Using the precise genome editing that CRISPR/Cas9 technology enables in cells used for tissue regeneration, tissue engineering has the potential to undergo a revolution. Using biomaterials and cells, tissue engineering aims to produce functioning tissues or organs that may take the place of the body’s sick or damaged tissues (Armstrong and Stevens, 2019). Getting a firm grip on the behavior of the cells utilized in the regeneration process is one of the main difficulties in tissue engineering. Scientists can accurately change DNA sequences in cells using the potent genome editing technology CRISPR/Cas9 (Gopal et al., 2020). In tissue engineering cells, certain genes may be altered using CRISPR/Cas9 to improve their capacity for regeneration, encourage differentiation into cell types, and optimize their behavior in response to signals. For instance, CRISPR/Cas9 might be used to change the genes that regulate stem cells’ differentiation and proliferation, which are frequently employed in tissue engineering applications (Zhang et al., 2017; Valenti et al., 2019). Scientists might influence the development of stem cells into cell types, such as bone or cartilage cells, that may be utilized to rebuild damaged tissues by manipulating the expression of these genes. Moreover, CRISPR/Cas9 has the potential to alter immune response-related genes, which could enhance the compatibility of transplanted tissues with the recipient’s immune system. This could lower the possibility of rejection and increase the effectiveness of tissue engineering treatments over time (Clément et al., 2017; Ryczek et al., 2021; Meissner et al., 2022).

Tissue engineering using CRISPR/Cas9 involves using genome editing to modify the genetic makeup of cells used for tissue regeneration. The goal is to enhance the regenerative potential of these cells and optimize their behavior in response to specific signals. One example of how CRISPR/Cas9 can be used in tissue engineering is in the development of organoids, which are three-dimensional structures that mimic the architecture and function of organs in the body (Driehuis and Clevers, 2017; Rodríguez-Rodríguez et al., 2019). By modifying specific genes in stem cells, researchers can direct their differentiation into specific cell types that can be used to create organoids that closely resemble real organs. In addition to organoid development, CRISPR/Cas9 can also be used to modify genes involved in immune responses. This could improve the compatibility of transplanted tissues with the recipient’s immune system, reducing the risk of rejection and improving the long-term success of tissue engineering procedures (Yin et al., 2016; Lehmann et al., 2019; Kim et al., 2020). CRISPR/Cas9 can also be used to modify genes involved in wound healing, such as those that control the migration and proliferation of cells. By enhancing the ability of cells to regenerate damaged tissues, CRISPR/Cas9 could improve the outcomes of tissue engineering procedures for injuries and diseases (Grath and Dai, 2019; Srifa et al., 2020; Li et al., 2023a).

Using CRISPR/Cas9 to change certain genes or regulatory components, it is possible to precisely design tissues. This can lessen the possibility of negative effects while improving the efficacy of created tissues. By altering the expression of genes involved in angiogenesis, CRISPR/Cas9 has been utilized, for instance, to create a functioning blood artery (Liu et al., 2019; Uddin et al., 2020). With the introduction of disease-causing mutations into cells, CRISPR/Cas9 may be used to simulate illnesses in a lab setting. This can be utilized to research the processes that underlie the development of diseases and to create novel treatments. For instance, the modeling of Huntington’s disease using CRISPR/Cas9 in stem cells has resulted in the discovery of novel therapeutic targets (Cai et al., 2016; Cribbs and Perera, 2017; Li et al., 2020). Gene-edited tissues may be generated using CRISPR/Cas9 and exploited for drug testing or transplantation. For instance, CRISPR/Cas9 was utilized to make gene-edited liver tissue that might be used to study drug metabolism and liver disease (Xu et al., 2020a). CRISPR/Cas9 technology can be used to change the genomes of immune and stem cells used in tissue engineering to improve their therapeutic potential. For instance, T cells may be modified using CRISPR/Cas9 to form chimeric antigen receptors (CARs), which can enhance their ability to detect and destroy cancer cells (Gao et al., 2019; Kim et al., 2021). Overall, the potential of CRISPR/Cas9 in tissue engineering is vast, and ongoing research is likely to uncover new ways to harness this powerful technology to create functional tissues and organs that can be used to treat a wide range of diseases and injuries.

A cationic polymer derived from chitin called chitosan has undergone substantial research as a drug carrier medium. It can create microparticles, hydrogels, and nanoparticles that may be utilized to deliver a range of medications, including proteins, peptides, and DNA (Cheung et al., 2015). Delivery methods based on chitosan have been utilized to treat infections, inflammatory disorders, and cancer. For instance, curcumin, a naturally occurring anti-cancer chemical, had its bioavailability and anti-tumor activity increased by a chitosan-based nanoparticle system (Huang et al., 2017). Because of their mucoadhesive qualities and capacity to improve drug absorption, chitosan-based delivery systems have shown promise in the administration of many medications, including anti-cancer therapies. Chitosan-based delivery systems for a variety of medicines, including polypeptides, polymers, and polynucleotides, have been created (Mohammed et al., 2017; Herdiana et al., 2021a). The effective creation of chitosan-based nanocarriers for the transport of capsaicin, an aqueous solubility anti-inflammatory medication. Chitosan has also been explored for gene delivery, with results demonstrating that it has a higher transfection effectiveness than other delivery methods (Cao et al., 2019; Herdiana et al., 2021b).

