Journal of Drug Delivery Science and Technology 92 (2024) 105338

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Journal of Drug Delivery Science and Technology

journal homepage: www.elsevier.com/locate/jddst

Review article

Principles of CRISPR-Cas9 technology: Advancements in genome editing

and emerging trends in drug delivery
Alaa A.A. Aljabali a, **, Mohamed El-Tanani b, Murtaza M. Tambuwala b, c, *
a
Faculty of Pharmacy, Department of Pharmaceutics & Pharmaceutical Technology, Yarmouk University, Irbid, 21163, Jordan
b
College of Pharmacy, Ras Al Khaimah Medical and Health Sciences University, Ras Al Khaimah, United Arab Emirates
c
Lincoln Medical School, University of Lincoln, Brayford Pool Campus, Lincoln, LN6 7TS, UK, United Kingdom

A R T I C L E I N F O A B S T R A C T

Keywords: The rapid advancement of CRISPR-Cas9 technology has instigated a profound transformation in genome editing
CRISPR-Cas9 with significant implications for fields like health, agriculture, and biotechnology. This review provides an
Genome editing overview of the historical significance and fundamental components of CRISPR-Cas9, notably the Cas9 protein
Nanoparticle-based delivery
and guide RNA, underscoring its pivotal role in genetic manipulation. It emphasizes CRISPR-Cas9’s preeminence
Personalized medicine
in the domain of precise genome editing, driving breakthroughs in personalized medicine, gene therapy, and
Breakthroughs in genome editing
agriculture. Of paramount importance is the integration of nanomaterials, encompassing lipid-based and poly­
meric nanoparticles, alongside viral vectors, serving as potent vehicles for CRISPR-Cas9, augmenting delivery
efficiency and precision. We explore strategies aimed at enhancing CRISPR-Cas9 through nanomaterials, while
also addressing ethical and regulatory considerations. In the expert opinion section, we offer a nuanced
perspective on the present state of the field, highlighting the potential for transformative progress in research and
therapy. CRISPR-Cas9 stands on the brink of unlocking new possibilities in genome editing, providing innovative
solutions to address pressing global challenges.

1. Introduction
Genome editing has emerged as a groundbreaking discipline in mo­
lecular biology, with the clustered regularly interspaced short palin­
dromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) system
reigning as a preeminent technology, attracting substantial attention
[1]. CRISPR-Cas9, through its remarkable precision and adaptability,
has ushered in a transformative era in genome editing. It has out­
performed earlier methods like zinc finger nucleases and transcription
activator-like effector nucleases (TALENs) in cost-efficiency and ease of
use, marking a significant advancement in this field. Its applications are
multifaceted, encompassing medical research, human gene therapy,
plant science, and crop improvement as shown in Fig. 1 [2]. The
CRISPR-Cas9 system comprises two fundamental components: the Cas9
protein and the guide RNA (gRNA). Functioning as a molecular scissor,
Cas9 cleaves the DNA at predetermined target sites, guided by the gRNA,
which aligns with the target DNA sequence. Initially derived from bac­
terial adaptive immune systems, this molecular machinery has been
harnessed for genome editing across various organisms, including
humans, owing to its simplicity and adaptability [3]. The genome
editing process utilizing CRISPR-Cas9 encompasses several pivotal steps.
The Cas9-gRNA complex is directed to the target DNA sequence, where
Cas9 induces a double-stranded DNA break. Subsequently, cellular
repair mechanisms introduce modifications at the target site through
either non-homologous end joining (NHEJ) or homology-directed repair
(HDR) pathways. This programmable and precise nature of
CRISPR-Cas9 enables accurate modifications of the genome, rendering it
invaluable for basic research, therapeutics, and crop enhancement [4].
CRISPR-Cas9 boasts numerous advantages over alternative genome
editing tools, driving its widespread adoption and impact. It exhibits
heightened efficiency and accuracy, mitigating off-target effects and
enhancing editing outcomes compared to older techniques like zinc
finger nucleases (ZFNs) and TALENs. The simplicity and
cost-effectiveness of CRISPR-Cas9 democratizes access for the scientific
community, facilitating gene function studies, disease modeling, and
potential therapeutic interventions [5]. However, CRISPR-Cas9 con­
fronts challenges that warrant careful consideration. Off-target effects,
where in Cas9 may inadvertently cleave unintended DNA sites, can lead

* Corresponding author. Lincoln Medical School, University of Lincoln, Brayford Pool Campus, Lincoln, LN6 7TS, UK, United Kingdom.
** Corresponding author.
E-mail addresses: alaaj@yu.edu.jo (A.A.A. Aljabali), eltanani@rakmhsu.ac.ae (M. El-Tanani), mtambuwala@lincoln.ac.uk (M.M. Tambuwala).

https://doi.org/10.1016/j.jddst.2024.105338
Received 19 October 2023; Received in revised form 29 December 2023; Accepted 3 January 2024
Available online 6 January 2024
1773-2247/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
A.A.A. Aljabali et al. Journal of Drug Delivery Science and Technology 92 (2024) 105338

to undesired genetic alterations. Research endeavors are concentrated
on bolstering target specificity through modified Cas9 variants and
improved gRNA design employing bioinformatic tools. Additionally, the
efficient delivery of CRISPR-Cas9 components into target cells and tis­
sues remains a hurdle that researchers are addressing by exploring
various delivery systems, including viral vectors and nanoparticles.
Ethical considerations are paramount, particularly concerning the
editing of the human germline, necessitating responsible usage and
robust regulatory frameworks [6]. In conclusion, CRISPR-Cas9 stands as
a powerful genome editing tool, with the Cas9 protein and gRNA
working harmoniously to precisely modify target DNA sequences. Its
potential in unraveling genetic mechanisms and developing therapeutics
is substantial. Nevertheless, challenges related to off-target effects, de­
livery efficiency, and ethical implications must be diligently addressed
through continued research and responsible implementation. By doing
so, the scientific community can fully harness the potential of
CRISPR-Cas9, propelling advancements across diverse fields and deep­
ening our understanding of biology [7].
Fig. 1 CRISPR-Cas systems represent a crucial genome editing tool
extensively applied across diverse organisms, including bacteria, yeast,
and tobacco. These systems are categorized into two classes: class 1,
featuring multiple effector nucleases, and class 2, characterized by a
single effector nuclease. Notably, class 2 includes type II Cas9 and type V
Cas12a, prominent for genome editing in eukaryotic cells. Beyond DNA
modification, CRISPR-Cas systems exhibit proficiency in RNA editing.
Within class 2, Type VI CRISPR-Cas13 systems utilize a single RNA-
guided Cas13 protein with ribonuclease activity to precisely cleave
target single-stranded RNA (ssRNA). Various editing methodologies,
such as base editing and prime editing as shown in Fig. 2, have been
developed. Base editing systems, incorporating dCas9 coupled with
cytosine deaminase (CBE) or adenosine deaminase (ABE), enhance the
efficiency of site-directed mutagenesis. Prime editing, a recent
advancement, employs a fusion protein comprising Cas9 nickase and
reverse transcriptase to directly inscribe new genetic information at a
specific DNA site [8–10].
1.1. CRISPR-Cas9 technology overview and its significance in genome
editing
The CRISPR-Cas9 technology represents a revolutionary break­
through in genome editing, powered by the coordinated action of its
essential components – the guide RNA (gRNA) and Cas9 endonuclease.
The gRNA, a succinct 20-nucleotide sequence, demonstrates high gene
specificity by binding to the target DNA through the protospacer adja­
cent motif (PAM) [3]. In conjunction with the Cas9 endonuclease, the
gRNA orchestrates a precise double-strand break in the target DNA,
strategically positioned three base pairs before the PAM sequence. This
break, commonly repaired through non-homologous end joining
(NHEJ), often results in insertion or deletion (indel) mutations, dis­
rupting gene function [11,12]. The historical foundation of
CRISPR-Cas9 research can be traced to its identification as an adaptive
immune defense mechanism in bacterial cells, serving as a robust de­
fense against foreign DNA. Scientists have adeptly utilized CRISPR-Cas9
for precise genome editing across diverse organisms, resulting in sub­
stantial advancements and a refined comprehension of its mechanisms.
Its applications extend into various domains, notably contributing to
improvements in agronomic traits [13]. This review aims to scrutinize
the pivotal role of nanomaterials in enhancing the delivery and efficacy
of CRISPR-Cas9. The efficient delivery of CRISPR-Cas9 components into
target cells is crucial for the success of genome editing endeavors.
Challenges related to cellular uptake and endosomal escape have stim­
ulated innovative strategies, with nanomaterials emerging as promising
solutions, offering controlled and targeted delivery systems. Their
distinctive attributes, including expansive surface area, tunable surface

