AAV Vector-Based CRISPR Delivery: Innovations and Challenges in Therapeutic Genome Editing

Advances in recombinant adeno-associated virus (rAAV)–based CRISPR delivery continue to redefine the landscape of in vivo genome editing. A recent review article titled “Therapeutic in vivo genome editing: innovations and challenges in rAAV vector-based CRISPR delivery”, published in Gene Therapy (2025) by Jin-Seok Gil, Soyeon Lee, and Taeyoung Koo, provides one of the most comprehensive and up-to-date analyses of this rapidly evolving field.

The authors—researchers from Kyung Hee University’s Department of Regulatory Science and Department of Pharmaceutical Sciences—synthesize a broad spectrum of scientific advances across CRISPR engineering, rAAV vector design, clinical translation, and capsid innovation. Genome editing technologies like CRISPR have transformed medicine by enabling precise modifications to DNA, offering hope for treating genetic disorders. When combined with rAAV vectors—known for their safety and efficiency in gene delivery—these tools become powerful for in vivo therapies, where editing occurs directly inside the body. Their review provides a comprehensive overview of the latest strategies, breakthroughs, and obstacles in using rAAV vectors to deliver CRISPR-Cas systems directly into the body for therapeutic genome editing.

CRISPR Mechanisms and Precision Tools

The paper introduces the integration of CRISPR systems with rAAV vectors as a game-changer in gene therapy. CRISPR, derived from bacterial immune systems, allows targeted DNA cuts, while rAAV vectors serve as safe delivery vehicles for these tools into living organisms.

CRISPR-Cas systems use a programmable Cas nuclease and a guide RNA (gRNA) to recognize a target sequence adjacent to a PAM and introduce a double-strand break (DSB) in DNA. The break is repaired either by non-homologous end joining, which generates indels for gene knockout, or by homology-directed repair if a donor template is provided for precise sequence correction or insertion. These core mechanisms underpin all AAV therapy designs that use CRISPR nucleases, base editors, or prime editors in vivo.​​

Beyond basic CRISPR, advanced tools like base editors (BEs) and prime editors (PEs) avoid DSBs for safer editing. Cytidine base editors (CBEs) convert C to T, adenine base editors (ABEs) change A to G, and PEs use reverse transcriptase for versatile changes. To reduce dependence on DSBs, CBEs and ABEs convert single bases using deaminase enzymes fused to nCas9, while prime editors combine nCas9 with a reverse transcriptase and a prime-editing guide RNA to support small insertions, deletions, and substitutions without donor DNA. These platforms are highly attractive cargoes for AAV therapy but significantly stress AAV packaging capacity and thus drive advanced AAV construction and dual-vector strategies. For AAV manufacturing and AAV packaging service providers, these tools create demand for customized vector manufacturing workflows that can reliably support large and complex CRISPR payloads.​​

Why rAAV for in vivo CRISPR?

CRISPR genome editing has transformed the treatment potential for genetic and rare diseases. Ex vivo CRISPR therapies, such as the FDA-approved Casgevy for sickle cell disease, have validated the therapeutic benefit of gene editing. However, many tissues—such as retina, CNS, liver, and muscle—require direct in vivo delivery of CRISPR reagents, which demands a safe and efficient vector.

rAAV vectors have become the dominant platform for in vivo CRISPR delivery because they combine:

• Excellent safety profile
• Broad tropism
• Long-term episomal expression
• Low immunogenicity

These advantages, paired with advances in AAV manufacturing and vector production, have positioned rAAV as the leading choice for next-generation genomic medicines.

However, the limited AAV packaging capacity (≈4.7 kb) influences AAV construction choices and drives innovation in AAV capsid engineering, dual-vector systems, and compact Cas ortholog development.​​

AAV capsid engineering enhances tropism, directing vectors to organs like the liver or retina. AAV production and AAV manufacturing processes are crucial here, with AAV packaging services optimizing cargo loading. Viral vector manufacturing scales up production, while custom AAV and AAV construction enable bespoke designs for overcoming size limits, broadening therapeutic potential in disorders like retinitis pigmentosa.