Nucleic acids and other medicinal compounds, including those, have been thoroughly explored for use as delivery systems using chitosan. Delivering CRISPR/Cas9 gene editing tools is one of the chitosan’s most intriguing uses (Caprifico et al., 2022). With the use of the potent gene-editing tool CRISPR/Cas9, genes in the genome may be precisely targeted and altered. Nevertheless, because the CRISPR/Cas9 components are bulky and negatively charged, they have a difficult time crossing the cell membrane, making their distribution into target cells a significant barrier (Qiao et al., 2019; Khademi et al., 2022). It has been demonstrated that chitosan-based delivery methods may successfully overcome this obstacle and distribute CRISPR/Cas9 into target cells (Wilbie et al., 2019).

Chitosan is often utilized as a cationic polymer to bind with the negatively charged CRISPR/Cas9 components in chitosan-based CRISPR/Cas9 delivery systems, which mainly include the creation of nanoparticles. Cells may quickly absorb the resultant nanoparticles and transfer them to the nucleus, where the CRISPR/Cas9 components can carry out their gene-editing task (Givens et al., 2018; Zhang et al., 2018; Rouatbi et al., 2022). The CRISPR/Cas9 combination was loaded with CsNPs by researchers to treat pulmonary arterial hypertension (PAH). The bone morphogenic protein receptor II (BMPR2) gene was proposed as the key gene implicated. BMPR2 displays functional alterations or decreased expression, which causes lung cells to proliferate less and undergo more apoptosis (Figure 3A). As a result, fibroblasts were used for the CRISPR/Cas9 transfection for altering BMPR2. The findings suggested that utilizing pure CsNPs for transfection of cells resulted in a considerable reduction in BMPR2 mRNA expression and an increase in cell proliferation. Site-specific and regulated distribution might be enabled accessible by encapsulating the surface of PLGA NPs with chitosan in order to enhance electrostatic attraction with the negatively charged membranes of the cells (Figure 3B). Additionally, using the CRISPR/Cas9 plasmid distribution technique for addressing the human embryonic kidney cell line (HEK-293), the replication of green fluorescent protein (GFP) was 80% suppressed. Even though the formation of protein corona was induced because the size of NCs increased by 1.3-fold and their transfection ability by about 20% in several cancer cell lines, the coating of AGNPs with Cs conferred a higher protection of the plasmid in the presence of serum proteins (Figure 3C). This was because the coating protected the plasmid from enzymatic degradation. (Tuder et al., 2009; Caprifico et al., 2022; Kabwe et al., 2022).

Numerous studies have shown that CRISPR/Cas9 delivery methods based on chitosan are efficient in a variety of cell types and animal models. For instance, chitosan nanoparticles may effectively transfer CRISPR/Cas9 to human lung cancer cells, which resulted in a substantial decrease in cancer cell viability (Xu et al., 2021; Huang et al., 2022a). Several formulations and preparation techniques have been used to create chitosan-based delivery systems for CRISPR/Cas9. One method uses chitosan nanoparticles, which may be created by ionic gelation, polyelectrolyte complexation, or coacervation techniques. To increase these nanoparticles’ stability, cellular absorption, and gene editing effectiveness, targeted ligands, and surface modifications can be added (Vauthier et al., 2013; Ghadi et al., 2014). Chitosan hydrogels or films, which may be loaded with CRISPR/Cas9 components and delivered directly to target tissues or cells, are a different strategy. These formulations can enhance the bioavailability and retention of the gene editing components and offer a prolonged release of those components (Mahmudi et al., 2022).

Another biopolymer that has been extensively employed in drug delivery applications is alginate. Drugs including antibiotics, anti-inflammatories, and growth hormones have all been delivered using hydrogels made of alginate. Applications for tissue engineering, such as the regeneration of bone and cartilage, have also made use of alginate-based systems (Lee and Mooney, 2012; Abourehab et al., 2022). There have been many studies on the use of alginate-based delivery techniques in biochemical regeneration and cellular encapsulation. Alginate has also been used to administer medications, and the results point to a delayed drug release and greater therapeutic efficacy (Zhang et al., 2021a). Alginate-based delivery systems for a range of pharmaceuticals, including proteins, peptides, and small molecules, have been developed. Making hydrogels with an alginate base that work well to deliver insulin continuously (Li and Mooney, 2016; Tomić et al., 2023).

These methods are being investigated for a variety of applications, such as skin regeneration, cancer treatment, and genetic editing. To enclose and distribute CRISPR/Cas9 components, such as plasmid DNA or ribonucleoproteins (RNPs), to target cells, alginate-based delivery approaches have been utilized (Wang et al., 2022). According to one study, the delivery of CRISPR/Cas9 RNPs to lung cancer patient cells via an alginate-based delivery method led to precise genetic alterations. An alginate-based polymer may be able to carry CRISPR/Cas9 RNPs to a murine model’s retina, enabling precise genetic manipulation and a possible cure for retinal illnesses, according to another study. Target cells may be effectively supplied utilizing alginate-based nanoparticles and CRISPR/Cas9 (Burnight et al., 2018; Behr et al., 2021; Caprifico et al., 2022).

