Delivery of Cas9/sgRNA Ribonucleoprotein Complexes via Hydroxystearyl Oligoamino Amides

Abstract

The programmable endonuclease activity and simple usage of CRISPR/Cas9 have revolutionized the field of genome editing. The binding of single guide RNA (sgRNA) by the Cas9 protein results in the formation of negatively charged ribonucleoprotein (RNP) complexes. The presence of this functional complex inside cells is imperative for the intended specific genome modifications. The direct intracellular delivery of Cas9/sgRNA RNP complexes is of great advantage. In this work, a compound library of sequence-defined oligo­(ethylenamino) amides containing structural motifs for stable nanoparticle formation, cellular uptake, and endosomal release was used for the screening and development of suitable Cas9 RNP delivery vehicles. Lipid-containing oligoaminoamides (lipo-OAAs) were identified as the most efficient carriers for intracellular Cas9/sgRNA delivery and gene disruption. Fluorescence correlation spectroscopy measurements indicated that the lipo-OAAs only interact with sgRNA-loaded Cas9 protein, which suggests exclusive ionic interaction with the negatively charged RNPs. The type of contained fatty acid turned out to have a critical impact on the knock out efficiency: the presence of one hydroxy group in the fatty acid dramatically changes the properties and performance of the resulting Cas9/sgRNA lipo-OAA complexes. The lipo-OAA-containing hydroxy-stearic acid (OHSteA) was superior to the analogues with saturated or unsaturated fatty acids without hydroxylation; it formed smaller and more defined nanoparticles with Cas9/sgRNA and improved the cellular uptake and endosomal release, which altogether resulted in an increased nuclear association and the highest gene knock out levels. The efficient and adaptable delivery platform has high potential for the future development of therapeutics based on precise genome modifications.

Nanotechnologies for Intracellular Protein Delivery: Recent Progress in Inorganic and Organic Nanocarriers

“…Recently, a library of new lipid‐containing oligoaminoamides was synthesized to allow cas9‐RNP complexation by electrostatic interactions. [ 112 ] Interestingly, the presence of one hydroxyl group in the fatty acid was evidenced as crucial to form stable well‐defined nanoparticles and increase cellular uptake, endosomal escape, as well as nuclear delivery. A GFP reporter gene was used as a read‐out to assess the knock out efficiency of cas9RNP targeting this gene.…”

Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing

“…A library of sequence-defined oligo(ethylenamino) amides (OAAs) containing structural motifs were reported for Cas9 RNP delivery 175 . Among the designed OAAs, lipid-containing OAAs (lipo-OAAs) possess superior efficiency in Cas9 RNP delivery.…”

“…Illustration of T-shape lipo-OAAs with different fatty acids, in which lipo-OAA-containing OHSteA was superior to others in higher genome editing efficiency. Reduced with permission form 175 . Copyright 2020, American Chemical Society.…”

Non-Viral Targeted Nucleic Acid Delivery: Apply Sequences for Optimization

“…Kuhn et al [ 134 ] screened an analogous T-shape lipo-oligomer library containing saturated stearic acid, mono-/bis-unsaturated and amide functionalized or hydroxylated stearic acid lipid (OHSteA) moieties for Cas9/sgRNA RNPs delivery. Tyrosine tripeptides and terminal cysteines were adopted due to their beneficial effect in polyplex stabilization found in the context of pDNA and siRNA delivery.…”