AAV Vectors for CRISPR-mediated in Vivo Genome Editing
The clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9-based RNA-guided DNA endonuclease is transforming biomedical research and has already become a popular genome-editing tool for interrogating in vivo gene function. Adeno-associated viral (AAV) vector is a preferred delivery vehicle for tissue-directed gene therapy, which packages the CRISPR/Cas9 system (AAV-CRISPR) and can significantly modify endogenous disease-relevant genes with high efficiency. As a first-class supplier in the gene therapy, Creative Biolabs is committed to state-of-the-art AAV vector design for targeted gene editing and particularly offers versatile AAV-CRISPR services with low immunogenicity and long-term gene expression for incurable diseases.
Overview
The CRISPR-based genome-editing system has thoroughly revolutionized the gene-editing technology owing to its simplicity in target design, high efficiency, affordability, versatility, and multiplexing capability. In this system, Cas9 endonuclease generates double-strand DNA breaks (DSBs) at desired locations of genome, directed by a single guide RNA (sgRNA) comprised of 20 nucleotides. The commonly used gene-editing strategy can be performed in mammalian cells by co-expressing Cas9 nuclease with chimeric sgRNA.
AAV particles are a leading candidate for the delivery of CRISPR/Cas9 to somatic tissues in humans. They provide major advantages over other viral vectors for basic research and disease treatments, including a very mild immune response and almost no side effect. Moreover, AAV remains primarily episomal under transduction, avoiding random integration of viral genic materials into the host genome. It has been reported that the CRISPR/Cas9 packaged in AAV vectors could permanently modify multiple functional genes in the retina, livers, heart, brain, and skeletal muscle-related disorders.
AAV-CRISPR System for in Vivo Genome Editing at Creative Biolabs
AAV-mediated gene transfer has been widely used in the CRISPR/Cas9 system, showing promising therapeutic efficacy with good safety in a number of animal models and human clinical trials. The CRISPR/Cas9 complex can be introduced into the cells in the forms of DNA, mRNA, or protein. The combined use of CRISPR/Cas9, AAV serotypes, tissue-specific minimal promoters, and different routes of administration can promote the development of efficient and precise in vivo genome editing.
Figure 2. CRISPR/Cas9-based in vivo genome editing by using small Cas9 orthologues, different routes of AAV administration, tissue-specific minimal promoters, and AAV serotypes. (Lau, 2017)
Recent studies of smaller Cas9 orthologues enabled the packaging of Cas9 nuclease and its synthetic sgRNA into a single AAV delivery vehicle for strong in vivo genome editing. For most Cas9 genes, the expression of both Cas9 and sgRNA is beyond the packaging capacity of the single AAV vector. Therefore, Creative Biolabs has designed two classes of AAV vectors, of which, one vector is in charge of the Cas9 expression while the other comprises sgRNA expression cassettes and DNA sequences for reporter genes or donor templates for homology-directed repair. So far, the CRISPR/Cas9 system delivered with these two AAV vectors has been implemented successfully to correct gene mutations in many different diseases.
Furthermore, the DNA encoding Cas9 and sgRNA is transferred into the cell through AAV expression vectors. Through microinjection, electroporation, or nucleofection, or liposome-mediated transfection, the delivery format of active Cas9 protein/gRNA ribonucleoprotein complex has lower off-target effects and speeded gene editing.
Advantages
- Low immunogenicity and toxicity induced by CRISPR components and AAV vehicles
- Stable transgene expressions, avoiding random integration of viral genic materials into the host genome
- A good safety profile of AAV-CRISPR system and great efficacy in animal models and human clinical trials
- Efficient packaging and precise delivery of AAV-CRISPR vector for in vivo genome editing in specific diseases
With the advent of CRISPR-based genome-editing techniques, AAV particle becomes one of the most suitable and accessible viral vectors to package, transfer, and express CRISPR elements for targeted genes. As an expert in vector design, Creative Biolabs offers abundant viral vector design services to achieve the great potential of AAV-CRISPR system in biological studies and therapeutics. For more information, please feel free to contact us.
Reference
- Lau, C.H.; et al. (2017). In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease. F1000Res. 6: 2153.
