CRISPR/Cas9 genome editing technology has introduced valuable versatility in the design of pre-clinical animal models for functional genomics research. Accuracy, timing, and flexibility with CRISPR component delivery is especially critical in neuroscience and behavior research. The production of interpretable data often relies on genetic manipulation at specific stages of development or behaviors.1 Stereotaxic techniques have long been used for investigations of the brain, and their combination with site-specific infusion apparatuses are essential to efficient delivery for targeted genome editing.
The RNA-guided endonuclease, Cas9, of the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) bacterial adaptive immune system, is the crux of this genetic engineering tool. In short, a pre-designed guide RNA (gRNA) directs Cas9 to generate site-specific DNA double-stranded breaks (DSBs) that are mended by the cell’s own repair mechanisms in the presence or absence of an exogenous DNA template, creating a genetically-modified organism.2
The translational potential of CRISPR/Cas9 in genomic medicine is limited by efficient delivery of the active complex, particularly if donor DNA is also required. Nevertheless, two promising applications of the technology exist. First, those centered on non-homologous end joining (NHEJ) could permanently silence disease-causing genes due to the frameshift mutations that occur during repair of the protein-coding, Cas9-specified region of the organism’s genome.3 Second, those centered on the homology-directed repair (HDR) mechanism could correct disease-causing genetic mutations to their wild-type sequence.3 The latter approach is dependent on the presence of an exogenous DNA template composed of the correct sequence for incorporation at the Cas9-generated DSB. Therefore, efficient in vivo delivery of HDR-based therapeutics is particularly challenging. Stereotaxic techniques, traditionally utilized to precisely create lesions for the implantation of electrodes or microdialysis probes, are also essential for the accurate delivery of CRISPR-based therapeutics to the brain of a living animal.
It’s all in the Delivery!
CRISPR/Cas9 technology has only recently been adapted for in vivo tissue targeting. This is accomplished using viral vectors for delivery, of which the most effective and frequently used is the adeno-associated virus (AAV). AAV is capable of both nuclear entry and site-specific integration within the animal genome, resulting in sustained expression of the genetic modification with every cell division.4,5 However, AAV has its limits; much of the human population is immune to infection.5,6
Most recently, gold nanoparticle vehicles (i.e., CRISPR-Gold) have been successfully used in mice for direct component delivery into cells via endocytosis.3 In this system, Cas9, gRNA, and donor DNA are wrapped around a gold nanoparticle of just 15 nm diameter in size. Endocytosis is facilitated by the endosomal disruptive polymer poly(N-(N-(2-aminoethyl)-2-aminoethyl) aspartamide), or PAsp(DET) for short.3
Regardless of the delivery vehicle employed, generation of a neurologically relevant animal model via CRISPR genetic engineering requires a highly precise, efficient, and flexible physical delivery technique.
CRISPR on the Brain?
The surgical methods used for accessing the brain of an animal are well established and based on its anatomical atlas. As an automated delivery system, infusion syringe pumps coupled with a stereotaxic framework allow for the continuous or timed delivery of CRISPR components. In essence, cells making up any region of the brain can be stably genetically manipulated at any stage of development.1 This is both fundamental to spatiotemporal control and the generation of reproducible results.
1. A. Cetin, et al., “Stereotaxic gene delivery in the rodent brain,” Nat Prot 1:3166-3173, 2007.
2. P. Singh, et al., “A mouse geneticists practical guide to CRISPR applications,” Genetics 199:1-15, 2015.
3. K. Lee, et al., “Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair,” Nat Biomed Eng DOI: 10.1038/s41551-017-0137-2, 2017.
4. S. Mali, “Delivery systems for gene therapy,” Indian J Hum Genet 19:3-8, 2013
5. D. Shyam, et al., “Gene therapy using adeno-associated virus vectors,” Clin Microbio Rev 21:583-593, 2008.
6. L.E. Dow, “Modeling disease in vivo with CRISPR/Cas9,” Trend Mol Med 21:609-621, 2015.
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This article is brought to you by Chemyx, Inc. Syringe Pumps by Chemyx are used in top-level biomedical, pharmaceutical, chemical, and petrochemical research, offering highly precise, consistent, and reproducible fluidic delivery. Chemyx pump devices orchestrate the performance of different technologies that make modern research into novel materials, drugs, and energy resources possible. www.chemyx.com