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Kanu, G.A.;  Parambath, J.B.M.;  Odeh, R.O.A.;  Mohamed, A.A. Gold Nanoparticle-Mediated Gene Editing. Encyclopedia. Available online: (accessed on 13 July 2024).
Kanu GA,  Parambath JBM,  Odeh ROA,  Mohamed AA. Gold Nanoparticle-Mediated Gene Editing. Encyclopedia. Available at: Accessed July 13, 2024.
Kanu, Gayathri A., Javad B. M. Parambath, Raed O. Abu Odeh, Ahmed A. Mohamed. "Gold Nanoparticle-Mediated Gene Editing" Encyclopedia, (accessed July 13, 2024).
Kanu, G.A.,  Parambath, J.B.M.,  Odeh, R.O.A., & Mohamed, A.A. (2022, November 10). Gold Nanoparticle-Mediated Gene Editing. In Encyclopedia.
Kanu, Gayathri A., et al. "Gold Nanoparticle-Mediated Gene Editing." Encyclopedia. Web. 10 November, 2022.
Gold Nanoparticle-Mediated Gene Editing

Gold nanoparticles (AuNPs) have gained increasing attention as novel drug-delivery nanostructures for the treatment of cancers, infections, inflammations, and other diseases and disorders. They are versatile in design, synthesis, modification, and functionalization. This has many advantages in terms of gene editing and gene silencing, and their application in genetic illnesses. 

CRISPR/Cas9 system zinc finger nucleases TALENs gene editing

1. Introduction

Inorganic functional nanomaterials have emerged as reliable and adaptable nano scaffolds for gene delivery [1][2]. The synthesis of numerous primary proteins in microbes and living cells is the focus of cutting-edge recombinant techniques that have replaced outdated methods and the current trends in bio-nanotechnology. For example, “The Human Genome Project” and its developments in molecular genetics in high-throughput techniques have enabled us to decipher the genetic background of numerous diseases and discover novel therapeutic targets [3]. More focus has been placed on nanomedicine, which has enormous future potential to improve nucleic acid-based treatments, such as gene editing, gene silencing, and viral vectors. These have the potential to significantly advance the treatment of cancer and genetic diseases, such as acute immunodeficiency and Parkinsonism [4].
In principle, gene therapy relies on delivering recombinant synthesized nucleic acids to insert, delete, edit, or silence the gene or gene sequences, as opposed to drugs which produce more susceptible phenotypes. As a result, it is possible to restore cell function in monogenic illnesses or give cells new capacities. However, there are many obstacles to overcome to attain this goal, specifically, the nonspecific capture of gene delivery vehicles by the liver reticuloendothelial system and endosomal trapping, which leads to a marked reduction in the delivery efficiency to the target tissues/cells [5]. Oligonucleotides (e.g., antisense oligonucleotides) and polynucleotides (e.g., messenger RNA) have a relatively limited half-life in physiological fluids due to the presence of endo- and exonucleases [6][7], and this is the major challenge in ensuring they reach their target organs/tissues/cells without degradation.
The construction of safe, biocompatible nano-carriers that can conjugate with genetic materials to transfer therapeutic agents to different cells, tissues, or organs is the most difficult engineering problem encountered while constructing an effective delivery system [8]. Although viral-based nanocarriers are highly effective and widely utilized in immunization, researchers have worked tirelessly to avoid utilizing viruses due to their intrinsic immunogenicity, difficult synthetic pathways, and protein pre-modification [8]. Synthetic methods with minimal or low immunogenicity, high biocompatibility, ease of manufacture, a programmed target ability, and the capacity to be injected numerous times [9][10][11] are judiciously viewed as promising alternatives.
Gold nanoparticles (AuNPs) have been analyzed in detail for decades and are the most developed alternative for a selection of medical applications, such as sensing, imaging, catalysis, therapies, diagnostics, medication, and gene delivery [12][13]. AuNPs have unique features that set them apart from their alternatives [14]. Firstly, AuNPs can be coated with cationic molecules to modulate the surface charge of nanostructures and enhance DNA binding via an electrostatic interaction due to their physicochemical properties. Supporting studies have demonstrated the efficiency of AuNPs as DNA carriers [15][16]. Secondly, the excellent photophysical properties of AuNPs are crucial in bio-diagnostic testing. Thirdly, their simple surface chemistry enables them to function as synthetic antibodies with binding interactions that can be accurately adjusted by varying the density of the binding molecules in their shells. Therefore, gold nanotechnology could make biomedicine a successful route for improving drug delivery by enabling targeted, diagnostic, and therapeutic capabilities that can be chemically customized for a particular condition.