Fig. 1. Delivery methods and applications of CRISPR-Cas components. The illustration shows diverse applications and delivery methods of CRISPR-Cas components,
showcasing their pivotal role in advancing medicine and biotechnology. The schematic illustrations emphasize their applications in genetic disease corrections, drug
development, biofuel production, and plant biotechnology. The image was thoughtfully crafted using Biorender.com, illustrating the transformative potential of
CRISPR-Cas technology across various domains. The image was created with Biorender.com.

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A.A.A. Aljabali et al. Journal of Drug Delivery Science and Technology 92 (2024) 105338

Fig. 2. Genomic navigation through prime editing in crispr-cas9: explore the groundbreaking proficiency to accurately insert, delete, or substitute genetic sequences
with unparalleled precision, heralding a paradigm shift in the landscape of targeted genome engineering.

modifications, and multifunctionality, position nanomaterials as highly compared to other genome editing tools as shown in Table 1.
suitable candidates for augmenting CRISPR-Cas9 delivery efficiency.
Nanomaterials, encompassing lipid-based nanoparticles, polymeric
2.1. Explanation of the CRISPR-Cas9 system and its components (Cas9
nanoparticles, and viral vectors, exhibit tremendous potential in
protein, guide RNA)
enhancing CRISPR-Cas9 delivery. They provide protection to the
CRISPR-Cas9 components from degradation, facilitate cellular uptake,
The CRISPR-Cas9 system stands as a groundbreaking genome editing
and enable endosomal escape. Furthermore, nanomaterials can be
tool, relying on two critical components: the Cas9 protein and the guide
tailored with targeting ligands, bolstering specificity, and diminishing
RNA (gRNA). A deeper understanding of this system can be attained by
off-target effects. By harnessing nanomaterials, researchers can sur­
further exploring the structure and function of these components [3].
mount the delivery challenges entwined with CRISPR-Cas9 and elevate
The Cas9 protein, originating from bacterial immune systems, assumes
its efficiency, thereby broadening its applications across diverse bio­
the role of molecular scissors, executing precise DNA cleavage at specific
logical systems [14–16]. In conclusion, the CRISPR-Cas9 technology has
target sites. Comprising two nuclease domains accountable for DNA
spearheaded a transformative era in genome editing, furnishing a pre­
strand cleavage and a recognition domain that interacts with the gRNA,
cise and efficient mechanism for genetic manipulation. Nanomaterials
Cas9 undergoes a structural alteration when it binds to both the gRNA
represent a beacon of hope in optimizing CRISPR-Cas9 delivery, sur­
and the target DNA sequence, culminating in the creation of a
mounting existing barriers, and refining genome editing approaches.
double-strand break (DSB) at the designated location [21,22]. The
This review endeavors to explore recent advancements and strategies
gRNA, serving as a guiding beacon, directs the Cas9 protein to the
involving nanomaterials for CRISPR-Cas9 delivery, illuminating their
desired location within the genome for editing. This synthetic RNA
potential in shaping the future landscape of genome editing [17].
molecule features a scaffold region and a customizable guide sequence.
The guide sequence is intricately designed to complement the target
2. Principles of CRISPR-Cas9 technology
DNA sequence, facilitating Cas9’s binding and DNA cleavage as shown
in Fig. 3. Practical insights into gRNA selection and design, incorpo­
In the last ten years, CRISPR-Cas9 technology has gained widespread
rating considerations of target specificity, the protospacer adjacent
recognition as a groundbreaking influence in the domain of genome
motif (PAM) sequence, and effective gRNA design criteria, will signifi­
editing. It has acted as a catalyst, sparking a revolution in the field of
cantly aid researchers in comprehending and harnessing this vital
molecular biology [18–20]. This comprehensive review endeavors to
component [23,24]. By harnessing the Cas9 protein’s cutting prowess
furnish a thorough analysis of CRISPR-Cas9 technology, delving into its
and the gRNA’s specificity, the CRISPR-Cas9 system empowers precise
fundamental principles, encompassing its constituent components, the
genome modifications.
intricacies of the genome editing process, and its unparalleled strengths
The genome editing process using CRISPR-Cas9 unfolds through
several steps. Firstly, the CRISPR-Cas9 components are introduced into
Table 1 the target cells, commonly via viral vectors or direct injection. Within
Comparison of CRISPR-Cas9 with other genome editing tools. the cells, Cas9 and the gRNA form a complex that navigates the genome,
Criteria CRISPR-Cas9 TALENs ZFNs seeking the target DNA sequence based on gRNA complementarity [25,
26]. Upon locating the target site, the Cas9 protein triggers a precise
Efficiency High Moderate to High Moderate to High
Specificity High Moderate Moderate
double-strand break (DSB) at that locus. DSB repair encompasses two
Ease of Use Relatively Easy Moderate Complex primary pathways: non-homologous end joining (NHEJ) and
Design High Moderate Moderate homology-directed repair (HDR). NHEJ, recognized for its propensity
Flexibility for errors, frequently produces minor insertions or deletions (indels) at
Off-Target Possible, but can Possible, but can be Possible, but can
the DSB location. In contrast, HDR relies on a template DNA molecule to
Effects be minimized minimized be minimized
Cost Cost-effective Relatively Expensive facilitate accurate modifications [11,27]. Comprehending the implica­
Effectiveness Expensive tions of indel mutations, including frame-shift mutations and disrup­
Availability and Widely Available Moderate Limited tions in protein translation, provides valuable insights into the
Adoption and Adopted Availability and Availability and downstream effects of genome editing.
Adoption Adoption
When emphasizing the merits of CRISPR-Cas9 over alternative