The research discovered that the CRISPR/Cas9 system could be adequately delivered to targeted in vitro cultivating cells using alginate nanoparticles that were loaded with the Cas9 protein and sgRNA (single guide RNA) (Wan and Ping, 2021). In research that has been released, scientists have demonstrated that alginate hydrogels can increase the efficacy of CRISPR/Cas9 administration by halting the degradation of Cas9 protein and sgRNA by restriction enzymes in plasma. For specific applications, CRISPR/Cas9 delivery on alginate may be tailored (Guo et al., 2018; Farheen et al., 2022). In a study, researchers modified the alginate nanoparticles’ surface using a “click” chemical technique to better target and distribute CRISPR/Cas9 to certain cell types. Investigations on CRISPR/Cas9 delivery utilizing alginate have been conducted on animals (Alallam et al., 2020). In a recent study, it was shown that alginate nanoparticles loaded with the Cas9 protein and sgRNA could effectively transport the CRISPR/Cas9 system to target cells in the lungs of mice (Kim et al., 2020). Overall, these investigations indicate that alginate-based delivery methods may be useful for CRISPR/Cas9 gene editing applications, but more study is required to tailor the delivery system for uses and assess its effectiveness and safety in vivo.

A natural protein called collagen is present in large amounts in the extracellular environment of several tissues, including cartilage, skin, and bone. Because of its biological properties and capacity to promote tissue repair and cell proliferation, it has been utilized in the creation of systems for delivering drugs. Small compounds, peptides, and growth factors have all been delivered using collagen-based methods (Gelse et al., 2003; Dong and Lv, 2016; Naomi et al., 2021). Collagen-based delivery methods have been employed by investigators to successfully deliver CRISPR/Cas9 to target cells in vitro and in vivo. In one research, CRISPR/Cas9 was successfully delivered via a collagen-based delivery method to treat mice with Duchenne muscular dystrophy (Sharma et al., 2021; Javaid et al., 2022). Another research demonstrates that CRISPR/Cas9 may be delivered to neuronal cells via a collagen-based delivery method, possibly leading to novel treatment options for neurological illnesses (Abdelnour et al., 2021). Collagen-based delivery methods have also demonstrated potential in lowering CRISPR/Cas9 off-target effects, a significant problem in gene therapy. The development of collagen-based delivery methods for the effective and targeted administration of CRISPR/Cas9 to target cells still faces obstacles (Maeder and Gersbach, 2016; Li et al., 2022a).

In an investigation, researchers have developed a collagen-based hydrogel that may deliver CRISPR/Cas9 to the brain to treat neurological problems. Another study increased the efficiency of gene editing and improved the targeting of CRISPR/Cas9 to specific liver cells while reducing off-target effects by using a growth factor delivery technique. To treat skin diseases, CRISPR/Cas9 has also been administered via collagen-based delivery systems (Cota-Coronado et al., 2019; Zhou et al., 2022). In a scientific report, epidermolysis bullosa, a hereditary skin condition, was successfully treated using a hydrogel made of collagen that was utilized to transport CRISPR/Cas9 to skin cells. Xenografts made from biallelic corrected induced pluripotent stem cell (iPSC)-derived recessive dystrophic epidermolysis bullosa (RDEB), keratinocytes (KCs) and fibroblasts (FBs) were immunofluorescently labeled for C7, epidermal development markers such keratin 14, keratin 10, loricrin, filaggrin, and vimentin, and they were compared to iPSC-derived xenografts made from WT and mutant cells 2 months after grafting (Figures 4A, B). The fact that the xenografts from iPSCs that had their genes corrected generated C7 and looked exactly like WT skin suggests that the iPSC-derived iKCs and iFBs had their protein functionality recovered. The COL7A1 mutation in exon 19 was corrected in 10% of the selected clones by biallelic correction and in 40% of the clones through monoallelic correction. About 80% of the selected clones showed Cas9 activity. The hereditary mutation (c.2470insG) in exon 19 of COL7A1 was corrected by 58% biallelic and 42% monoallelic correction, whereas the heterozygous mutation (c.2470insG/c.3948insT) in exons 19 and 32 of COL7A1 was corrected by 19% biallelic and 48% monoallelic correction, respectively (Figure 4C). (Jacków et al., 2019). The effectiveness and security of CRISPR/Cas9 gene editing might be enhanced by collagen-based delivery methods, and current research in this area is expected to result in future developments (Prodinger et al., 2019; du Rand et al., 2022; Wu et al., 2017). Collagen-based therapeutic approaches must nevertheless be improved to distribute CRISPR/Cas9 precisely and effectively to target cells. In conclusion, collagen-based CRISPR/Cas9 delivery systems represent a fascinating and promising area of research with potential applications in the treatment of several inherited disorders (Kang et al., 2022).

Gelatin is a protein made from collagen that has been employed in tissue engineering and medication delivery. Proteins, peptides, and medicines have all been delivered using gelatin-based delivery methods. They have also been employed in the creation of scaffolds for tissue engineering, such as those used to regenerate cartilage and skin. (Thein-Han et al., 2009; Afewerki et al., 2018). Gelatin may be used to administer a wide range of drugs, including proteins, peptides, and small compounds. The efficient synthesis of gelatin-based solid lipid nanoparticles for the delivery of the widely used chemotherapy drug paclitaxel (Ma et al., 2020; Wong et al., 2020).