2. CRISPR/Cas9 System

Genetic disorders previously believed to be incurable may soon be treated, thanks to the “CRISPR-Cas9” system’s ease of use, versatility, precision, and site specificity. The discovery of clustered regularly interspaced short palindromic repeats, the CRISPR-associated nuclease system (CRISPR/Cas9), and the transition of genome engineering from bacterial to mammalian cells have represented a substantial change. This method of genome engineering has been used not only in different cell lines but also in human primary and stem cells. Moreover, in vivo experiments have used zebrafish and mice to investigate gene functions, cancer models, and gene therapy techniques [17][18][19][20][21][22][23]. Thus, the CRISPR/Cas9 method has the potential to alter how genetic diseases are treated [24][25]. Recently, treatment of the most common genetic diseases, such as cancer, have been focused on CRISPR/Cas9-based genetic approaches. However, there are a few more on the list, such as Duchenne’s muscular dystrophy, cystic fibrosis, Leber congenital amaurosis, thalassemia, sickle cell disease, and Huntington’s disease, which could be cured or treated using similar genetic approaches [26][27].
There are two main components of a well-designed CRISPR-Cas9 system; the first is a single-guide RNA (sgRNA), and the second is Cas9 endonuclease. If necessary, the system can also include a donor template for homologous repair. Together, these two elements create active ribonucleoprotein (RNP) complexes that may pinpoint a precise genomic region where double-strand breaks (DSBs) are created because of editing [28][29][30]. The application targeted delivery system specific to the disease, especially the CRISPR-Cas9 technique, requires an efficient carrier method or molecule for sufficient cellular uptake and protection against the degradation of the protein that finally leads to the on-/off-target effects of a particular disease [31]. Chemical methods or synthetic transfection carriers have the advantage that their properties may be tailored to meet the needs of delivering CRISPR systems in gene correction. Additionally, they help overcome the limitations of biosafety, loading, and packaging capacities. Non-viral vectors, in comparison with viral vectors, possess low immunogenicity, lack endogenous virus recombination, reduce restrictions in delivering larger genetic payloads, and are simpler to manufacture on a large scale. Non-viral vectors may offer enticing prospects for CRISPR-Cas9 delivery. This is the reason why non-viral vectors are considered an alternative for CRISPR-Cas9 gene therapy translational studies [32][33][34].
The shortest co-delivery method for Cas9 protein and sgRNA was developed by Mout et al., representing the first use of materials based on AuNPs in genome editing [35]. The proposed Cas9 protein was used to interact with cationic arginine-AuNPs, sgRNA, and a negatively charged glutamate peptide tag. AuNPs and delivery payloads interacted to create nano assemblies that could quickly bind to cell membranes and release compressed Cas9 protein and sgRNA cargo into the cytoplasm. Up to 90% of target genes were successfully transfected using this method, which also had good gene editing efficiencies, such as adeno-associated virus integration site 1 (AAVS1) (29%) and phosphatase and tensin homolog (PTEN) (up to 30%) in in vitro investigations.