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A.A.A. Aljabali et al.
Fig. 3. The CRISPR-Cas9 system meticulously reveals its sequential processes, ranging from target identification to meticulous gene alteration. Delve into the cellular
repair mechanisms, observing the expeditious Non-Homologous End Joining (NHEJ) pathway as it swiftly rectifies DNA breaks, serving as a conduit for genetic
advancements and consequential transformative applications.
genome editing tools, it is crucial to address potential limitations or
challenges. A comparative analysis with other technologies like zinc
finger nucleases (ZFNs) and transcription activator-like effector nucle­
ases (TALENs) can illuminate the superior qualities of CRISPR-Cas9,
including its flexibility in targeting, user-friendliness, and scalability
[28,29]. Furthermore, addressing the concept of off-target effects and
presenting strategies to minimize them through gRNA design, speci­
ficity, and experimental optimization will provide a balanced perspec­
tive on the limitations of CRISPR-Cas9 [30]. In conclusion,
incorporating the suggested improvements will render this review a
comprehensive resource, offering a clearer understanding of the
CRISPR-Cas9 system, its advantages, and potential limitations. Conse­
quently, researchers can wield this powerful genome editing tool more
effectively and responsibly, further advancing the frontiers of molecular
biology. The comprehensive understanding of the CRISPR-Cas9 system
and its components, as discussed in Section A, lays the groundwork for a
thorough SWOT analysis (Strengths, Weaknesses, Opportunities, and
Threats) of CRISPR-Cas9 technology [2,31]. The following SWOT
analysis highlights the transformative potential of this genome editing
tool while critically examining its limitations and challenges (see
Table 2).
3. Efficient delivery systems: unlocking the potential of CRISPR-
Cas9 gene editing
3.1. Nanoparticle-based delivery systems
Lipid-based nanoparticles have garnered substantial interest as
highly efficient carriers for Cas9 delivery. Composed of lipids, such as
cationic lipids or lipid-like materials, these nanoparticles offer a multi­
tude of advantages. These entities act as protective carriers, encapsu­
lating both Cas9 and guiding RNA molecules. This shielding effectively
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Journal of Drug Delivery Science and Technology 92 (2024) 105338
guards them against degradation and aids in their efficient delivery into
the intended target cells. Moreover, lipid-based nanoparticles boast
tunable properties, enabling optimization of crucial factors such as size,
surface charge, and surface functionalization, thereby enhancing
cellular uptake and enabling precise target-specific delivery [32–35]. To
gain a comprehensive understanding of this approach, a meticulous
examination of both its advantages and limitations is warranted. Delving
into the intricacies of various lipid carriers and their impact on delivery
efficiency, stability, and biocompatibility would illuminate the nuances
of lipid-based nanoparticle formulations. Addressing challenges such as
the immunogenicity of lipid-based nanoparticles and the need to opti­
mize lipid-to-nucleic acid ratios would provide a more comprehensive
analysis [36]. On a parallel front, polymeric nanoparticles have emerged
as promising contenders for targeted genome editing as shown in Fig. 4.
Employing biodegradable and biocompatible polymers, these nano­
particles adeptly encapsulate Cas9 and guide RNA molecules, safe­
guarding them throughout the delivery process and enabling controlled
release. The selection of polymers significantly influences critical fac­
tors, including payload release kinetics, cellular uptake efficiency, and
biocompatibility [37–39]. Expanding the discussion to encompass
various types of commonly used polymers, their degradation kinetics,
and their impact on payload release and cellular uptake would foster a
deeper understanding of this delivery strategy. Furthermore, empha­
sizing the paramount importance of nanoparticle size, surface charge,
and surface functionalization for achieving targeted genome editing
would contribute to a more comprehensive and insightful analysis.
3.2. Viral vectors as nanocarriers
Adeno-associated viruses (AAVs) have emerged as highly promising
viral vectors for in vivo CRISPR-Cas9 delivery, attracting significant in­
terest in the scientific community. These non-pathogenic viruses exhibit
remarkable efficiency in infecting target cells and delivering the
A.A.A. Aljabali et al. Journal of Drug Delivery Science and Technology 92 (2024) 105338

Table 2 vivo applications. Expanding the analysis to encompass a comparative

SWOT analysis of CRISPR-Cas9 technology: Exploring strengths, weaknesses, examination of these delivery systems and addressing potential chal­
opportunities, and threats in genome editing. lenges will enable a deeper understanding of their strengths and limi­
Aspect Strengths Weaknesses tations. Further research and development in this burgeoning field hold
CRISPR-Cas9 - Precision of guide RNA for - Risk of off-target effects,
immense promise for revolutionizing gene editing and propelling the
Efficiency targeted DNA modifications impacting accuracy frontiers of personalized medicine. Careful consideration of delivery
Delivery - Advancements in viral - Challenges inin vivo strategies and vector selection will be instrumental in harnessing the full
Methods vectors and nanoparticles applications, requiring potential of CRISPR-Cas9 in therapeutic applications, ushering in a new
for targeted delivery further development
era of precision medicine [14,45].
Versatility in - Enables simultaneous - Potential immune responses
Targeting targeting of multiple genes against Cas9 protein, CRISPR holds immense potential in reshaping personalized medicine
for complex manipulations necessitating investigation by addressing disease-causing genetic mutations. Notably, CRISPR/
and delivery optimization Cas9, coupled with next-generation sequencing (NGS), is applied in the
Established - Well-studied and optimized - Challenges inin vivo delivery personalized treatment of cancer [46,47]. This involves using
Protocols system with established methods, requiring
protocols and techniques refinement
CRISPR/Cas9 to disable or repair genes implicated in cancer develop­
Cost- - Cost-effective compared to - Time-consuming and ment, while NGS identifies patient-specific genetic mutations [8]. The
Effectiveness traditional tools expensive large-scale acquired genetic information becomes the foundation for personalized
manufacturing of Cas9 com­ treatment plans that precisely target the unique genetic anomalies
ponents, necessitating
within each patient’s cancer cells. Another promising avenue is the use
streamlined production
of saturation genome editing, employing CRISPR to systematically edit
Aspect Opportunities Threats every nucleotide in a gene to distinguish between benign and pathogenic
Delivery Methods - Advancements in viral - Challenges inin vivo mutations. Additionally, CRISPR-based assays, such as engineering T
vectors and nanoparticles applications, requiring cells for CAR-T cancer therapy and addressing inherited blindness,
for targeted delivery further development
exemplify the potential of CRISPR in advancing precision medicine [48].
Versatility in - Enables simultaneous - Potential immune responses
Targeting targeting of multiple genes against Cas9 protein,
for complex manipulations necessitating investigation 4. Enhancing CRISPR-Cas9 efficiency with nanomaterials
and delivery optimization
Established - Well-studied and - Challenges in in vivo delivery
The incorporation of nanomaterials stands as a key development in
Protocols optimized system with methods, requiring
established protocols and refinement the quest to optimize the efficiency of CRISPR-Cas9 gene editing.
techniques Leveraging the distinctive properties intrinsic to nanoscale materials,
Cost- - Cost-effective compared to - Time-consuming and researchers have identified opportunities to enhance both the activity
Effectiveness traditional tools expensive large-scale and specificity of Cas9, concurrently improving the delivery of Cas9 and
manufacturing of Cas9 com­
guide RNA into target cells. This thorough review delves into two crucial
ponents, necessitating
streamlined production facets: the use of nanomaterials for the optimization of gene editing tools
Opportunities for - Potential for gene therapy - Continuous optimization of and strategic surface engineering methods targeting enhanced cellular
Therapeutics and treatment of genetic protocols and techniques for uptake and endosomal escape [34,37]. Efficiency and specificity are
diseases enhanced efficiency and
pivotal considerations for the success of Cas9 nuclease activity in
safety
Research - Ongoing research to - Competition from genome editing. Nanomaterials, with their nuanced nanoscale modifi­
Advancements improve specificity and alternative genome editing cations, emerge as a promising platform for achieving these funda­
accuracy technologies (e.g., base mental goals. Recent advancements in nanotechnology play a critical
editing, prime editing), role in refining the precision of genome editing while minimizing
necessitating continuous
off-target effects. The functionalization of Cas9 with nanoparticles, like
advancements
gold nanoparticles or quantum dots, underscores the potential to
amplify stability, enzymatic activity, and DNA binding affinity [49,50].
essential Cas9 and guide RNA payloads [40,41]. AAVs offer a host of Recent advancements in nanotechnology have paved the way for opti­
advantages, including the ability to achieve long-term gene expression, mizing the activity and specificity of the CRISPR-Cas9 gene editing tool.
making them ideal for sustaining therapeutic effects over extended pe­ Supporting this concept with specific studies is vital to maintain scien­
riods. However, a more comprehensive exploration of the underlying tific rigor [51]. Crucially, gold nanoparticles, functionalized with
mechanisms of AAV infection and their potential limitations, such as single-stranded DNA, have demonstrated efficacy in enhancing Cas9
AAV immunogenicity, would further enrich the analysis. Understanding binding to target sites, resulting in improved DNA cleavage efficiency
the host immune response to AAVs is of utmost importance for their [52]. Graphene oxide (GO) nanosheets, distinguished by their expansive
successful application in gene therapy, as pre-existing immunity in some surface area and compatibility with biological systems, have emerged as
individuals may impact therapeutic outcomes [42]. On a parallel front, promising carriers for CRISPR components. Upon modification with
lentiviral vectors present a compelling alternative for CRISPR-Cas9 de­ specific targeting molecules and the Cas9 protein, GO nanosheets enable
livery, offering stable integration and long-term gene editing capabil­ precise delivery of CRISPR elements to specific cell types. The negatively
ities. These vectors possess the capacity to deliver larger payloads in charged nature of GO nanosheets facilitates electrostatic interactions
comparison to AAVs, rendering them suitable for applications necessi­ with the positively charged Cas9 protein and guide RNA, promoting
tating the delivery of large DNA fragments or multiple genes. However, their cellular uptake. The attachment of cell-specific targeting molecules
it is imperative to discuss the process of lentiviral integration and to GO nanosheets further enhances the selectivity of CRISPR delivery to
associated risks, such as the potential for insertional mutagenesis, to desired cell groups [53,54]. Mesoporous silica nanoparticles (MSNs),
provide readers with a well-rounded perspective. The risk of unintended investigated as carriers for CRISPR-Cas9 components, offer expansive
genomic modifications resulting from lentiviral integration should be surface areas and customizable pore dimensions, facilitating the effec­
diligently considered when evaluating their suitability for gene editing tive integration of Cas9 protein and guide RNA. The adaptability of
therapies [43,44]. In conclusion, nanomaterials play a pivotal role in MSNs extends to their customization with targeting ligands, allowing
enabling efficient CRISPR-Cas9 delivery. Both lipid-based nanoparticles precise delivery of CRISPR elements to specific cell types for refined
and polymeric nanoparticles offer distinct advantages, while viral vec­ genome editing [55,56]. Single-walled carbon nanotubes (SWCNTs),
tors such as AAVs and lentiviral vectors provide unique features for in characterized by a high aspect ratio and inherent cellular permeability,