To cure Duchenne muscular dystrophy in a mouse model, CRISPR/Cas9 has been delivered via gelatin nanoparticles, which has improved muscle function and raised dystrophin expression. The key enzyme neuronal nitric oxide synthase (nNOS), which is a part of the dystrophin-glycoprotein complex that regulates muscle function, was able to localize to the sarcolemmal region as a result of the induction of dystrophin expression (Figure 5). The detected dystrophin levels within muscles remained sustained, leading to notable gains in specific force-producing ability as well as protection from contraction-induced damage (Bengtsson et al., 2017).

Gelatin hydrogels were used as a delivery mechanism to target the carcinogenic KRAS gene by CRISPR/Cas9 in pancreatic cancer cells in vitro, which led to the reduction of cell proliferation and colony formation (Kim et al., 2018). Improved airway epithelial function and a decrease in bacterial load were the results of delivering CRISPR/Cas9 via gelatin-based nano complexes in a pig model of cystic fibrosis. To deliver CRISPR/Cas9 to glioblastoma cells in vitro, gelatin nanoparticles functionalized with tumor-targeting peptides have been created. This reduces cell survival and promotes tumor development (Zhou et al., 2019; Piotrowski-Daspit et al., 2022).

Gelatin-based delivery systems for CRISPR/Cas gene editing have become a viable tool for therapeutic uses because of their biomedical applications, good biocompatibility, and capacity to incorporate and preserve genetic information (Xu et al., 2020b). Gelatin nanoparticles equipped with Cas9 protein and guide RNA were shown to effectively transfer the elements to human embryonic kidney cells (HEK293T) and trigger gene editing, demonstrating the higher effectiveness with which gelatin nanoparticles can deliver CRISPR/Cas components to target cells (Rouet et al., 2018; Andrée et al., 2022).

It has been demonstrated that gelatin hydrogels can boost the long-term stability of the Cas9 protein and guide RNA by protecting it from nuclease degradation. Substantial levels of synthetic biology were also maintained by the gelatin hydrogels in HEK293T cells, demonstrating that gelatin hydrogels may improve the toughness and stop CRISPR/Cas components from deteriorating (Kim and Liu, 2019; Boucard et al., 2022; Li et al., 2022b). Another study created gelatin-based microcapsules that could deliver the Cas9 protein and RNA guide to certain mice organs, including the hepatic and pulmonary. Given how effectively the microparticles edited the genes in these tissues, gelatin-based microparticles might also successfully transport CRISPR/Cas components to specific tissues in vivo (Wang et al., 2016; Sahu et al., 2019). According to different research, gelatin-based nanoparticles have been created that can be tailored to deliver CRISPR/Cas to certain cells. Gelatin-based nanoparticles may be designed to target certain cells for CRISPR/Cas delivery, according to the investigators’ in vitro and in vivo experiments showing the nanoparticles could effectively carry Cas9 protein and guide RNA to cancer cells (Naeem et al., 2021; Xu et al., 2021; Saw et al., 2022).

Because of its physiological differences, high functional properties, and capacity to create hydrogels, silk protein, generated from the silkworm, has been explored as a way of delivering pharmaceuticals (Huang et al., 2018). Silk-based distribution techniques have been developed for a variety of medications, including proteins, peptides, and small compounds. Silk-based hydrogels are being developed for the prolonged release of bone morphogenetic protein-2, a growth factor that stimulates bone repair (Yucel et al., 2014; Nguyen et al., 2019).

It has been demonstrated that silk fibroin nanoparticles effectively transport CRISPR/Cas9 to human glioblastoma cells and human embryonic kidney cells (HEK293T) (U87). The research established both biocompatibility and lack of toxicity of the silk fibroin nanoparticles in the cells. Gene editing was also demonstrated to be successful when CRISPR/Cas9 was supplied using silk fibroin nanoparticles (Zhao et al., 2015). In a different scenario, silk nanoparticles were used to administer CRISPR/Cas9 for genome - editing in the retina. Silk nanoparticles, according to the study’s findings, may effectively transport CRISPR/Cas9 to retinal pigment epithelium (RPE) cells both in vitro and in vivo. The research demonstrated that the silk nanoparticles were biocompatible and had no adverse effects on the cells (Salman et al., 2022). A hydrogel based on silk fibroin was created recently for the delivery of CRISPR/Cas9 for cartilage gene editing. The scientists discovered that the silk fibroin-based hydrogel could effectively transport CRISPR/Cas9 to the chondrocyte cells both in vitro and in vivo. The research revealed that the hydrogel made of silk fibroin was biocompatible and had no harmful effects on the cells (Yu et al., 2023a).

A study showed that silk fibroin nanoparticles may successfully edit genes in vitro and in vivo by effectively delivering CRISPR/Cas9 complexes into cells. In a different study, silk-based nano complexes were developed for the delivery of the CRISPR/Cas9 ribonucleoprotein (RNP) into cells. The authors established in vitro and in vivo effective gene editing with little off-target side effects (Wei et al., 2014). In the current research, a peptide was used to functionalize silk fibroin nanoparticles to improve their uptake by cells and endosomal escape. The authors successfully delivered CRISPR/Cas9 RNP into cells and successfully carried out both in vitro and in vivo genome engineering experiments (Esser et al., 2021; Pirota et al., 2023). The most recent developments in silk-based gene editing delivery technologies, including CRISPR/Cas9, were detailed in a review paper. The biodegradability, adjustable mechanical characteristics, and biocompatibility of silk fibroin were emphasized by scientists as advantages for gene transfer (Kochhar et al., 2021).

