Optimizing Next-Generation Disease Models with CRISPR

When studying the biological processes that underpin and cause disease, it’s important that an appropriate model is utilized to understand how the disease develops and to test potential treatment options. A disease model is an animal or cells that display the biological and pathological processes that are observed in the actual human disease state. Genome editing has allowed for the creation of more accurate cellular or animal models in which to study the molecular and genetic mechanisms that drive pathological processes, identify potential therapeutic strategies, and discover how patients will respond to certain drugs.

The recent development of the CRISPR-Cas9 system has revolutionized gene editing, enabling researchers to make precise and efficient changes to genomic sequences relatively easily in virtually any cell or organism through the introduction of a double-strand break at the target site. This article looks at how CRISPR-based gene editing is being used in the generation of in vivo and in vitro models to better mimic human disease and reflect the genetic drivers that govern specific pathologies.

Engineering genomes to understand biological function

The introduction of specific genetic modifications with genome engineering has proven to be a powerful tool for a variety of applications, including basic research, drug discovery, biotechnology, and biomedical research. Previously, genome engineering relied on complicated targeting and selection constructs to facilitate homologous recombination (HR) where homologous DNA fragments are used as a template to facilitate gene addition, replacement, or inactivation. HR is inherently inefficient in mammalian cells—but work demonstrating that the introduction of a double-strand break (DSB) into DNA can increase HR events significantly increased our ability to make specific gene modifications. This discovery drove the utilization of several genome-editing technologies such as ZFNs, TALENs, and, more recently, the RNA-based CRISPR-Cas9 gene-editing system, which has exploded into the biological space and has been rapidly adopted.

CRISPR-Cas9—a versatile tool for genome editing

An important reason behind the almost universal adoption of CRISPR as a gene-editing tool is its inherent simplicity and flexibility. The system has been developed to require only two components—a short guide RNA (sgRNA), which directs the Cas9 endonuclease to the target site to introduce a DSB. Once a DSB has been introduced in the presence of a donor DNA template, this activates the HR cellular DNA repair pathway to introduce specific sequences or mutations at the target site. Alternatively, the error-prone non-homologous end joining (NHEJ) pathway can be used to introduce a frameshift mutation and induce specific gene knockout.

As well as gene editing, CRISPR can also be used for regulation of endogenous gene expression with the use of a catalytically inactive version of Cas9 (dCas9) tethered to regulatory regions. When recruited by sgRNAs to target specific promoters, this can trigger gene silencing (CRISPRi) or activation of gene expression (CRISPRa). The RNA-based nature of the CRISPR system means that it can be performed in multiplex, and so can be used to target multiple genes simultaneously.

Modeling for the next generation

In addition to becoming the go-to gene-editing tool, CRISPR has been used to generate alternative in vivo and in vitro models for a range of different diseases. Here are a few examples:

• By injecting sgRNAs and Cas9 mRNA into single-cell embryos, CRISPR can rapidly and efficiently create genetically modified animal models, decreasing the time and complexity in generating transgenic animal models when one or several genes have been modified.^1,2
• CRISPR-Cas9 has been successfully used for in vivo gene editing with the use of tissue-specific adeno-associated viral vectors (AVVs) to target specific cells in native tissues. A cardiac-specific transgenic mouse model was created by introducing Cas9 containing plasmids into mouse zygotes, with Cas9 expressed exclusively in heart cardiomyocytes under the control of the Myh6 promoter. When sgRNAs targeting Myh6 within an AAV vector were introduced, cardiac-specific gene modification at the Myh6 locus was induced resulting in hypertrophic cardiomyopathy.³ This approach can therefore be used in existing disease models and removes the need for germline modified mutant strains.
• CRISPR-Cas9 has been used in the generation of ^{in vitro} organoid tumor models with the introduction of modifications to tumor suppressor genes and oncogenes.⁴ Tumor organoids have also been created in vivo—Roper et al., used a combination of colonoscopy and mucosal injection to establish CRISPR-engineered tumor organoids in the distal colon of mice by delivering viral vectors containing CRISPR components.⁵
• The ability to perform gene editing in multiplex allows for combinatorial targeting of oncogenes or tumor suppressor genes in order to create precision cancer models—Heckl et al., created a leukemia model by targeting several inactivated oncogenes simultaneously, including Tet2, Runx1, Dnmt3a, Nf1, Ezh2, and Smc3.⁶
• Neurological disorders are inherently difficult to model in animals due to significant genetic differences and human-specific cell types. CRISPR has been used along with human induced pluripotent stem cells (iPSCs) to create an accurate model of Alzheimer’s disease (AD).⁷ Patient-derived iPSC-neurons have been shown to also display the AD disease phenotype, and so CRISPR has been employed to correct disease-associated mutations thereby creating an isogenic cell line pair.

Interrogating biological systems with CRISPR

A major challenge in disease model studies is finding an appropriate model that is both physiologically relevant and experimentally tractable—there is always a trade-off between the ease of use, relevancy as a proxy for studying human disease, and cost implications of the chosen model. CRISPR-Cas9 gene editing now allows researchers to make precise, targeted changes to genomic sequences relatively cheaply and easily and so is proving to be a powerful tool for the creation of precise disease models. CRISPR Cas9 can be used to alter specific genes, either by knockout, replacement, or regulation of gene expression to specifically mirror the genetic changes that feature in disease states providing researchers with a powerful tool to study the biology driving disease and identify potential therapeutic targets.

Key Takeaways

• Genome-editing technologies such as CRISPR-Cas9 have improved the creation of accurate disease models with the ability to make targeted and specific changes to the genome to reflect the genetics that underpin disease pathology.
• CRISPR-Cas9 gene editing is allowing researchers to perform genome engineering relatively easily, requiring only basic molecular biology skills.
• Transgenic animal models can now be created rapidly in almost any species, with injection of CRISPR components into single-cell embryos.
• While traditional animal models have yielded many valuable insights, they have limitations as some biological processes such as brain development and response to certain drugs is specific to the human body. CRISPR-Cas9 gene editing can be used with emerging technologies to create alternative model systems, such as human induced pluripotent stem cells and 3D cell culture.

References

1. Shen BZhang JWu H, et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res 2013;23:720–3.

2. Shao YGuan YWang L, et al. CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos. Nat Protoc 2014;9:2493–512.

3. Carroll, K. J. et al. A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9. Proc. Natl Acad. Sci. USA 113, 338–343 (2016).

4. Matano, M. et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015).

5. Roper, J. et al. Colonoscopy-based colorectal cancer modeling in mice with CRISPR-Cas9 genome editing and organoid transplantation. Nat. Protoc. 13, 217–234 (2018).

6. Heckl, D. et al. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat. Biotechnol. 32, 941–946 (2014).

7. Israel, Mason A et al. Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells. Nature vol. 482,7384 216-20. 25 Jan. 2012.