CRISPR therapeutic tools for complex genetic disorders and cancer (Review)

Abstract

One of the fundamental discoveries in the field of biology is the ability to modulate the genome and to monitor the functional outputs derived from genomic alterations. In order to unravel new therapeutic options, scientists had initially focused on inducing genetic alterations in primary cells, in established cancer cell lines and mouse models using either RNA interference or cDNA overexpression or various programmable nucleases [zinc finger nucleases (ZNF), transcription activator-like effector nucleases (TALEN)]. Even though a huge volume of data was produced, its use was neither cheap nor accurate. Therefore, the clustered regularly interspaced short palindromic repeats (CRISPR) system was evidenced to be the next step in genome engineering tools. CRISPR-associated protein 9 (Cas9)-mediated genetic perturbation is simple, precise and highly efficient, empowering researchers to apply this method to immortalized cancerous cell lines, primary cells derived from mouse and human origins, xenografts, induced pluripotent stem cells, organoid cultures, as well as the generation of genetically engineered animal models. In this review, we assess the development of the CRISPR system and its therapeutic applications to a wide range of complex diseases (particularly distinct tumors), aiming at personalized therapy. Special emphasis is given to organoids and CRISPR screens in the design of innovative therapeutic approaches. Overall, the CRISPR system is regarded as an eminent genome engineering tool in therapeutics. We envision a new era in cancer biology during which the CRISPR-based genome engineering toolbox will serve as the fundamental conduit between the bench and the bedside; nonetheless, certain obstacles need to be addressed, such as the eradication of side-effects, maximization of efficiency, the assurance of delivery and the elimination of immunogenicity.

Figure 1

CRISPR system mechanism of action. The main action of the CRISPR system is mediated by the Cas nuclease. This nuclease is recruited to DNA by the orientation of the PAM motif and generates double-strand breaks in DNA sequence, matching the broken genomic region with a single guide RNA. Following this, non-homologous end joining or homologous mediated repair mechanisms are conducted to restore the nucleotide sequence induced by double-strand breaks, causing the anticipated genomic alterations. CRISPR, clustered regularly interspaced short palindromic repeats; PAM, protospacer adjacent motif; DSBs, double-strand breaks; NHEJ, non-homologous end joining; HDR, homology directed repair.

Figure 2

The use of two distinct repair pathways in performing different modifications. In the NHEJ mechanism, the ends of the DNA are chemically ligated back together with a small insertion or deletion at the site of the break. The NHEJ mechanism is usually employed in cases of gene disruption (small deletions or insertions), inversions, duplications or deletions whereas the HDR mechanism is used for deletions, base mutations, insertions and replacements. In HDR, a donor DNA molecule matches with the genomic sequence flanking the site of the double-strand break and thus it can be integrated into the genome at the site of the break, introducing new genetic information into the genome. NHEJ, non-homologous end joining; HDR, homology directed repair; sgRNA, single-chimeric guide RNA.

Figure 3

Cas9 nuclease as a therapeutic model for the treatment of genetic disorders. In the case of monogenic recessive disorders such as cystic fibrosis, sickle cell anemia, hereditary tyrosinemia or Duchenne muscular dystrophy, the target mutation is repaired with the aid of Cas9. In this manner, the protein derived from corrected gene can be developed in native conditions. In the case of dominant-negative disorders in which the target gene is represented by one allele (haploinsufficiency phenomenon), the CRISPR system seems to be the most advantageous method in inactivating the mutated allele. In other instances, the elimination of duplicated regions could be accomplished through Cas9 nuclease and NHEJ mediated repair, whereas therapeutic benefit has also been observed by introducing protection mutations in mitotic tissues in complex diseases. Finally, CRISPR system has been employed for the modification of T cells, especially with CAR or artificial TCRs, with the aim to introduce modified cells into the body of cancer patients. CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR-associated protein 9; NHEJ, non-homologous end joining; CAR, chimeric antigen receptor; TCR, T-cell receptor. The single asterisk (*) indicates cystic fibrosis, sickle cell anemia or Duchenne muscular dystrophy. The double asterisks (**) indicate transthyretin-related hereditary amyloidosis or dominant forms of retinitis pigmentosum.

Figure 4

The use of CRISPR-edited organoids. CRISPR gene editing can be used to generate organoids for drug target validation, mechanistic analysis and patient stratification studies, as well as high-throughput pooled or high-content arrayed screens. CRISPR, clustered regularly interspaced short palindromic repeats; PAM, protospacer adjacent motif; iPSCs, induced pluripotent stem cells.

Figure 5

Modified T cell fight against immune HIV variants. In general, T cells recognize and eliminate the HIV-infected cells. However, some cells express variant HIV epitopes that help them to accomplish immune escape. In this context, the CRISPR-edited infected cells can revert to their normal state, recruiting T cells in order to abrogate the HIV challenge. HIV, human immunodeficiency virus; CRISPR, clustered regularly interspaced short palindromic repeats.

Figure 6

(A) Generation of genetically modified mouse models harboring either eliminations or insertions or chromosome translocations through transfecting Cas9 with single or multiple sgRNAs. (B) Generation of screens using Cas9 and pool of sgRNAs libraries. (C) Targeted mutagenic screens through viral delivery of Cas9 and targeted sgRNA libraries. Generating organoids to predict the response of patient to administration of potential drugs. sgRNA, single-chimeric guide RNA; Cas9, CRISPR-associated protein 9; iPSCs, induced pluripotent stem cells.