Clinical Progress with rAAV–CRISPR

The first in vivo rAAV–CRISPR trial (EDIT-101) uses an AAV5 vector to deliver SpCas9 and dual gRNAs to photoreceptors in CEP290-associated Leber congenital amaurosis type 10. Delivered subretinally, EDIT-101 excises a mutation to restore splicing, achieving acceptable safety and evidence of functional rescue in a subset of patients. The BRILLIANCE trial improved vision in most participants but was paused for partnering. These data validate rAAV-based AAV therapy for in vivo editing but also highlight challenges: modest efficacy, small patient populations, and the need to refine vector manufacturing and AAV production processes to improve dose–response and safety. Lessons from such trials are now influencing AAV service offerings, including custom AAV serotype selection and CRISPR-optimized AAV packaging.​​ Viral vector manufacturing and AAV manufacturing handle scale-up, while custom AAV construction adapts for rare diseases, highlighting challenges like low editing rates

Development of rAAV Vector Platforms for In Vivo CRISPR Delivery

1. All-in-One rAAV Vectors Employing Compact Nucleases

This strategy focuses on reducing the size of the genome editing components, so they fit into a single rAAV vector—an “all-in-one” solution—a major advantage for AAV packaging services and scalable viral vector manufacturing. This is often achieved by utilizing naturally hypercompact Cas variants, such as Cas9 from Staphylococcus aureus (SaCas9, = 3.2 kb) or the ultra-compact Cas12f.

Other examples include:

• CjCas9
• Nme2-ABE8e
• IscB and TnpB ultra-compact nucleases

These miniaturized Cas proteins reduce genome size and improve AAV production yields, since vectors with optimized genome lengths are easier to manufacture and package at high titers.

2. Dual rAAV Vectors for Full-Length CRISPR Delivery

Dual rAAV systems allow delivery of full-length Cas9 with multiple gRNAs and/or large donor templates via two coordinated vectors. Preclinical work in dystrophin restoration demonstrates that dual rAAV9 vectors can mediate targeted exon integration and restore functional transcripts and protein in DMD models after early systemic dosing. These designs are more demanding for AAV therapy programs and AAV production service teams because they require precise titer control, co-formulation strategies, and careful characterization of AAV packaging efficiency for both components.​​

Figure 1. Single- and Dual-rAAV Vector Strategies

3. Dual rAAV Vectors for Split-CRISPR Delivery

These systems deliver halves of a large Cas protein that later reassemble via either protein trans-splicing or RNA trans-splicing. For even larger genome editors like PEs and BEs, which are too big even for the dual vector approach, trans-splicing technologies allow the Cas effector gene to be split and reconstituted inside the target cell.

1. Protein Trans-Splicing rAAV Vectors (Figure 2)

This advanced AAV construction method relies on self-catalyzing protein elements called inteins to reassemble the full-length enzyme after the two separate vector components are delivered and translated.

• How it works: Cas9 is split into N- and C-terminal fragments, each packaged into separate rAAVs. Upon co-expression, intein-mediated splicing reassembles the functional protein.
• Therapeutic use: Successfully applied in ALS, Niemann-Pick disease, and retinal disorders.
• Manufacturing note: Requires high-precision AAV construction and stringent quality control to ensure proper splicing and avoid immune responses to intein fragments.

Protein trans-splicing rAAV vectors’ performance depends on co-transduction efficiency, intein splicing kinetics, tissue context, and balanced expression of each half, all of which must be factored into AAV therapy design and AAV production planning. This increases the importance of advanced viral vector manufacturing capabilities, including precise AAV packaging control for each split component and sensitive potency assays.​​

Figure 2. Protein Trans-Splicing rAAV Vectors Encoding Split-CRISPR Molecules

1. RNA Trans-Splicing rAAV Vectors (Figure 3)

These vectors rely on splice donor (SD) and splice acceptor (SA) sites to assemble full-length mRNA molecules after transcription. They avoid nonfunctional protein fragments and offer more flexible split sites.

• How it works: Uses endogenous spliceosomes to ligate separate mRNA transcripts into a full-length coding sequence.
• Advantage: Avoids potential immunogenicity of intein domains.
• Example: In DMD mice, RNA trans-splicing rAAVs achieved functional dystrophin restoration.

In addition, trans-splicing ABE vectors have also reduced tau pathology in MAPT-driven tauopathy models, and restored Rpe65 function and visual responses in LCA models after subretinal AAV therapy. These studies show that even modest editing levels (a few percent) can be therapeutically significant when combined with tissue-specific AAV capsid engineering and optimized AAV production. RNA trans-splicing designs highlight the need for highly customized AAV construction and analytics to confirm concatemer and splicing behavior in relevant tissues.​​

Figure 3. RNA Trans-Splicing rAAV Vectors Encoding Split-CRISPR Molecules

1. mRNA trans-splicing rAAV-CRISPR

The REVeRT (Reconstitution via mRNA Trans-splicing) system (Figure 4) represents an advanced mRNA trans-splicing platform, enabling efficient reconstitution across multiple tissues without relying on ITR-mediated genomic recombination. Dual rAAV vectors transcribe separate pre-mRNAs that contain complementary binding domains (BDs). These BDs hybridize, bringing the engineered SD and SA sequences into close proximity to facilitate the RNA splicing event, producing a full-length Cas effector mRNA. This innovation highlights the growing sophistication of AAV service vector manufacturing and custom AAV design capabilities.