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A.A.A. Aljabali et al.
Fig. 4. CRISPR nestled within polymeric nanocarriers exhibits versatility. These carriers are customizable, featuring biocompatible coatings and targeting moieties to
precisely guide the vesicle to specific sites. Moreover, the carriers possess an activatable coating, safeguarding Cas9 during circulation, and leverage positively
charged polymers for efficient endosomal escape. Additionally, they can be tailored with self-antigens to mitigate immune recognition. Image was generated by Bior
ender.com.
have garnered attention as promising carriers for CRISPR components.
Engineered SWCNTs, endowed with specific functionalities, interact
effectively with Cas9/guide RNA complexes, facilitating their transport
across cell membranes and presenting a prospective avenue for height­
ened efficiency in genome editing [57]. Furthermore,
nanocarrier-mediated co-delivery of Cas9 protein and guide RNA has
emerged as a promising strategy to enhance CRISPR-Cas9 editing effi­
ciency [58]. Lipid-based nanoparticles, exemplified by their effective
encapsulation of Cas9-gRNA complexes, enable simultaneous intracel­
lular delivery, resulting in improved gene editing outcomes [59]. In
summary, the integration of nanomaterials into CRISPR-Cas9 technol­
ogy signifies a dynamic frontier with substantial potential for trans­
formative progress in genome editing. The reviewed studies underscore
the intricate interplay between nanomaterials and CRISPR components,
offering insights into strategies that enhance efficiency, specificity, and
delivery precision. These advancements contribute to a broader under­
standing of the technology and open avenues for innovative solutions to
current challenges in gene editing.
4.1. Surface modifications to enhance nanoparticle stability and
bioavailability
The strategic modification of nanoparticle surfaces emerges as a key
approach to augment their stability and bioavailability, thereby
enhancing their efficacy as CRISPR-Cas9 carriers. A notable example lies
in the surface functionalization of polymeric nanoparticles with cell-
penetrating peptides, facilitating robust cellular uptake and efficient
delivery of Cas9-gRNA complexes into target cells [60–62]. Overcoming
impediments to delivery stands as a critical factor for successful
CRISPR-Cas9 gene editing. Notably, advancements in pH-responsive
nanoparticles represent a pioneering solution, displaying substantial
improvements in gene editing efficiency by enabling escape from
endosomes [38,63]. Recognizing the constraints linked to the applica­
tion of nanomaterials in CRISPR-Cas9 technology is paramount. It is
imperative to confront potential issues such as cytotoxicity or immune
reactions provoked by specific nanoparticles. An all-encompassing
comprehension of these limitations will serve as a compass for
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Journal of Drug Delivery Science and Technology 92 (2024) 105338
ongoing research and the creation of secure and efficient nanomaterials
for gene editing purposes. Acknowledging constraints associated with
nanomaterial application in CRISPR-Cas9 technology is imperative,
requiring consideration of issues like cytotoxicity or immune reactions.
A comprehensive understanding of these limitations guides ongoing
research toward the creation of secure and efficient nanomaterials for
gene editing purposes [64]. To conclude, nanomaterials exhibit signif­
icant potential for optimizing CRISPR-Cas9 gene editing through
tailored modifications and innovative strategies [65–67].
5. Applications of CRISPR-Cas9 technology
CRISPR-Cas9 technology has emerged as an extraordinarily potent
tool for genetic engineering, with extensive applications spanning
human genetic diseases and agriculture. This section aims to furnish a
comprehensive overview of its potential in these domains while also
delving into the associated challenges and ethical considerations [8,68,
69].
5.1. Human genetic diseases and therapeutic interventions
Precision Correction of Disease-Causing Mutations: CRISPR-Cas9
stands as a transformative tool, showcasing its profound capacity to
precisely target and rectify disease-causing mutations within the human
DNA. This revolutionary approach holds significant promise for treating
a myriad of genetic disorders. One illustrative example is the application
of CRISPR-Cas9 in addressing Sickle cell anemia [8]. This hereditary
condition, caused by a mutation in the HBB gene, leads to the production
of abnormal hemoglobin. CRISPR-Cas9 allows for precise modification
of the HBB gene, correcting the genetic anomaly responsible for Sickle
cell anemia. Successful applications of CRISPR-Cas9 have been docu­
mented, affirming its efficacy in providing targeted therapies for a wide
range of genetic conditions [70,71]. Moreover, CRISPR/Cas9 has suc­
cessfully rectified numerous gene mutations associated with retinitis
pigmentosa, a genetic disorder characterized by gradual vision loss.
CRISPR/Cas systems have also been employed to create models for
diseases and rectify mutations responsible for monogenic diseases.
A.A.A. Aljabali et al.
Notably, these systems exhibit promise in addressing genetic conditions
that conventional approaches struggle to reach [72,73].
Advancing Gene Therapy and Personalized Medicine: CRISPR-Cas9
spearheads the evolution of gene therapy and personalized medicine,
offering a groundbreaking approach to targeted gene manipulation. This
technology facilitates the insertion, deletion, or modification of specific
genes, unlocking potential treatments for various genetic diseases. In the
context of personalized medicine, CRISPR-Cas9 opens avenues for
tailoring treatments based on an individual’s unique genetic profile. A
notable instance involves the application of CRISPR-Cas9 in correcting
mutations associated with cystic fibrosis, a genetic disorder affecting the
respiratory and digestive systems. By precisely modifying the CFTR
gene, which is responsible for this condition, CRISPR-Cas9 holds
promise in developing personalized therapies tailored to the genetic
makeup of each patient. However, it is imperative to acknowledge the
challenges associated with ensuring the safety and efficacy of gene
therapies. Addressing concerns such as the precise delivery of CRISPR-
Cas9 components, mitigating off-target effects, and considering im­
mune responses is crucial for the success of these therapeutic in­
terventions [74,75].
5.2. Agriculture and food security
The profound impact of CRISPR-Cas9 on agriculture and global food
security is unequivocal. Through precise plant genome modification,
CRISPR-Cas9 stands as a revolutionary force, augmenting pivotal crop
traits—yield, nutritional content, and disease resistance. The notable
achievements of researchers, exemplified in Fig. 5 across diverse crops
like rice, wheat, and tomatoes, underscore the technology’s revolu­
tionary potential. Specifically, targeted modifications in genes associ­
ated with pathogen susceptibility have yielded robust and disease-
resistant crop varieties, exemplifying CRISPR-Cas9’s promise in culti­
vating resilient and high-yielding crops [76,77]. In agricultural domain
implementations, a meticulous examination of regulatory frameworks
and ethical considerations is imperative. Despite the substantial bene­
fits, divergent regulations governing genetically modified organisms
(GMOs) and gene-edited crops necessitate thoughtful scrutiny. Striking a
delicate balance between the imperative for innovative crop improve­
ment and judicious risk management is crucial in implementing
CRISPR-Cas9 in agriculture. Furthermore, ethical considerations,
encompassing potential environmental impacts and equitable benefits
distribution, demand thorough examination to ensure the responsible
