or the endocome will be directed to lysosomal degradation.

While mosquitoes provided an ideal system for testing P2C-Cas9 activity, it may be possible to apply this technology to other insects, offering new promise for insect genome editing in species where egg collection and microinjection is difficult or impractical.

Chaverra-Rodriguez D, Macias VM, Hughes GL, Pujhari S, Suzuki Y, Peterson DR, Kim D, McKeand S, Rasgon JL. Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing. Nat. Commun. 9, 3008 (2018).

Basu S, Aryan A, Overcash JM, Samuel GH, Anderson MAE, Dahlem TJ, Myles KM, Adelman ZN. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti. Proc. Natl. Acad. Sci. 112, 4038–4043 (2015).

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