Figure 4. mRNA trans-splicing rAAV-vectors for delivering split-CRISPR components

Capsid engineering for targeted delivery

Native AAV serotypes exhibit biased tropism—strong liver targeting and variable muscle and CNS transduction—which can be limiting for extrahepatic genome editing. Library-based directed evolution has produced capsids like AAV-PHP.B and AAVMYO that improve CNS or skeletal muscle delivery while reducing off-target liver transduction, and rational design and machine-learning approaches (e.g., myoAAV, Fit4Function capsids) are further tuning multi-trait performance and cross-species predictability. These advances directly feed into AAV capsid engineering pipelines in viral vector manufacturing and AAV service organizations, enabling sponsors to match capsid, promoter, and CRISPR modality to desired tissue and safety profile for a given AAV therapy.​​

High-fidelity CRISPR platforms

To mitigate off-target editing, high-fidelity Cas9 variants (eSpCas9, SpCas9-HF1, efSaCas9) and improved base editors (ABE8e-V106W, eCBE) incorporate mutations that reduce non-specific DNA interactions while maintaining on-target efficiency. Prime editing fidelity is being refined via pegRNA engineering, such as mismatch-containing mpegRNAs that lower indel formation without sacrificing editing rates. These high-fidelity platforms are particularly important for in vivo AAV therapy because persistent expression after AAV packaging can amplify off-target effects, so AAV construction and AAV manufacturing strategies increasingly integrate HF CRISPR variants as standard options in custom AAV projects.​​

Immunological challenges

Despite relatively low innate immunogenicity, rAAV vectors can trigger humoral and cellular immunity, especially in the presence of pre-existing neutralizing antibodies to AAV capsids. CRISPR components themselves, particularly bacterial Cas nucleases, can elicit T-cell responses that clear edited cells and reduce durability of AAV therapy. Countermeasures include engineering less immunogenic Cas variants, self-eliminating AAV designs that limit Cas expression, capsid engineering to evade neutralizing antibodies, transient immunosuppression, and exploration of non-viral delivery for certain indications, all of which influence how AAV manufacturing and AAV production services design dosing regimens and release criteria.​​

Safety considerations and regulatory context

High-dose systemic rAAV administration has been associated with serious adverse events and fatalities in some gene therapy trials, often related to immune-mediated toxicities and liver injury. These events have led to increased regulatory scrutiny, emphasizing careful dose escalation, long-term follow-up, and more rigorous risk–benefit assessments for AAV therapy, especially when combined with permanent CRISPR editing. As a result, viral vector manufacturing groups are prioritizing robust AAV production, improved characterization of vector genomes, and integrated AAV packaging quality controls to minimize empty/full heterogeneity and off-target biodistribution.​​

Conclusion

The remarkable progress in addressing the AAV packaging constraint—through compact Cas nucleases, dual vectors, and sophisticated trans-splicing mechanisms—is rapidly accelerating the translation of CRISPR systems into viable AAV therapy options. These innovative AAV construction strategies are paving the way for the treatment of numerous diseases previously considered intractable.

Despite the therapeutic promise, challenges remain, primarily concerning immune responses to both the rAAV vectors (pre-existing neutralizing antibodies) and the foreign bacterial-derived CRISPR enzymes. Efforts to combat these issues, including high-fidelity Cas variants, immune-evading AAV capsids, and transient immunosuppression protocols, are central to ensuring the long-term safety and durability of these treatments.

The innovations summarized in the paper collectively reshape expectations for AAV packaging, AAV production, and custom AAV design in clinical programs.​​

• All-in-one compact systems are highly compatible with current AAV manufacturing platforms and can streamline AAV production service offerings.
• Dual-vector and trans-splicing systems demand more sophisticated vector manufacturing strategies, including matched titers, co-formulation, and specialized AAV analytical release assays.
• Capsid and CRISPR engineering innovations require close collaboration between investigators and AAV service providers to coordinate AAV construction, AAV capsid engineering, and regulatory-grade viral vector manufacturing.

Overall, rAAV–CRISPR platforms are driving the need for integrated AAV therapy solutions that combine advanced biology with scalable, high-quality AAV manufacturing and AAV packaging service capabilities.

References:

https://www.nature.com/articles/s41434-025-00573-2