and sustainable deployment of this groundbreaking technology [78–81].
In conclusion, the potential of CRISPR-Cas9 technology extends limit­
lessly in both the field of human genetic diseases and agriculture. Its
precision in genome editing opens transformative possibilities for
Fig. 5. Plant genome editing initiates with an elite variety exhibiting disease susceptibility. Through the application of CRISPR technology, the resultant plant or
crop undergoes targeted modifications, transforming into an elite variety endowed with disease-resistant traits.
7
Journal of Drug Delivery Science and Technology 92 (2024) 105338
addressing genetic disorders and revolutionizing crop traits. However,
realizing these prospects requires addressing challenges related to de­
livery methods, mitigating off-target effects, navigating diverse regula­
tory landscapes, and comprehensively addressing ethical
considerations. Sustained research, collaborative efforts, and open dia­
logue are indispensable in overcoming these obstacles, thereby fully
harnessing the boundless benefits of CRISPR-Cas9 to propel advance­
ments in human health and foster sustainable agriculture [72].
The extensive utilization of CRISPR-Cas9 technology has unques­
tionably brought about a revolutionary transformation in the field of
genome editing. However, akin to any formidable tool, its exceptional
capabilities are accompanied by a set of challenges and limitations that
necessitate thoughtful consideration for its optimal and responsible
utilization. In this section, we will delve into some of the key challenges
and limitations that are intricately linked with CRISPR-Cas9 technology
[6,82]. Table 3 summarises the major challenges and limitations of
CRISPR-Cas9 technologies.
6. Challenges and considerations in CRISPR-Cas9 genome
editing
6.1. Off-target effects and specificity concerns
The major concern surrounding CRISPR-Cas9 technology revolves
around the potential occurrence of off-target effects, challenging its
intended precision in homing in on specific DNA sequences. The
inherent risk lies in the plausible manifestation of unintended genetic
modifications at sites closely resembling the desired target sequence,
underscoring the critical importance of addressing off-target effects due
to their capacity to induce inadvertent changes in the genome, thereby
harboring the potential for unanticipated consequences [51,75,88]. To
tackle this challenge, conscientious efforts are actively underway to
enhance the specificity of CRISPR-Cas9, recognizing the imperative of
mitigating off-target effects. Noteworthy strategies have been formu­
lated for this purpose, with a significant emphasis on deploying
high-fidelity Cas9 variants and meticulously optimizing guide RNA
design to curtail off-target activity. Additionally, the integration of so­
phisticated computational algorithms and bioinformatics tools equips
researchers with the capability to predict potential off-target sites,
facilitating the judicious selection of target sequences with minimal risk
of unintended effects. These innovative approaches collectively epito­
mize a concerted endeavor to ensure the secure and precise application
of CRISPR-Cas9 in genetic editing and therapeutic interventions. This
steadfast commitment aligns with the ethical mandates of responsible
and meticulous genome engineering, thereby underscoring the dedica­
tion to advancing the technology with due diligence [91,92].
A.A.A. Aljabali et al.
Table 3
Summary of the challenges and limitations of CRISPR-Cas9 technologies.
Challenge/Limitation Description
Off-target effects and specificity • Challenge: CRISPR may unintentionally
issues modify genes like the target, leading to
undesired changes.
• Solution: Scientists use improved guide
RNA design, bioinformatics tools, and high-
fidelity Cas9 enzymes to enhance speci­
ficity [30].
Delivery efficiency and cell/tissue • Challenge: Efficiently delivering CRISPR
specificity components to target cells is crucial.
• Solution: Researchers explore viral vectors,
nanoparticles, and various strategies to
enhance delivery precision [83].
Large DNA fragment insertion • Challenge: Inserting large DNA fragments
using CRISPR faces technical complexity.
• Solution: Scientists explore alternative
delivery methods and enhance homology-
directed repair mechanisms [84,85].
Immune response and safety • Challenge: Viral vectors in CRISPR
concerns associated with viral applications may trigger immune responses
vectors and safety concerns.
• Solution: Strategies include using gutted
vectors, non-integrating vectors, and
rigorous preclinical testing for safety [86].
Delivery to specific tissues/organs • Challenge: Precise delivery to specific
tissues within an organism is challenging.
• Solution: Scientists explore tissue-specific
promoters, targeting ligands, and diverse
delivery methods [87].
Ethical considerations and • Challenge: CRISPR raises ethical questions
regulatory challenges and requires strong regulatory frameworks.
• Solution: Addressing ethical challenges
involves continuous public engagement,
strong guidelines, and risk assessment [78,
79].
Off-target detection methods • Challenge: Accurately detecting off-target
effects is critical for safety.
• Solution: Ongoing efforts focus on
improving detection methods like high-
throughput sequencing and in silico tools
[88].
Delivery to challenging cell types • Challenge: Efficient delivery to challenging
cell types (neurons, stem cells) is complex.
• Solution: Researchers develop specialized
delivery systems tailored to specific cell
types [55,83,87].
Control of gene expression and • Challenge: Achieving precise control over
spatiotemporal regulation gene expression is crucial.
• Solution: Strategies include inducible and
reversible CRISPR systems, optogenetic
tools, and synthetic biology approaches
[49,89].
Delivery to non-dividing cells • Challenge: Delivering CRISPR to non-
dividing cells presents unique challenges.
• Solution: Research focuses on alternative
repair mechanisms and cell-cycle-
dependent promoters for non-dividing cells
[59,90].
6.2. Immune response and safety considerations associated with viral
vectors
In a myriad of CRISPR-Cas9 applications, viral vectors serve as the
go-to delivery vehicles, proficiently ushering the requisite CRISPR
components into target cells. While viral vectors have proven them­
selves as effective gene delivery tools, legitimate concerns emerge
regarding immune responses and potential safety risks inherent in their
deployment [40,86]. Viral vectors possess the inherent capability to
elicit immune responses within the host organism, setting off cascades of
immune activation that may lead to undesirable effects. Furthermore,
the prospect of insertional mutagenesis looms, wherein the integration
of viral vectors into the host genome carries the inherent risk of dis­
rupting essential genes or regulatory elements. These valid safety
8
Journal of Drug Delivery Science and Technology 92 (2024) 105338
concerns emphasize the indispensability of rigorous testing and metic­
ulous evaluation of viral vector-based CRISPR applications, with a
paramount focus on ensuring their safety and the judicious minimiza­
tion of potential risks. In this light, researchers must exercise vigilance
and embrace comprehensive safety assessment protocols, embodying
the essence of responsible science to advance the secure and sound
implementation of CRISPR-Cas9 technology employing viral vectors
[14,43,84].
6.3. Ethical considerations and regulatory challenges
The extraordinary potential unlocked by CRISPR-Cas9 technology
gives rise to profound ethical considerations and regulatory challenges.
Manipulating the genetic code of living organisms raises fundamental
