CRISPR-Cas9 Technology for the Creation of Biological Avatars Capable of Modeling and Treating Pathologies: From Discovery to the Latest Improvements

Abstract

This is a spectacular moment for genetics to evolve in genome editing, which encompasses the precise alteration of the cellular DNA sequences within various species. One of the most fascinating genome-editing technologies currently available is Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated protein 9 (CRISPR-Cas9), which have integrated deeply into the research field within a short period due to its effectiveness. It became a standard tool utilized in a broad spectrum of biological and therapeutic applications. Furthermore, reliable disease models are required to improve the quality of healthcare. CRISPR-Cas9 has the potential to diversify our knowledge in genetics by generating cellular models, which can mimic various human diseases to better understand the disease consequences and develop new treatments. Precision in genome editing offered by CRISPR-Cas9 is now paving the way for gene therapy to expand in clinical trials to treat several genetic diseases in a wide range of species. This review article will discuss genome-editing tools: CRISPR-Cas9, Zinc Finger Nucleases (ZFNs), and Transcription Activator-Like Effector Nucleases (TALENs). It will also encompass the importance of CRISPR-Cas9 technology in generating cellular disease models for novel therapeutics, its applications in gene therapy, and challenges with novel strategies to enhance its specificity.

Keywords: CRISPR-Cas9; disease modeling; gene therapy; genome editing.

Figure 1

Genome editing tools’ mechanism of action and the pathways involved in DSBs repair of targeted endogenous DNA. DSBs are either repaired by NHEJ or HDR repair pathways. In the absence of a donor template, the NHEJ repair pathway will process the ends of the broken targeted double-stranded DNA by either inserting or deleting nucleotides, thus disrupting the open reading frame by generating frameshift mutations leading to the loss of the gene’s functionality. In the presence of a donor template, the HDR repair pathway will precisely incorporate the donor template DNA in homology with the neighbor lesion sequences based on homologous recombination. All three genome-editing tools (ZFNs, TALENs, and CRISPR-Cas9) generate DSBs that are either repaired by NHEJ or HDR depending on the aim of the research.

Figure 2

Comparison of genome editing tools. Comparison between homologous recombination (HR), ZFNs, TALENs, and CRISPR-Cas9 in terms of advantages, drawbacks, and key aspect features.

Figure 3

Key features of CRISPR-Cas9-based acquired immune response in Streptococcus pyogenes. Adaptation. The first step involves the recognition and cleavage of the foreign viral DNA by specific Cas proteins (Cas2 and Cas3) into smaller fragments and their incorporation into the spacer region of the bacterial genome. Expression. The second step involves the expression of the CRISPR locus to generate pre-cRNA, which is afterwards processed by specific RNases (RNase III) to generate mature crRNA. Interference. The third step involves the assembly of the mature crRNA with the tracRNA, which recruits the Cas9 endonuclease to form the CRISPR-Cas9 complex system. Taking the advantage of crRNA’s homology with the viral DNA sequence, the target viral DNA is chopped by Cas9 endonuclease to inactive the virus.

Figure 4

Direct injection of Cas9 mRNA and sgRNAs into single-cell mouse embryos for genome editing. The generation of transgenic mouse models can be achieved by collecting eggs from a super-ovulated female mouse followed by in vitro fertilization by the sperms of a male mouse. The fertilized egg then undergoes a direct injection of the CRISPR-Cas9 machinery. After several days, the developed transgenic zygote is reintroduced into a host female mouse to become pregnant and produce a transgenic mouse utilized for modeling the disease of interest.

Figure 5

Viral and non-viral in vivo genome editing strategies for therapeutic applications based on CRISPR-Cas9 machinery. Viral strategies such as adeno-associated virus and non-viral strategies such as liposomes, polymers, and peptides can deliver CRISPR-Cas9 machinery for in vivo genome editing for therapeutic applications.

Figure 6

Use of patient-derived induced pluripotent stem cells (iPSCs) with CRISPR-Cas9 for pediatric neurological disorder modeling and for treatment. Patient’s somatic cells can be reprogrammed to generate iPSCs where they can be genetically modified and corrected by CRISPR-Cas9 followed by their re-differentiation into corrected neurons. Characterization of the disease phenotype can be achieved by various techniques including immunocytochemistry, electrophysiology, and gene expression analysis. These corrected neural cells can rescue the disease phenotype of the patient by undergoing autologous transplantation of corrected cells.

Figure 7

Various disease modeling applications achieved by CRISPR-Cas9 technology. CRISPR-Cas9 can be utilized to model various diseases including neurological, immunological, infectious, cardiac, ocular, and cancerous diseases.

Figure 8

Cas9 nickase is a variant of the wild-type CRISPR-Cas9 system-based genome editing. The Cas9 nickase (Cas9n) harboring a mutation in either HNH (H840A) or RuvC (D10A) domains can generate a single nick instead of a DSB at the target site of interest. Dimeric Cas9n with two different guide RNAs can be fused to generate double nicks, one at each part of the target DNA, thus yielding DSBs with staggered ends implementing enhanced specificity with extremely reduced off-target effects.

Figure 9

dCas9 is a variant of the wild-type CRISPR-Cas9 system acting as a key modular system for the recruitment of transcriptional regulators. dCas9, which is catalytically inactive, can be utilized for transcriptional regulation processes. dCas9 can recruit various transcriptional regulators acting either positively (activators) or negatively (repressors) at the transcriptional level. For instance, VP64 fused to dCas9 can activate transcription, whilst for KRAB, it can inhibit transcription.

Figure 10

Dimeric RNA-guided FokI nucleases (RFNs) is a variant of the wild-type CRISPR-Cas9 system-based genome editing. To ensure highly efficient specificity with reduced off-target events, RFNs were constructed by merging dCas9 coupled to FokI with a guide RNA, which is catalytically inactive and can be utilized for transcriptional regulation processes. dCas9 can recruit various transcriptional regulators acting either positively (activators) or negatively (repressors) at the transcriptional level. For instance, VP64 fused to dCas9 can activate transcription, whilst for KRAB, it can inhibit transcription.