External stimuli-responsive nanoparticles for spatially and temporally controlled delivery of CRISPR-Cas genome editors

Abstract

The CRISPR-Cas9 system is a powerful tool for genome editing, which can potentially lead to new therapies for genetic diseases. To date, various viral and non-viral delivery systems have been developed for the delivery of CRISPR-Cas9 in vivo. However, spatially and temporally controlled genome editing is needed to enhance the specificity in organs/tissues and minimize the off-target effects of editing. In this review, we summarize the state-of-the-art non-viral vectors that exploit external stimuli (i.e., light, magnetic field, and ultrasound) for spatially and temporally controlled genome editing and their in vitro and in vivo applications.

Figure 1 |. Schematic illustration for nanoparticle-enabled in vivo delivery of genome editors, from nanoparticle fabrication to genome editing in the nucleus.

a | Nanoparticle preparation and administration. CRISPR genome editors (e.g., DNA, mRNA, sgRNA, and Cas9 RNP) are first encapsulated in the non-viral nanoparticles, which can then be administrated through either systemic or local injections. b | Extracellular barriers for delivery. The nanoparticles encapsulating genome editors must minimize the recognition and clearance by immune cells and protect payloads from enzymatic degradation by nucleases and proteases. After extravasation from the bloodstream or local administration, the nanoparticle must also diffuse through the extracellular matrix and get internalized by target cells via endocytosis. c | Intracellular barriers for delivery. The endocytosed nanoparticles need to escape from endosomes/lysosomes and then release payloads into the cytoplasm. The released DNA and mRNA have to undergo transcription and/or translation to express Cas9 RNPs, which enter the nuclease for genome editing. d | Genome editing mechanisms. Genome editing happens after Cas9 nuclease binds the target gene sequence and generates a dsDNA break, via NHEJ, MMEJ, or HDR. NHEJ is an error-prone pathway that induces gene deletion or gene insertion. MMEJ generates large deletions and insertions. HDR occurs in the presence of a donor DNA template to allow precise genome editing.

Figure 2 |. Schematic illustrations of photo-responsive CRISPR-Cas9 delivery nanosystems.

a | Cell-penetrating peptide-conjugated gold nanoparticles were complexed with Cas9/sgRNA plasmids, which were then coated with cationic lipids to yield a nanosystem. b | Gold nanorods were coated with PEI and complexed with RNP-expressing plasmid. The plasmid contained a heat-inducible promoter, HSP70, thus the elevated local temperature induced by external light can switch on the expression of Cas9/sgRNA. c | SPPF was constructed by sequentially conjugating alkyl side chains, polyethylene glycol (PEG) chains, and fluorinated PEI to the backbone of the initial semiconducting polymer. d | pSPN polymer backbone was able to generate singlet oxygen under NIR irradiation, whereas PEI brushes were conjugated through a thioketal moiety which can be cleaved by singlet oxygen. e | T-CC-NP had a core-shell structure. The micellar core was formed by NTA-PEG-PCL to encapsulate a photosensitizer Ce6. The shell was constructed by His-tagged Cas9 RNP, which binds to the NTA moiety through nickel coordination, and the Cas9 RNP was then coated with iRGD-PEG-pAsp(DAB). f | The liposome system delivering the Cas9 RNP was constructed by the lipid bilayer incorporated with a clinically used photosensitizer, verteporfin. g | The NaYF₄:Yb/Tm UCNP was coated with a silica shell, where Cas9 RNPs were covalently conjugated via o-nitrobenzyl ester linkages. The UCNPs-Cas9 conjugates were thereafter complexed with PEI. h | Cas9/sgRNA plasmids were complexed with NaYF₄:Yb/Tm UCNPs with a bridging layer of the charge-reversal polymer.

Figure 3 |. A schematic illustration of the magnetic responsive CRISPR-Cas9 delivery nanosystem.

MNP-BVs were formed by the complexation of BVs with MNPs. The MNPs were covalently conjugated with cell-penetrating peptide-PEG to facilitate complexation with BVs via electrostatic interactions.

Figure 4 |. Schematic illustrations of ultrasound-responsive CRISPR-Cas9 delivery nanosystems.

a | Cas9 RNPs are immobilized onto the gold nanowire (i.e., nanomotor) surface through disulfide bonds. b | The Cas9 RNP was first encapsulated in ~100 nm nanoliposomes via a film hydration method. The nanoliposome was then conjugated to the sulfur hexafluoride-filled microbubble via a disulfide linkage, resulting in a microbubble-nanoliposome complex. c | LPHNs with a PLGA core and a lipid coating were used to encapsulate Cas9 RNP-expressing plasmids. LPHNs were then decorated with cRGD and conjugated to octafluoropropane-filled microbubbles through biotin-avidin interactions.