Creating cell and animal models of human disease by genome editing using CRISPR/Cas9

Abstract

A set of unique sequences in bacterial genomes, responsible for protecting bacteria against bacteriophages, has recently been used for the genetic manipulation of specific points in the genome. These systems consist of one RNA component and one enzyme component, known as CRISPR (“clustered regularly interspaced short palindromic repeats”) and Cas9, respectively. The present review focuses on the applications of CRISPR/Cas9 technology in the development of cellular and animal models of human disease. Making a desired genetic alteration depends on the design of RNA molecules that guide endonucleases to a favorable genomic location. With the discovery of CRISPR/Cas9 technology, researchers are able to achieve higher levels of accuracy because of its advantages over alternative methods for editing genome, including a simple design, a high targeting efficiency and the ability to create simultaneous alterations in multiple sequences. These factors allow the researchers to apply this technology to creating cellular and animal models of human diseases by knock‐in, knock‐out and Indel mutation strategies, such as for Huntington's disease, cardiovascular disorders and cancers. Optimized CRISPR/Cas9 technology will facilitate access to valuable novel cellular and animal genetic models with respect to the development of innovative drug discovery and gene therapy.

Spontaneous and Induced Animal Models for Cancer Research

“…Through CRIPR/Cas9 technologies, researchers can generate reliable models of human disease due to likelihood of including simultaneous targeted modifications within multiple sequences. This feature allows for generating authentic animal models capable of incorporating the heterogenous genetic makeup of human disease that most of the time is initiated by cumulative genetic modifications [99]. There are several early successful transgenic models for cancer research created through the incorporation of CRISPR/Cas9 system: lung cancer knock-in model via a mice model with Cas9 dependent on Cre that in the moment of sgRNA administration targets TP53, Lkb1 and K-RAS, causing loss of function mutations for the first two genes and driver oncogenic mutations for the third gene that in the end determines the apparition of lung adenocarcinoma [100] and pancreatic cancer model with somatic mutations generated with CRISPR/Cas9 and retrograde viral vector delivery [101].…”

Non-Viral Targeted Nucleic Acid Delivery: Apply Sequences for Optimization

“…Precise genome modifying nucleases, such as CRISPR-associated nuclease Cas9 have been harnessed to create a DNA double-strand break (DSB) for site-specific genome editing [ 129 , 130 , 131 , 132 ]. Among different intracellular delivery of genome-editing agents in the form of gene expression constructs based on DNA [ 23 ] or mRNA [ 132 ] or Cas9 protein/sgRNA RNP formulations, Cas9/sg RNPs [ 23 , 131 ] were considered to be the most effective tool for the genome engineering duo to their immediately genome editing process without transcription or translation and risk of spontaneous genome integration.…”

Applying gene‐editing technology to elucidate the functional consequence of genetic and epigenetic variation in Alzheimer’s disease

“…However, despite its DNA binding specificity at a single nucleotide level and immense targeting capacity, TALEN guided gene editing can be costly and time intensive (29). In contrast, the CRISPR-Cas9 system offers a simple, cost-effective, multifunctional platform that has led to the generation of new model systems in vitro and in vivo in various research areas (23,83). As a result, this technology has the realistic potential to elucidate the functional consequence of genetic and epigenetic signatures in AD, thereby establishing whether these associations are secondary to pathogenesis or causal in disease development.…”