Targeted medication delivery to cancer cells that overexpress hyaluronan receptors has been accomplished using hyaluronic acid-based delivery systems. It has been demonstrated that hyaluronic acid nanoparticles improve the effectiveness of anticancer medications like paclitaxel and doxorubicin (Maruyama, 2011; Lee et al., 2020). Sustained-release medication delivery systems have been created using cellulose derivatives such as methylcellulose and hydroxypropyl methylcellulose. With better drug release profiles and fewer side effects, these devices have been employed to administer medications including diclofenac and metformin (Hoare and Kohane, 2008; Wadher et al., 2011; Roy et al., 2013).

The CRISPR/Cas delivery mechanism hyaluronic acid (HA) has lately gained popularity. HA, a natively abundant glycoprotein found in the extracellular matrix of many tissues, is safe and well-tolerated in humans (Kelkar et al., 2016). In addition, HA is a top choice for the targeted delivery of CRISPR/Cas to cancerous cells because of its high affinity for CD44, a cell surface receptor that is upregulated in many cancerous cells (Wang and Jia, 2016; Xu and Li, 2020). The efficacy of HA-based delivery methods for CRISPR/Cas has been shown in numerous investigations. One research, for instance, delivered CRISPR/Cas9 to ovarian cancer cells both in vitro and in vivo using HA nanoparticles, which significantly inhibited tumor development (Vaghari-Tabari et al., 2022). Another study successfully achieved effective gene editing and the activation of death by delivering CRISPR/Cas9 to human lung cancer cells in vitro using HA-coated polymeric nanoparticles (Fang et al., 2022).

As a potential CRISPR/Cas delivery method, HA-based hydrogels have also been examined in other investigations. One experiment, for instance, used a hydrogel based on HA to deliver CRISPR/Cas9 to mice’s retinas while preventing retinal degeneration (Yang et al., 2021). A system of hyaluronic acid-based nanoparticles was used in one study to deliver CRISPR/Cas9 to cancer cells. The researchers showed that the CRISPR/Cas9 system could be effectively delivered to cancer cells in vitro using hyaluronic acid-based nanoparticles, resulting in precise gene editing and the induction of death (Liang et al., 2015; Qiu et al., 2016; Lin et al., 2022).

Another study administered CRISPR/Cas9 to treat osteoarthritis using a hyaluronic acid hydrogel. The research showed that the CRISPR/Cas9 system could be successfully introduced into mesenchymal stem cells in vitro via the hydrogel, resulting in gene editing and cartilage regeneration (Figure 6) (Mehta et al., 2021; Kim and Guilak, 2022). A recent study demonstrated how to deliver CRISPR/Cas13a to the SARS-CoV-2 RNA using hyaluronic acid-based nanoparticles. The researchers showed that the CRISPR/Cas13a system could be successfully introduced into SARS-CoV-2-infected cells in vitro through hyaluronic acid-based nanoparticles, resulting in precise RNA cleavage and inhibition of viral growth (Chen et al., 2022; Deol et al., 2022).

Branched cationic biopolymers generated from lactose are one class of biopolymers that have demonstrated potential as CRISPR/Cas9 delivery vehicles. These biopolymers are created from the disaccharide lactose, which is found in large quantities in milk (Qi et al., 2020). The positive charge and cationic nature of the lactose-derived biopolymers enable them to associate with negatively charged nucleic acids like DNA and RNA. These biopolymers’ branching structure offers the CRISPR/Cas9 complex several locations for attachment, enhancing delivery effectiveness. The lactose-derived biopolymers are also safe for in vivo applications since they are biocompatible and biodegradable (Moradali and Rehm, 2020; Xavier et al., 2021; Shtykalova et al., 2023).

Highly branched-charged biomaterials generated from lactose have recently been investigated as possible CRISPR/Cas9 delivery systems. Researchers demonstrated how lactose-derived branching cationic biopolymers may effectively carry CRISPR/Cas9 into cells, leading to considerable gene editing (Zhuo et al., 2021). Its effectiveness was ascribed by the researchers to the biopolymer’s capacity to create stable nanoparticles that can enter cell membranes (Sundar et al., 2010). Researchers examined the biocompatibility and toxicity of lactose-derived branched cationic biopolymers in vitro and in vivo in another investigation. The biopolymer was found to be non-toxic and did not cause any major inflammatory reaction, indicating that it might be a safe and effective CRISPR/Cas9 delivery vehicle (Rouatbi et al., 2022).

In a published paper, researchers showed how targeting receptors may be incorporated into lactose-derived branching cationic biopolymers to specifically transport CRISPR/Cas9 to cancer cells. The researchers’ in vitro and in vivo tests revealed extensive use of genetic engineering strategies and tumor growth inhibition (Kong et al., 2021; Chakraborty and Sarkar, 2022). In a thorough study that has been published, researchers asserted the potential of lactose-derived branched cationic biopolymers as CRISPR/Cas9 delivery vehicles for therapeutic purposes. The scientists talked about the biocompatibility, non-toxicity, targeting, and ability of the biopolymer to avoid issues with CRISPR/Cas9 distribution in vivo (Jie et al., 2021).

