Near-infrared optogenetic engineering of photothermal nanoCRISPR for programmable genome editing

Abstract

We herein report an optogenetically activatable CRISPR-Cas9 nanosystem for programmable genome editing in the second near-infrared (NIR-II) optical window. The nanosystem, termed nanoCRISPR, is composed of a cationic polymer-coated Au nanorod (APC) and Cas9 plasmid driven by a heat-inducible promoter. The APC not only serves as a carrier for intracellular plasmid delivery but also can harvest external NIR-II photonic energy and convert it into local heat to induce the gene expression of the Cas9 endonuclease. Due to high transfection activity, the APC shows strong ability to induce a significant level of disruption in different genomic loci upon optogenetic activation. Moreover, the precise control of genome-editing activity can be simply programmed by finely tuning exposure time and irradiation time in vitro and in vivo and also enables editing at multiple time points, thus proving the sensitivity and inducibility of such an editing modality. The NIR-II optical feature of nanoCRISPR enables therapeutic genome editing at deep tissue, by which treatment of deep tumor and rescue of fulminant hepatic failure are demonstrated as proof-of-concept therapeutic examples. Importantly, this modality of optogenetic genome editing can significantly minimize the off-target effect of CRISPR-Cas9 in most potential off-target sites. The optogenetically activatable CRISPR-Cas9 nanosystem we have developed offers a useful tool to expand the current applications of CRISPR-Cas9, and also defines a programmable genome-editing strategy toward high precision and spatial specificity.

Upconversion optogenetic micro-nanosystem optically controls the secretion of light-responsive bacteria for systemic immunity regulation

“…The key to its operation lies in the efficiency of optical signal response and the gene expression effect of downstream destination. Obviously, the first link is the construction of lightsensitive engineered plasmid 21,22 . It converts light signals such as electromagnetic waves, into intracellular signals that regulate the expression of the target product.…”

Engineering a far-red light–activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors

“…We first delivered the minicircle iterations of the FAST system alongside a PLK1 -targeting sgRNA minicircle vector when the tumors had reached 80 to 100 mm 3 ; note that we also injected transfection reagent, a cationic polymer-coated nanoparticle (APC), ( 41 ) to facilitate the transfection of tumor cells in situ. Subsequently, FRL illumination was delivered to the xenograft-bearing mice via LED for 4 hours each day for 7 days ( Fig.…”

“…The in vivo DNA delivery reagent APC is a cationic polymer-coated nanoparticle composed of biocompatible polystyrene sulfonate and β-cyclodextrin–PEI ( M w , 25 kDa) and prepared, as previously reported ( 41 ). First, the seed solution was prepared by adding freshly prepared 600 μl of NaBH 4 (10 mM) into 5-ml mixture of HAuCl 4 ·3H 2 O (0.5 mM) and cetyltrimethylammonium bromide (CTAB; 0.1 M) and incubated at 30°C for 30 min.…”

CRISPR/Cas9: A powerful genome editing technique for the treatment of cancer cells with present challenges and future directions

“…The emerging evidence processed that the non-viral delivery method has superiority than viral-vectors and plasmid DNA methods due to high packing ability, fewer adverse-effects, high accuracy, and spatiotemporal specificity. The different types of material, including graphene oxide, liposome, metal framework, gold nanoparticles, and cationic polymers, have shown promising results in the delivery of CRISPR/Cas9 components [ 96 , 97 ]. Recently, the cationic polymers (see glossary) have received considerable attention due to the effective delivery method during the treatment of cancer cells by the CRISPR/Cas9 tool.…”