The advent of CRISPR has not only marked a new era for the in vitro genome editing, giving us a powerful tool to better understand the fine mechanisms at the base of molecular biology but has also revolutionized the “personalized” or “precise” medicine therapy. Many diseases in fact, hide a genetic origin and following the central dogma of molecular biology, we can precisely edit those mutations that cause the disease to restore the expression of the “healthy” form of the protein. As recently reported by three separate research groups [
38‐
40], gene therapy using CRISPR technology for Duchenne muscular dystrophy is ongoing clinical trials showing very positive results both in adult muscle differentiated cells and muscle stem cells. CRISPR approach for treatment of Cystic Fibrosis as well, despite being in its early stages, is very promising. Indeed, stem cells can be corrected by delivering the therapeutic agents into the airways or, alternatively, generating CFTR-corrected stem respiratory epithelial cells and subsequently be administered to the patients [
41]. Methods for efficient delivery and expression of CRISPR-Cas system components will undoubtedly need to be optimized for each particular cell-type or organism to be modified. Collectively, these advances will be important for research use and therapeutic applications. Strategies for shifting the balance away from NHEJ-mediated indel mutations and toward HDR-driven alterations remain a priority. Although high rates of HDR can be achieved with the CRISPR and single-stranded DNA oligonucleotides, competing mutagenic NHEJ also occurs simultaneously. One of the drawbacks to developing an approach to improve the HDR:NHEJ ratio is that inhibition of NHEJ is likely to be poorly tolerated by most cells, given its central role in normal DNA repair. For therapeutic applications seeking to exploit HDR, reduction or elimination of competing NHEJ will be crucially important. Another promising therapy is to eliminate viral infection from the host genome using CRISPR. Human Papillomaviruses (HPVs) are today the main responsible for cervical carcinoma and anogenital cancers. Two research groups in 2014 used for the first time CRISPR to induce indel mutations in the viral genes encoding for E6 and E7 proteins in human cells [
42,
43]. These proteins, inactivating respectively p53 and pRb, drive infected cells to an abnormal proliferation leading to tumor transformation. CRISPR capability to perform a selective knock out of viral genes is being use in Hepatitis B virus (HBV) as well as in HPVs. Indeed, several researchers designed sgRNAs targeting the HBV core and HBsAg proteins to reduce HBV-related symptoms and to treat HBV-associated disease. The most recent one, is the approach used by Zhen et al. [
44] who targeted the HBsAg and HBx-encoding region of HBV, both in vitro and in vivo. HBsAg levels in the cultures media of cells and in the sera of mice were reduced as well as the HBV DNA levels and HBsAg protein expression in mouse livers. Epstein-Barr virus (EBV) is another etiologic agent capable to drive cancer-leading mutations, causing the Burkitt’s lymphoma and the nasopharyngeal carcinoma. While Wang and Quake designed several sgRNAs targeting the nuclear antigens EBNA1, EBNA3C and the latent membrane protein 1 (LMP-1) [
45], Yuen et al. [
46] used two sgRNAs targeting the promoter region of BART miRNA, to reduce the proliferation and to promote the decline in viral load as well as restoration of the apoptosis pathway in infected cells. As demonstrated by Wang et al. [
47], Cas9 can knockout viral sequences of HIV-1 in mammalian cells causing the activation of NHEJ repairing system and generating some indel mutations that are potentially lethal for the virus. So while in some cells the virus is easily eradicated, in others some indel mutations are refractory to recognition by the same gRNA as a result of changing the target DNA sequences, leading to the emergence of replication competent viruses that are resistant to Cas9/gRNA. More recently, Chaoran et al. used an all-in-one adeno-associated virus (AAV) vector to deliver multiplex sgRNAs targeting four different viral structural genes and the Staphylococcus aureus-derived Cas9, demonstrating that this strategy greatly reduces the potential of HIV-1 escape and increases the possibility of HIV-1 excision despite the continuous mutations in the clinical HIV-1 patients’ population [
48]. Many other therapies are currently ongoing to cure disease such as haemoglobinopathy, β-thalassaemia, Leber congenital amaurosis, haemophilia giving hope to all patients that could not count on alternative therapy ways [
49‐
51]. Even on cancer research there are some encouraging studies. In the last few years, research focused especially on those types of cancer that are untreatable with standard chemo- or radiotherapies and in particular the lung cancer, which is one of the most fatal and, even if a lot of efforts have been spent, is still the main cause of cancer-related deaths. Immunotherapy has emerged as a promising way to treat lung cancer using therapeutic vaccines and targeting the specifically cytotoxic T lymphocyte associated protein 4 and programmed death receptor 1 (PD-1) pathways [
52]. Lu You, an oncologist at Sichuan University’s West China Hospital in Chengdu, started testing modified immune T cells treating non-small cell lung cancer for patient whose chemotherapy, radiation therapy and other treatments have failed [
53]. These T cells, which are PD-1 KO, once reintroduced in patients will home in the tumor activating the immune response and hopefully eradicating tumor cells. Indeed, PD-1, also known as CD279, and its pathway is involved in T-cells regulation and autoimmunity, so knocking it out lead to a forced activation of the immunity system, deleting the brakes that limit the immune response [
54]. Similar trials with PD1‑knockout T cells for prostate, bladder cancer, as well as renal cell carcinoma, are also being initiated [
55]. Other anticancer immune therapies are recently emerged founded on the production of next-generation chimeric antigen receptor (CAR) T cells [
56]. These cells, which express tumor-targeting receptors, have shown promise in the treatment of various leukaemias, lymphomas and solid cancers. CARs include an extracellular binding domain which recognizes an antigen that is highly specific and strongly expressed on tumor cells, and an intracellular chimeric signalling domain that activates the T cells upon receptor engagement. This mechanism promotes T cell-mediated killing of tumor cells (Table
1).