Several studies have concentrated on creating biopolymer-based CRISPR/Cas9 delivery systems as a potential replacement for viral vectors, which have drawbacks and safety issues. A lactose-derived branching cationic biopolymer known as lactose-grafted polyethyleneimine (Lac-PEI) is one intriguing possibility (Li et al., 2018; Iqbal et al., 2023). Target cells can effectively receive CRISPR/Cas9 plasmids using Lac-PEI. Researchers demonstrated in a study that Lac-PEI-mediated CRISPR/Cas9 delivery caused a considerable suppression of the target gene in human lung cancer A549 cells and human embryonic kidney (HEK293T) cells (Ryu et al., 2018).

According to a study, targeting peptide-coated Lac-PEI nanoparticles may effectively deliver CRISPR/Cas9 to breast cancer cells both in vitro and in vivo. Lac-PEI can be changed to increase delivery effectiveness and lessen toxicity (Nie et al., 2022). Researchers transformed Lac-PEI in a study that was published with the addition of a zwitterionic polymer, which increased the effectiveness of its gene transport and decreased both in vitro and in vivo cytotoxicity (Liu et al., 2022a). Multicarrier gene editing is now possible with Lac-PEI, wherein the researchers created a Lac-PEI-based CRISPR/Cas9 system in a study that allowed three genes to be edited in HEK293T cells simultaneously (Rohiwal et al., 2020). Furthermore, the development of more efficient and secure genome editing therapeutics shows promise with the use of lactose-derived branched cationic biopolymers for CRISPR/Cas9 delivery. To completely describe their features, enhance the effectiveness of their distribution, and improve their usage in therapeutic interventions, more study is required.

Bacteria that experience inconsistent reproduction synthesize polyhydroxyalkanoates (PHA), a class of organic, biodegradable polyesters that operate as internal carbon- and energy-storing components. They have greater biodegradability, biocompatibility, and the capability to produce harmless byproducts, making them suitable replacements for implantable devices such as sutures, repair patches, slings, orthopedic pins, scaffold, stents, and adherence barriers (Lee, 1996; Guzik, 2021). Despite their great features as indicated above, unmodified PHAs are inappropriate for use in many biological applications due to the presence of large aggregates, which leads to poor biomechanical properties, poor thermodynamic stability, excessive hydrophobicity, and a sluggish disintegration rate (Ray and Kalia, 2017; Riaz et al., 2021). PHAs’ intrinsic hydrophobicity hinders them from being employed in biomedical applications as many biomedical devices require greater hydrophilicity (Pulingam et al., 2022).

PHAs are now being researched for their effectiveness as a CRISPR/Cas-based biomedical research framework. The CRISPR/Cas system is a potent genomic tool that enables precise editing of DNA sequences but getting it to cells correctly and effectively is still a big barrier (Jacinto et al., 2020). For instance, positively charged amino acids and negatively charged carboxyl groups can facilitate cell uptake of the CRISPR/Cas system. PHAs can encapsulate these functional groups and may also be modified to decay at predetermined rates, which is essential for managing the duration of CRISPR/Cas activity (Imamoğlu et al., 2022; Liu et al., 2022b).

It has been demonstrated that PHA-based nanoparticles may successfully distribute CRISPR/Cas systems in vitro and in vivo. One research, for instance, showed that Cas9 and sgRNA-loaded poly (3-hydroxybutyrate-co-3-hydroxy hexanoate) (PHBHHx) nanoparticles could effectively transport the CRISPR/Cas system to tumor cells in vitro and a mouse tumor model (Kılıçay et al., 2011; Chang et al., 2014). PHA scaffolds can also be used to distribute CRISPR/Cas systems locally. According to the study, gene modification was readily achieved in a bone-deficient mice model employing poly (3-hydroxybutyrate-co-3-hydroxy valerate) (PHBV) scaffolds loaded with Cas9 and sgRNA (Lv et al., 2015; Qin et al., 2018). Some traits can be included in PHAs to increase their potency as CRISPR-Cas delivery vehicles. For example, PHA nanoparticles may be functionalized with targeting ligands to improve their selectivity for specific cell types. A targeting peptide was added to PHA nanoparticles in a recently published study to enable the delivery of Cas9 protein and guide RNA to mouse liver cells in a targeted manner (Shrivastav et al., 2013; Prakash et al., 2022). PHAs may be used with other materials to create blended packaging solutions that have additional advantages. For instance, researchers have developed PHA-based hydrogels (embedding the Cas9 protein and guide RNA) that may be injected at a target site for permanent and precise genetic editing. Researchers developed a PHA-based hydrogel that, when implanted at the site of the tumor in mice, significantly inhibited the growth of tumors (Liu et al., 2021b).

PHAs have the potential to be used as a CRISPR/Cas delivery method, according to one published research, wherein to improve cellular absorption, the researchers created a PHA-based nanoparticle that was functionalized with amino groups. The CRISPR/Cas system was also included in the nanoparticle, and its capacity to trigger gene editing in human cells was examined. The outcomes demonstrated that the PHA-based nanoparticle efficiently and effectively introduced the CRISPR/Cas system into the cells and triggered gene editing (Allemailem et al., 2022; Xi et al., 2022). It is now possible to regulate the expression of endogenous or heterologous genes via the advent of transcriptional and/or configurable hereditary circuits, such as T7 polymerase-based expression vectors, programmable T7-based polymeric transposable elements, the RiboTite system, vector technology, and CRISPR-Cas tools. CRISPR/Cas9 has also been successfully used to change many genes simultaneously. CRISPR interference effectively directs metabolic flow toward PHA synthesis (Lv et al., 2015; Chen and Jiang, 2018).