questions concerning the boundaries of genetic manipulation and the
potential ramifications of altering natural life processes [79]. A persis­
tent subject of debate revolves around the ethical implications of
employing CRISPR-Cas9 for germline editing, where heritable changes
to the genetic material can be transmitted to future generations. The
intricacies surrounding germline alterations mandate meticulous
consideration to preclude unintended consequences and to ensure the
utmost responsibility in its application [78]. Furthermore, the regula­
tory landscape encompassing CRISPR-Cas9 technology is in a perpetual
state of flux, with different countries and jurisdictions adopting varying
approaches to its oversight. Establishing unambiguous guidelines and
regulations to govern the application of CRISPR-Cas9 becomes impera­
tive, ensconcing ethical use, responsible implementation, and an un­
wavering commitment to human and environmental safety [80]. In
conclusion, while CRISPR-Cas9 technology brims with immense promise
across diverse applications, it is not bereft of challenges and limitations.
Tackling concerns about off-target effects, enhancing system specificity,
attenuating immune responses, and mitigating safety risks associated
with viral vectors assumes paramount significance. Moreover, navi­
gating the intricate interplay of ethical considerations and regulatory
frameworks is of the utmost consequence. By deftly confronting these
challenges, we can unlock the full potential of CRISPR-Cas9, charting a
course toward transformative advancements in genetic research, ther­
apeutics, and agriculture, all while steadfastly ensuring its safe and
ethically responsible implementation [6,15].
6.4. Delivery mechanisms and cellular uptake optimization
The success of genetic editing and therapeutic interventions using
CRISPR-Cas9 hinges on the proficient delivery of its components into
target cells. Although viral vectors are widely employed for this purpose,
ongoing research explores alternative delivery methods and improve­
ments in cellular uptake mechanisms. Non-viral delivery systems,
including nanoparticles and electroporation, offer potential advantages
such as reduced immunogenicity and enhanced precision. Moreover,
optimizing the interplay between CRISPR components and cellular
machinery holds promise for augmenting editing efficiency. Addressing
challenges related to cell type-specific delivery and minimizing off-
target effects during the delivery process remains crucial for
advancing the applicability and safety of CRISPR-Cas9 technology [37,
59,93].
7. Future perspectives and emerging trends
The field of CRISPR-Cas9 genome editing has witnessed astounding
progress, culminating in transformative changes within the domain of
gene manipulation. This section endeavours to present an exhaustive
analysis and critical assessment of recent developments in CRISPR-Cas9
technology, concurrently exploring prospective trajectories and
emerging trends [94]. A pivotal facet of discussion is the rapid evolution
of CRISPR-Cas9 technology itself. Researchers have achieved notable
headway in augmenting the system’s efficiency and specificity,
A.A.A. Aljabali et al.
diligently addressing concerns pertaining to off-target effects and de­
livery challenges. Integration of high-fidelity Cas9 variants and
fine-tuning guide RNA design has significantly contributed to the
minimization of off-target activity. Moreover, computational algorithms
and bioinformatics tools play an indispensable role in predicting po­
tential off-target sites, serving as guiding beacons for the judicious se­
lection of target sequences with nominal off-target effects [95].
Additionally, the adoption of nanomaterial-based strategies as delivery
systems has emerged as a propitious avenue for enhancing the efficacy of
CRISPR-Cas9. Nanoparticle-based carriers, exemplified by lipid-based
nanoparticles and polymeric nanoparticles, have demonstrated
remarkable potential in safeguarding Cas9 and guiding RNA molecules
during delivery, augmenting cellular uptake, and facilitating
target-specific gene editing. Furthermore, the promise of viral vectors,
including adeno-associated viruses (AAVs) and lentiviral vectors, looms
large in the context of in vivo applications, empowered by their capacity
to confer long-term gene expression and accommodate larger payloads
[93,96]. CRISPR-Cas9’s applications span various domains, including
human genetic diseases, agriculture, and beyond. Its effectiveness at the
DNA level, particularly in correcting disease-causing mutations, holds
potential for targeted therapies in genetic disorders. Within agriculture,
the potential for CRISPR-Cas9 to revolutionize crop improvement,
endowing precise modifications to plant genomes to elevate yield,
nutritional content, and disease resistance, inspires optimism. More­
over, technology casts its transformative shadow across a plethora of
domains, from cancer research and regenerative medicine to environ­
mental conservation [97]. The success of utilizing CRISPR-Cas9 for ge­
netic editing and therapeutic applications relies heavily on the effective
delivery of its components into target cells. While viral vectors are
commonly used for this purpose, ongoing research is actively exploring
alternative delivery methods and enhancements in cellular uptake
mechanisms. Non-viral delivery systems, including nanoparticles and
electroporation, present potential advantages such as reduced immu­
nogenicity and improved precision. Furthermore, the optimization of
the interplay between CRISPR components and cellular machinery
shows promise in enhancing editing efficiency. Addressing challenges
associated with cell type-specific delivery and minimizing off-target
effects during the delivery process remains imperative for advancing
the applicability and safety of CRISPR-Cas9 technology [98]. By
assimilating opposing viewpoints and meticulously evaluating extant
literature, this section aspires to provide a nuanced and
all-encompassing overview of the present state and prospects of
CRISPR-Cas9 technology. Through tireless research and collaborative
synergy, the full potential of CRISPR-Cas9 can be harnessed, ushering in
momentous advancements in genetic research, therapeutics, and agri­
culture, all while steadfastly ensuring its responsible and ethically
conscientious implementation [99,100].
Over the last decade, the strides made in augmenting the efficiency,
precision, and versatility of CRISPR-Cas9 have been nothing short of
extraordinary. The advent of innovative Cas9 variants, encompassing
high-fidelity and enhanced specificity variants, stands as a testament to
the tireless pursuit of reducing off-target effects and elevating the ac­
curacy of gene editing. Moreover, the advent of base editing and prime
editing techniques has ushered in an era of boundless possibilities for
CRISPR-Cas9, eliminating the reliance on DNA double-strand breaks and
permitting precise modifications with unparalleled finesse. These
groundbreaking advancements have propelled CRISPR-Cas9 to the
forefront of therapeutic interventions and fundamental research pur­
suits, assuming a pivotal role in transforming the landscape of genetic
manipulation [101]. Yet, amidst this laudable progress, it is incumbent
upon us to acknowledge the persistent challenges tethered to
CRISPR-Cas9 technology. Despite commendable progress in alleviating
off-target effects through enhanced Cas9 variants, the continued
concern about such effects highlights the essential need for a thorough
investigation into potential long-term consequences, ensuring the safety
and efficacy of CRISPR-Cas9-based therapies. Overcoming the challenge
9
Journal of Drug Delivery Science and Technology 92 (2024) 105338
of precisely delivering CRISPR-Cas9 components to specific cells and