Table 1
Novel gene editing-based therapies
Duchenne Muscular Dystrophy | Dystrophin | |
Cystic Fibrosis | Cystic Fibrosis Transmembrane Conductance Regulator | |
HPV | E6 & E7 | [42;43] |
HBV | HBsAg & HBx | |
EBV | EBNA1, EBNA3C & LMP-1 | |
| BART miRNA | |
AIDS | Gag/Pol - Rev/Env | |
| LTR-1, LTR-3, GagD & PolB | |
β-thalassaemia | β-/γ-globin | |
Leber congenital amaurosis | CEP290 | |
Haemophilia | F9 | |
Non-small Cell Lung Cancer | PD-1 | |
Prostate Carcinoma | PD-1 | |
Bladder Carcinoma | PD-1 | |
In addition to the use described above, CRISPR-Cas system has the potential to be used to regulate endogenous gene expression or to label specific chromosomal loci in living cells or organisms. Catalytically inactive or “dead” Cas9 (dCas9) can be recruited by gRNAs to specific target DNA sites [
57]. Targeting of dCas9 to promoters was initially shown to repress gene expression in both Escherichia coli and human cells [
58‐
60]. It has also been demonstrated that an EGFP-dCas9 fusion can be used to visualize DNA loci harboring repetitive sequences, such as telomeres, with a single gRNA or non-repetitive loci using 26 to 36 gRNAs covering a 5-kb region of DNA [
61]. This imaging system provides a powerful tool for studying chromosome dynamics and structure. It is also really interesting to see whether dCas9 fusions to histone modifiers and proteins involved in altering DNA methylation, can also be used to perform targeted “epigenome editing”. Indeed, it was recently found that many epigenetic factors are involved in multiple types of cancer such as glioblastoma, chondrosarcoma and osteosarcoma [
62‐
64], so targeting epigenetic regulatory enzymes may be one suitable way to dysregulate tumor maintenance. As reported by Chen et al. [
65], dCas9 could be fused to transcriptional activation domain such as VP64, VP64-p65-Rta, Kruppel-associated box (KRAB), activating or repressing selectively the target gene expression depending on the strength of activator or repressor used and the target transcription start site. In this way dCas9 might also be used to interfere with transcriptional elongation, directing to the non-template strand of a gene, resulting in 10- to 300- fold repression of mRNA transcription or when directed to a region which was initially occupied by RNA polymerase, dCas9 could also inhibit transcription initiation. CRISPR was also used to recreate mutations commonly identified in patients’ tumors and assess their effects in a cell line, to better understand the tumor-driving mutations, their phenotypic effects and then to identify new anticancer agents [
66]. As reported by Matano et al. and Drost et al., it is possible to introduce a series of single mutations to transform an intestinal human organoid to an invasive carcinoma [
67,
68] demonstrating that four mutations (APC, KRAS, SMAD4 and TP53) were mandatory to drive this process and that APC and TP53 loss was sufficient to induce chromosomal instability. Transducing a non-metastatic mouse lung cancer cell line with a lentiviral CRISPR/Cas9 library, targeting thousands of protein-coding genes and hundreds of microRNA precursors and inoculating these cells in immunodeficient mice, Chen et al. formed several growing tumors and lung metastases. The consequent deep sequencing analysis of the randomized KO tumor cells enabled the identification of several genes, whose inactivation may trigger tumor growth and invasion [
69]. The main discovery of this kind of approach is that the selection of new tumor markers does not imply previous knowledge of the different functional hallmarks acquired throughout tumorigenesis. Another intriguing way to exploit CRISPR system would be to target tumor markers directly inside tumor site. In such a way it might be possible to overcome the genetic mutations leading tumor proliferation and metastatic capacity. However, in this approach the main problem is that every single tumor bears different mutation so it would require a very precise and personalized CRISPR therapy and another problem is the delivery system, because trying to make a selective gene knock out only in tumor cells is practically impossible with today technologies. An alternative target in cancer treatment for CRISPR might be the miRNAs expression. Indeed, miRNAs are involved in the regulation of a plethora of cellular physiological and pathological processes in a selective tissue-specific way [
70], so the selective knock out of a single miRNA can lead to a powerful modulation of many genes at the same time. As demonstrated by Chang et al. the CRISPR-mediated knock out of miR-17, miR-200c and miR-141 in two colon cancer cell is actually possible and the repression could be stably maintained unaltered for a long term period [
71]. So, it might be also possible to modulate miRNA expression with CRISPR technology in tumor-associated immune cells, regulating their recruitment and activation in the tumor microenvironment, waking up the immune system against tumor cells as yet demonstrated with the PD-1 KO [
72]. The challenge for the future will be for sure to find a secure and safe way to delivery CRISPR machinery only in the tumor site and to inhibit its proliferative and metastatic ability. However, this system even if fascinating is not error free as yet reported above and so potential ethical concerns related to the impact of targeted nucleases upon cells germline are under discussion. Moreover, CRISPR application in vivo and especially in human has a very low working efficiency, demonstrating that we need to further improve our knowledge of this gene editing tool to create really efficient and safe therapy for all the disease related to a genetic mutation. Methods for expanding the targeting range of RNA-guided Cas9 will be important for inducing precise HDR or NHEJ events as well as for implementing multiplex strategies, including paired nickases.