In research, CRISPR/Cas9 ribonucleoprotein (RNP) complexes were shown to be transported into human cells using PHA nanoparticles. The study found that PHA nanoparticles effectively activate targeted gene editing and transport CRISPR/Cas9 RNP complexes in primary T cells and neurons (Cheng et al., 2022). Researchers also considered PHA-based nanomaterials for the in vivo delivery of CRISPR/Cas9 plasmids. The researchers successfully carried CRISPR/Cas9 plasmids to target cells and started gene editing in a mouse model using PHA nanoparticles. In addition to being studied as a delivery method for CRISPR-Cas systems, PHAs are also being investigated as a scaffold material for those systems (Zhang et al., 2022). In a different study, scientists demonstrated how to use a scaffold made of PHA and CRISPR/Cas9 plasmids to treat bone defects. The researchers found that the PHA-based scaffold could effectively distribute the CRISPR/Cas9 plasmids and promote bone regeneration in a rat model (Liu et al., 2023).

Exosomes are extracellular vesicles that may be separated from a variety of physiological fluids, including blood, urine, and saliva. They are released by practically all cell types. They are rapidly being researched for their prospective use as drug carriers for pharmacological medicines, including CRISPR/Cas gene editing tools. They play a significant part in signaling pathways (Muthu et al., 2021; Rajput et al., 2022). Potential therapeutic uses for exosome-based CRISPR/Cas delivery include the treatment of cancer, genetic abnormalities, and viral infections. According to researchers, for instance, exosomes can carry siRNA to cancer cells and prevent the development of tumors in mice; Cas9 protein and sgRNA to cure Duchenne muscular dystrophy in a mouse model (Kooijmans, et al., 2013; Ahmadi et al., 2023).

If CRISPR/Cas is administered via exosomes, gene therapy might undergo a radical transformation. By separating exosomes from diverse sources, including cells generated by patients, CRISPR/Cas components may be produced and directed toward specific genes or mutations connected to certain diseases (Zhu et al., 2023). Studies have demonstrated promising results for the delivery of CRISPR/Cas for the treatment of several diseases, including cancer and genetic disorders. Exosome-mediated CRISPR/Cas delivery is superior to other delivery methods, including viral vectors and lipid-based transfection. This is owing to exosomes’ capacity to identify certain tissues and shield the payload from destruction by proteolytic enzymes (McAndrews et al., 2021; Wan et al., 2022). CRISPR/Cas administration through exosomes has been utilized successfully in experimental animals to cure hereditary mutations. In a mouse model of Duchenne muscular dystrophy, for example, exosomes containing CRISPR/Cas were capable of targeting muscle fibers and fixing the dystrophin mutated gene, resulting in enhanced neuromuscular activity (Erkut and Yokota, 2022; Zhang et al., 2023).

Several studies have shown that exosomes may successfully transport CRISPR/Cas components, such as sgRNA and Cas9 protein, to target cells in vitro and in vivo. For example, researchers discovered that exosomes generated by dendritic cells may transport siRNA to T cells and reduce the expression of a target gene (Haney et al., 2015). Similarly, researchers also demonstrated that exosomes may transport Cas9 protein and sgRNA to target cells and produce targeted genome editing (Chen et al., 2019). By directing the distribution of the CRISPR/Cas components to particular cell types and preventing off-target effects, exosomes can increase the precision and safety of CRISPR/Cas gene editing. For instance, research has shown that mesenchymal stem cell-derived exosomes may carry siRNA to breast cancer cells specifically and decrease the expression of a target gene (Lamichhane et al., 2016). Similar to this, another research showed that exosomes may cause targeted genome editing by only delivering Cas9 protein and sgRNA to pancreatic beta cells while having no negative effects on the function of other cell types (Chen et al., 2021). When KPC689 cells were treated with exosomes containing the KrasG12D sgRNA1 and KrasG12D sgRNA2 but not with exosomes containing the vector control or untreated cells, the T7/Surveyor test revealed indications of effective gene editing (Figure 7) (McAndrews et al., 2021).

In the context of research on infectious diseases, exosome-based CRISPR/Cas9 delivery has been suggested as a potential strategy for producing innovative therapies for viral infections including HIV and hepatitis B. For instance, a 2018 study discovered that exosomes containing CRISPR/Cas9 may target and harm HIV proviral DNA in vitro, decreasing the virus’ ability to produce genetic material (Dubey et al., 2022; Mazurov et al., 2023). Like this, exosome-based CRISPR/Cas9 delivery was shown to effectively target the hepatitis B virus (HBV) genome in vitro and in vivo, leading to a reduction in viral replication and gene expression (Martinez et al., 2022). Moreover, tissue engineering applications using exosome-based CRISPR/Cas9 dissemination have been studied, notably in the context of regenerative medicine. For instance, exosomes containing CRISPR/Cas9 were able to accurately target and modify the genes governing osteogenic differentiation in human mesenchymal stem cells, improving bone creation in vivo (Guo and Huang, 2022) Like this, it was demonstrated that exosomal delivery of CRISPR/Cas9 enhanced the capacity of human umbilical vein endothelial cells to produce angiogenic factors, suggesting the possibility of their use in tissue engineering and wound healing (Liu et al., 2021c; Zhao et al., 2023).