tissues, especially in in vivo applications, remains a formidable task.
Successfully conquering these challenges is imperative for the smooth
translational integration of CRISPR-Cas9 technology into clinical prac­
tice [102]. The endeavor to elevate the prowess of CRISPR-Cas9 is far
from abating. The sustained dedication of scientists and researchers
worldwide to unravel its intricacies and surmount its hurdles bears
testament to the profound significance of this revolutionary technology.
As we tread the path towards harnessing CRISPR-Cas9’s full potential,
fortified by an unwavering commitment to precision, safety, and ethical
consciousness, we traverse the precipice of a new era in medicine and
science, where genetic editing stands poised to reshape the trajectory of
human health and scientific inquiry [94].
The emergence of nanomaterials as a highly promising and sophis­
ticated avenue to bolster the efficiency and delivery of CRISPR-Cas9
represents a pivotal turning point in the landscape of genome editing.
The strategic utilization of diverse nanoparticle-based systems,
including liposomes, polymeric nanoparticles, and viral vectors, has
culminated in the successful encapsulation, and safeguarding of CRISPR-
Cas9 components during the critical delivery phase. This ingeniously
harnessed technology not only bestows heightened stability and cellular
uptake upon CRISPR-Cas9 but also orchestrates the precise delivery to
designated cells or tissues, thus ushering in an era of unparalleled ac­
curacy in gene editing endeavors [50,60,89]. The advancements ach­
ieved in this domain are unquestionably praiseworthy, yet the crucible
of strategies employing nanomaterials requires unwavering scrutiny.
The looming specter of potential toxicity, immunogenicity, and
off-target effects associated with nanoparticles necessitates navigating
this uncharted territory with the utmost circumspection, underscoring
the need for a meticulous pre-clinical assessment of these concerns.
Moreover, the formidable technical hurdle of efficiently delivering
nanomaterials to specific cell types within the complex tapestry of bio­
logical environments necessitates an indefatigable pursuit of optimiza­
tion. Future research initiatives must resolutely prioritize the design of
nanoparticles, ensuring safety, and delivery efficiency at their core. By
meeting these challenges head-on, the full clinical potential of
CRISPR-Cas9 can be unveiled, heralding a transformative chapter in the
annals of gene editing and galvanizing a new era of scientific innovation
[15]. The far-reaching impact of CRISPR-Cas9 technology extends
beyond the domain of human therapeutics, ushering in a new era of
groundbreaking discoveries in basic research and agricultural innova­
tion. This unparalleled tool empowers scientists to navigate the intricate
web of gene function and unravel the complexities of disease mecha­
nisms with unprecedented accuracy and finesse. In agriculture, CRISP­
R-Cas9’s immense potential for targeted genetic modifications holds the
key to elevating crop yields, enhancing nutritional content, and forti­
fying crops against the onslaught of diseases. Furthermore, it possesses
the potential to address infectious diseases through the strategic engi­
neering of disease-resistant crops or the genetic modification of disease
vectors, marking a paradigmatic shift in the battle against agricultural
challenges [103]. However, even amidst this promising landscape, the
ethical and regulatory dimensions loom large, casting a discerning gaze
on the wide-scale implementation of CRISPR-Cas9. The ethical imper­
ative to navigate the treacherous waters of unintended consequences
and responsible genome editing cannot be overlooked. A resolute
exploration of the profound ethical implications, supplemented by in­
clusive and meaningful stakeholder engagement, is indispensable.
Robust regulatory frameworks must stand as sentinels to safeguard the
moral compass guiding the ethical and responsible utilization of
CRISPR-Cas9 [104]. In conclusion, the path ahead for CRISPR-Cas9
resonates with boundless promise and potential, as continuous strides
and the seamless integration of nanomaterial-based strategies fortify its
precision, delivery efficiency, and versatility across multifarious do­
mains. However, humility in the face of challenges is the hallmark of
true progress. Prying opens Pandora’s box of off-target effects, navi­
gating delivery limitations, peering through the veil of nanoparticle
A.A.A. Aljabali et al.
toxicity, and grappling with the profound ethical considerations beckon
forth our undivided attention. It is through embracing this compre­
hensive and balanced approach that the scientific community can
orchestrate a harmonious symphony, unlocking the full and trans­
formative potential of CRISPR-Cas9 while steering its compass true to
the shores of ethical and responsible application [105,106].
8. Expert opinion
The exploration of challenges and considerations in CRISPR-Cas9
genome editing, as previously outlined, serves to illuminate the vast
potential and concurrent limitations of this pioneering technology. In
this “Expert Opinion” section, we will embark on a deeper analysis of the
implications arising from these discoveries, contemplating CRISPR-
Cas9’s trajectory in practical applications, recognizing areas necessi­
tating enhancement, charting the course for future research, and fore­
casting the field’s evolution over the next half-decade.
1. Real-World Impact: The advancements achieved in CRISPR-Cas9
technology, as expounded upon in preceding sections, hold far-
reaching implications for real-world outcomes. A particularly
promising facet lies within the field of human therapeutics. The ca­
pacity to rectify disease-causing mutations at the genetic level opens
the door to personalized medicine. Envisaging a future where genetic
disorders, once deemed insurmountable, can be addressed through
CRISPR-Cas9-based therapies is compelling. However, it’s essential
to acknowledge that despite the rapid progress of this technology,
translating these breakthroughs into clinical practice confronts
multiple challenges. Ensuring the safety and efficacy of CRISPR-
based treatments, navigating complex ethical dilemmas, and estab­
lishing robust regulatory frameworks are imperative steps in mate­
rializing these innovations. Furthermore, the economic feasibility
and accessibility of such treatments require thorough deliberation.
2. Areas Requiring Enhancement: Notwithstanding the remarkable
strides made in CRISPR-Cas9 technology, there are noteworthy areas
that necessitate further refinement. Foremost among these chal­
lenges is the mitigation of off-target effects. While the utilization of
high-fidelity Cas9 variants and optimized guide RNA designs has
been beneficial, there remains room for improving the system’s
precision. Additionally, the efficient delivery of CRISPR components
to target cells, especially in in vivo applications, poses a substantial
hurdle. The development of advanced delivery systems capable of
precisely reaching specific cell types becomes paramount. Technical
constraints, such as the potential toxicity and immunogenicity of
nanoparticles, demand comprehensive attention to unlock the full
potential of nanomaterial-based strategies. Furthermore, the ethical
and regulatory dimensions, particularly those related to germline
editing, call for judicious consideration and guidance.
3. Prospects for Future Research: The potential for further research in
the domain of CRISPR-Cas9 technology is vast. Researchers are
ceaselessly committed to refining the technology’s precision, safety,
and efficacy. The ongoing exploration of base editing and prime
editing techniques holds the promise of even more accurate genome