Lee et al. developed a CRISPR–AuNPs vehicle for the direct transfer and quick discharge of Cas9 ribonucleoprotein and donor DNA repair templates for correcting gene alterations in model for studying Duchenne muscular dystrophy (MDX) mice [36]. AuNPs of 15 nm were first conjugated with thiol-modified oligonucleotides, and then crossed with donor DNA and loaded with a Cas9–RNP complex, and finally decked with a cationic polymer, poly{N-[N′-(2-aminoethyl)-2-aminoethyl]aspartamide}, PAsp (DET). PAsp (DET) on the exterior of CRISPR–AuNPs can cause endosomal distraction and enable endosomal escape at the end of cellular endocytosis, releasing it into the cytoplasm and causing a release of the Cas9–RNP complex and donor DNA from the CRISPR–AuNPs. An exterior coating of PAsp (DET) facilitates the endosomal escape. Increased intracellular levels (~10 mM) of glutathione enable the release of the Cas9–RNP complex and donor DNA from CRISPR–AuNPs. This made it possible to restore the functional wild-type sequence in MDX dystrophin-deficient mice, which reduced muscle fibrosis, restored dystrophin protein expression, and restored muscular function without completely altering inflammatory cytokines. CRISPR–AuNPs, used together, present a possible healing method for the active treatment of DMD patients by eliminating mutant genes.
A typical single-gene form of autism spectrum disorder was used in a mouse model of Fragile X Syndrome. Lee et al. used CRISPR–AuNPs as the carrier to transfer RNP to the brain and helped the mouse recover its high repetition of behaviors [37]. Following the intracranial injection of CRISPR–AuNPs targeting the mGluR5 gene in the striatum of wild-type or Fmr1 knockout mice, the local mGluR5 gene levels in the striatum were successfully reduced.
AuNPs have been studied in conjunction with their photothermal properties and the traditional benefits of lipid-based particles [38], in which cationic AuNPs were formed by joining short peptides. This compound accelerates cellular uptake by TAT-peptides of AuNPs. The polo-like kinase 1 (PLK-1)-targeting Cas9–sgRNA plasmid was then coupled with the cationic AuNPs. The cationic lipid mixture of 1,2-dioleoyl-3-trimethylammoniumpropane, DOPE dioleoyl phosphatidylethanolamine, and cholesterol was next placed onto the AuNP–plasmid, and the particles were further enhanced by adding 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) and 2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) to boost permanency. It was confirmed that PLK-1 disruption in melanoma cells was enhanced by light-irradiated AuNPs.
The flexible cationic AuNP platform known as arginine ArgNPs was developed for improved membrane transfer [35][39]. Engineers used Cas9 with a glutamate peptide tag and NLS because glutamic acid generates a negative potential around the Cas9 RNP to connect to the positive ArgNPs. In contrast to endocytosis, it was discovered that Cas9–ArgNPs were internalized by cells via a membrane-fusion-like procedure that required cholesterol. Furthermore, cytosol delivery was related to the chain length of the glutamate peptide used. In HeLa cells, Cas9–ArgNPs targeting the PTEN or AAVS1 genes caused a 30% indel, demonstrating gene editing. Another study showed the flexibility of this delivery method by using Cas9–ArgNPs to produce SIRP knockout RAW264. 7 macrophages to enhance the phagocytosis of cancer cells [40].
SpCas9–AuNCs were localized into cells through endocytosis and subjected to endosomal and lysosomal low pH, which caused the disassociation of the SpCas9–AuNCs as well as the proton sponge, resulting subsequently in the release of the SpCas9 into the cytoplasm [41]. The SpCas9–AuNCs were then used to see if they could edit the E6 gene in the HPV 18 genome in HeLa cells using an E6 sgRNA transfection, and the expression of HPV18 E6 protein was dramatically decreased after treatment with SpCas9–AuNCs.
Mout et al. designed the Cas9 protein to have a negative charge, resulting in a protein that resembled sgRNA electrostatically [35]. In this research, an oligo glutamic acid tag (E-tag) was fused to the N-terminus of a Cas9 protein, Cas9En (n: glutamic acids), and the sgRNAs were assembled with cationic arginine AuNPs (ArgNPs) and delivered directly to the cytoplasm and nucleus.