Lipid bilayers in the liquid phase comprise the globular entities known as liposomes. Since both nucleic acids and biological membranes have negative charges, nucleic acids cannot pass through the membrane owing to the attraction between both (Yip, 2020). In this regard, positively charged liposomes encase negatively charged nucleic acids, making it easier for the complexes to pass cell membranes and enter cells. DNA, mRNA (Cas9 and sgRNA), or protein can all be used to deliver the CRISPR/Cas9 system (RNP) (Pensado et al., 2014). The spherical liposomes, made of a lipid bilayer, may store both hydrophilic and hydrophobic medications. Polymeric nanoparticles, made of natural or synthetic polymers, can be engineered to release medications in response to various stimuli, such as pH or temperature changes. Viral shape and functionality can be mimicked by protein-based nanoparticles, such as virus-like particles (VLPs), which are made of proteins (Sun et al., 2014; Bulbake et al., 2017; Nsairat et al., 2022).

A mouse model of Duchenne muscular dystrophy (DMD) was employed in a study to deliver CRISPR Cas9 to the dystrophin gene using liposomes. The scientists demonstrated that the liposome-delivered CRISPR Cas9 could correct the dystrophin gene mutation and enhance mouse muscle function (McGreevy et al., 2015). According to a different study, CRISPR Cas9 was delivered using liposomes to target the β-catenin gene in colorectal cancer cells. The scientists proved that CRISPR Cas9 administered by liposomes might stop tumor development both in vitro and in vivo (Chen et al., 2017; Jubair et al., 2021).

A mouse model of hypercholesterolemia was employed in another work to deliver CRISPR Cas9 to the PCSK9 gene via liposomes. The scientists demonstrated that the liposome-delivered CRISPR Cas9 could drastically lower the mice’s blood cholesterol levels (Ding et al., 2014; Chadwick and Musunuru, 2017). In a mouse model of acute myeloid leukemia (AML), liposomes were employed to carry CRISPR Cas9 to target the CXCR4 gene in a recent study. The scientists demonstrated that CRISPR Cas9 supplied by liposomes might cause apoptosis in AML cells and dramatically increase the longevity of the mice (Hou et al., 2015; Freen-van Heeren, 2022; Li et al., 2022c).

Although animals retained their percentages of gene-modified cells across time (Figure 8A), the CRISPR-modified cells in the hu-PBMC NSG mouse model were well tolerated. Additionally, X4-tropic HIV-1 resistance in CXCR4-CRISPR humanized mice led to selective enrichment of CD4⁺ T cells in spleen tissue compared to non-CRISPR animals. Despite the fact that CRISPR-mediated disruption of CXCR4 was effective in lowering viremia and safeguarding CD4⁺ T cells in vivo (Figures 8B, C), researchers noticed the numbers of R5X4-CRISPR-modified CD4⁺ T cells were much lower than unmodified controls in the bone marrow. To halt Jurkat CD4⁺ T cells from expressing CXCR4 on their surface, researchers first tested the efficiency of various sgRNAs for each target through lentiviral vectors. Flow cytometry analysis revealed a significant reduction in surface CXCR4 expression, with 15.4% CXCR4+ cells transduced with CXCR4-CRISPR compared to 99.3% CXCR4+ cells in control-CRISPR cells (Figure 8D).

According to a scientific report, liposomes successfully delivered CRISPR/Cas9 to mice’s livers. The Cas9 mRNA and guide RNA (gRNA) targeting the PCSK9 gene were delivered by the investigators using a lipid nanoparticle (LNP) formulation. When contrasted with the control group, they discovered that the mice treated had much lower levels of PCSK9 protein and LDL cholesterol (Chadwick et al., 2017; Carreras et al., 2019). In another work, mRNA, and gRNA for Cas9 were delivered to primary human T cells using a dual-lipid nanoparticles method. In addition to effective gene editing, proliferation and differentiation, good cell vitality, and reduced toxicity were all noted by the researchers. Using the same delivery method, they also showed effective deletion of the target gene in vivo (Rosenblum et al., 2020; Han et al., 2022b).

Investigators designed a liposomal delivery mechanism in a study that targets the oncogenic Bcl-2 in leukemia cells using Cas9 and sgRNA. The technique was discovered to effectively transport Cas9/sgRNA and cause apoptosis in the leukemia cells, pointing to its potential as a leukemia treatment method (Li et al., 2020). A lipid nanoparticle-based delivery method for Cas9 mRNA and sgRNA targeting Angptl3 in mice was created by researchers in a different study. The technique was discovered to effectively carry the gene editing apparatus to the liver, leading to a reduction in Angptl3 expression and an improvement in the mice’s lipid metabolism (Qiu et al., 2021; Syaharani et al., 2021).

Another subject that has been studied is CRISPR/Cas9 delivery using cationic liposomes. In a recently published work, bio-reducible cationic lipids nanoparticles (CLN) were developed for the delivery of Cas9 and sgRNA (Li et al., 2019). Targeted mutagenesis knockout and correction were observed in vitro and in vivo, and this was due to the efficient transport of the gene editing machinery to cells via the bCLN. The administration of CRISPR/Cas9 using liposomes for cancer therapy has also been studied. (Zuris et al., 2015). The oncogene KRAS in pancreatic cancer cells was targeted using a liposomal delivery system developed by researchers for Cas9 and sgRNA. The system’s ability to deliver gene editing tools and cause cancer cells to die suggests that it may be useful as a pancreatic cancer therapy strategy (Won et al., 2021; Katti et al., 2022).