modifications, obviating the need for double-strand breaks. These
advances carry the potential to revolutionize the treatment of genetic
disorders, cancer, and infectious diseases. Additionally, the agricul­
tural sector stands to gain significantly from CRISPR-Cas9, with the
potential to augment crop yield, nutritional content, and disease
resistance. Subsequent research should be dedicated to perfecting
delivery methods, addressing off-target effects, and broadening the
scope of applications.
4. The Future Trajectory: The future of CRISPR-Cas9 research appears
promising, albeit necessitating a judicious approach. While tech­
nology continues to evolve and present enticing prospects, it is
imperative to acknowledge the persistent challenges. Off-target ef­
fects, delivery constraints, and ethical considerations will remain at
10
Journal of Drug Delivery Science and Technology 92 (2024) 105338
the forefront of discourse. Nevertheless, through unwavering dedi­
cation and interdisciplinary collaboration, the transformative po­
tential of CRISPR-Cas9 in the domains of biomedicine and
agriculture can be harnessed. We anticipate a future where CRISPR-
based therapies become more accessible, safe, and effective, ulti­
mately leading to improved human health outcomes.
5. Evolution of the Field: Looking ahead, it is plausible that CRISPR-
Cas9 will become a standard procedure in various fields within the
next five to ten years. The technology is likely to gain widespread
acceptance in clinical practice, offering innovative therapeutic ap­
proaches for a spectrum of diseases. The development of refined
delivery methods and augmented safety measures will be pivotal in
realizing this vision. Furthermore, we anticipate that CRISPR-Cas9
research will continue to progress, with a focus on expanding its
applications in fields such as regenerative medicine, environmental
conservation, and disease resistance in agriculture. However, the
ethical and regulatory landscape will evolve in tandem, ensuring that
CRISPR-Cas9 is used responsibly and ethically.
6. Precision Medicine: The imminent impact of CRISPR-Cas9 on human
therapeutics heralds a new phase in precision medicine. The tech­
nology’s undeniable capability to rectify disease-causing mutations
at the genetic level underscores its significance. However, tran­
sitioning from laboratory breakthroughs to clinical applications re­
quires meticulous coordination, incorporating safety assessments,
ethical scrutiny, and regulatory structures. The economic feasibility
and global accessibility of CRISPR-based therapies are pivotal con­
siderations, underscoring the necessity for a collaborative initiative
to bring these innovations to diverse populations.
In summation, the insights drawn from the examination of chal­
lenges and considerations in CRISPR-Cas9 genome editing shed light on
the promises and obstacles of this groundbreaking technology. The path
ahead necessitates a collective effort to address limitations, hone tech­
niques, and navigate intricate ethical and regulatory landscapes.
Through sustained research, collaboration, and an unwavering
commitment to safety and ethics, CRISPR-Cas9 stands poised to revo­
lutionize the fields of medicine and agriculture, offering innovative so­
lutions to some of the most pressing challenges of our time. Over the
next five years, significant progress is anticipated, bringing us closer to a
future where CRISPR-Cas9 assumes an integral role in our pursuit of
enhanced well-being and a more sustainable world.
9. Conclusion
In the last quinquennial, the CRISPR-associated protein 9 (Cas9)
technology has traversed a remarkable trajectory, emerging as an
unparalleled force in the field of genome editing. Its transformative
impact has radiated across diverse domains, transcending animal
research, medical frontiers, human gene therapy, and the frontiers of
plant biology, particularly in the augmentation of crop traits. The ca­
pacity to generate genetic knock-out mutants and the strides in multi­
plex genome editing methodologies have unfurled the full expanse of its
potential in plant-based research. The confluence of nanomaterials with
CRISPR/Cas9 has kindled a burgeoning fascination. The allure of
nanomaterials lies in their distinct attributes, showcasing a copious
surface area and customizable physical properties, rendering them
enticing carriers for ferrying CRISPR/Cas9 components to target cells.
Lipid nanoparticles, polymer nanoparticles, and mesoporous silica
nanoparticles have unfailingly encapsulated and facilitated the delivery
of CRISPR/Cas9 complexes, conferring a boost to cellular uptake and the
panache of gene editing efficiency. This symbiosis epitomizes the
panacea to surmount the formidable delivery challenges and scale the
heights of precision and efficacy in genome editing techniques. The vista
of the future casts an ethereal glow on CRISPR/Cas9 technology and
genome editing. Unyielding research endeavors strive to refine and
optimize the CRISPR/Cas9 system, commencing a relentless pursuit of
A.A.A. Aljabali et al.
overcoming the shadow cast by off-target effects, amplifying the celerity
of efficiency, and tailoring delivery stratagems to ensnare specific cell
types or tissues. The allure of therapeutic applications looms large,
enlivening a rhapsodic symphony that envisions treatments for genetic
disorders, cancer, and infectious diseases, unfurling a kaleidoscope of
personalized medicine and precision therapeutics. Yet, even as we stand
poised on the cusp of limitless possibilities, a constellation of challenges
demands our undivided attention, a clarion call to fulfil the pact of the
promise of CRISPR/Cas9. With unwavering vigilance, we must enshrine
the sacrosanct tenets of safety and specificity in genome editing tech­
niques, grapple with the deep-seated ethical considerations that un­
derpin the ethical contours of our choices, and erect robust regulatory
frameworks that stand as bastions to preserve the ethical integrity and
welfare of the masses. Interdisciplinary collaborations, an intricate
tapestry woven by the deft hands of scientists, clinicians, policymakers,
and regulators, shall provide the compass that guides us through the
labyrinthine landscapes and propels us to harness the transformative
impact of CRISPR/Cas9 in the epoch of biomedicine. In summary, the
past quinquennium has unfurled the petals of progress in CRISPR/Cas9
technology and genome editing, ushering in a new dawn in the fields of
molecular biology. Its radiance illuminates a constellation of opportu­
nities, embellishing the tapestry of scientific exploration, enriching the
canvas of agricultural advancement, and illuminating the firmament of
therapeutic applications. As we traverse the vistas of continued ad­
vancements, converge in interdisciplinary collaborations, and orches­
trate the confluence of nanomaterial integration, a vibrant symphony
reverberates, a cadence that heralds transformative discoveries, novel
therapies, and a reverberating symphony of improved human health
outcomes.
Financial & and competing interests disclosure
This manuscript received no financial support, and the authors
declare no competing interests.
Data sharing statement
No data generated from this manuscript is to be shared.
CRediT authorship contribution statement
Alaa A.A. Aljabali: Writing – review & editing, Writing – original
draft, Visualization, Investigation, Conceptualization. Mohamed El-
Tanani: Writing – review & editing, Writing – original draft. Murtaza
M. Tambuwala: Writing – review & editing, Writing – original draft,
Supervision, Project administration.
Declaration of competing interest
The authors declare no conflict of interest.
Data availability
No data was used for the research described in the article.
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