3. Zinc Finger Nucleases (ZFNs) and TALENs Gene Editing

ZFNs were first discovered in Xenopus oocytes as a component of transcription factor IIIa [42], and they were subsequently identified as a site-specific endonuclease for cutting DNA [43]. ZFNs have site-specific DNA binding properties. They possess a group of Cys2His2 zinc fingers (ZFs) created via the interaction between their ZF-domains and similar DNA regions. ZF proteins are a type of transcription factor that is connected to an endonuclease. Each ZF unit specifically identifies three DNA base pairs and generates base-specific connections interacting with the major groove of the DNA [44][45] through its helix residues.
They can bind with any triplet in its naturally occurring setting, which varies depending on tissue condition, protein context, and DNA sequence. Single zinc fingers, which have amino acids [46][47], are straightforward structures with an unusually high degree of functional flexibility and structural malleability. The effectiveness of HR has been greatly boosted by the introduction of ZFNs to create gene-specific DNA breaks [48]. A FokI type II RE creates the DNA cleaving domain, then dimerized to specifically target its corresponding regions [49]. Two exogenous protein domains are active: the first one is a restriction nuclease called FokI, and the second one is a sequence-specific zinc-finger transcription factor protein (ZFP). FokI, a restriction nuclease enzyme, detects trinucleotide DNA sequences, sequence specific ZFPs, and FokI. The nuclease domain, which is composed of a wide array of C2H2 zinc fingers, interacts with this domain, limiting the DNA-binding specificity of the ZFNs and directing them to their target site. The ZFN gene-repair process is as follows: identification of the full ZFN-binding site within the target GOI (gene of interest); design of a pair of ZFNs; testing of the ZFN pair for activity; identification of a targeting construct to produce the anticipated genomic modifications; and co-transfer of the ZFNs and the targeting vector to produce the cuts and insertion of the therapeutic transgene to the GOI [50]. Cellular targeting moiety and a cytotoxic protein payload, respectively, were genetically coupled to two forms of ZnF that recognize various DNA sequences. Using streptavidin-biotin chemistry, double-stranded DNA with many ZnF-binding sites was created and grafted onto the gold nanoparticles. To create the assembly of nucleoprotein nanoparticles, the ZnF-fused proteins and DNA-functionalized nanoparticles were co-incubated.
Like ZFNs, TALENs are naturally occurring protein nucleases that cause DNA DSBs by fusing a non-specific DNA cleavage domain with a sequence-specific DNA-binding domain. A highly conserved repeat sequence from transcription activator-like effector (TALE), a protein originally discovered in phytopathogenic Xanthomonas bacteria, that spontaneously alters gene transcription in host plant cells, makes up this “DNA-binding region” [69][70]. ZFNs and TALENs are structurally and functionally identical, with TALENs being more specific, although both contain the restriction endonuclease Fok [69]. To bind to a specific DNA sequence, TAL effector proteins must be engineered. TALE binds to DNA through a core section containing a collection of three-amino-acid (33–35) motifs. A typical TALE DNA-binding domain recognizes 1420 nucleotides, including the conserved thymine (T) base at the 5′ borders. The repeat-variable di-residues (RVDs) [70][71] are two hypervariable amino acids that determine TALE selectivity. The RVDs are conserved at positions 12 and 13 [72]. There are around 24 known distinct RVDs, and these RVD loops contain an amino acid repeat with a missing residue denoted with N* means, 33 [73] being the most prevalent. It is easier to assemble four basic components that can recognize the bases A, T, G, and C in TALENs than it is to assemble ZFN subunits that detect 64 different DNA trio or triplet pairings. ZFNs and TALENs, on the other hand, rely on highly precise protein–DNA interactions that allow for fewer mismatches [74].
By inserting an NHEJ/HDR-induced change in a coding region, TALENs were able to disrupt the genes NTF3 and CCR5 in human leukemia cells, indicating that TALENs are designed for endogenous gene cleavage [75]. Breaking a single allele from the Fms-related tyrosine kinase 3 (FLT3) gene using a site-specific TALEN lead to the creation of isogenic leukemia cell clones. The use of artificial TALENs in prostate cancer cells and androgen receptor (AR) target gene reorganizations are functionally categorized sources of resistance [75]. Besides studies revealed that the type of gene-editing technology is a potent and broadly appropriate platform for examining gene transmutations on a molecular basis. This technology has been used in cancer cells such as prostate cancer cells, breast cancer cells [76], and hepatocellular carcinoma cells (HCC) [77] to knock out the genes. However, due to the numerous limitations of ZFN and TALEN, there has been limited or no research on nanomedicine applications.


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