30 November 2021
Infectious diseases pose a major threat to public health and result in high morbidity and mortality. There is a need to rapidly diagnose and treat these infections to improve public health. For that purpose, a detailed understanding of interaction and host and pathogen (viruses, bacteria parasites fungi) is required
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) are genomic loci present in bacteria and archaea as their adaptive immune system that is coupled with different CRISPR associated proteins (Cas) to protect the bacteria against invading bacteriophages. The CRISPR sequences are made up of short DNA segments of the previously invading viral genomes, which upon subsequent infection, are used to detect the same viral genome and destroy it. The Cas proteins are nucleases that utilize CRISPR sequences as their guide to specifically target the foreign genome and cut it into different segments to destroy it.
CRISPR Cas9 system has now been recently implemented in human medicine for genomic editing, development of diagnostic tools for infectious diseases.
CRISPR are loci present in the bacterial genome as memory units. The spacer sequences are derived from the genome of the invaders that are integrated into the bacterial genome and later on recalled on reinfection from the same invader to guide the Cas proteins to remove the invader from the bacterial cells.
Two classes of CRISPR-Cas systems have been discovered so far that include 6 types and subtypes. Type 1, 3, and 4 are included in class one, which use various Cas proteins to act against the foreign pathogen, while types 2, 5, and 6 are included in the class 2 CRISPR-Cas system that utilizes a single type of Cas protein for action against the invaders. CRISPR Cas system works through different stages i.e., adaptation, crRNA maturation (CRISPR RNA), and interference.
During adaptation foreign invading genome is recognized and a part of it is selected as protospacer sequence, processed, and integrated into the host cell genome into CRISPR array. This spacer sequence, upon re-infection from the same invader is used to guide the Cas proteins to recognize it and cut it into different segments to inactivate the invader’s genome. To avoid autoimmunity (Destruction of the bacterial genome or CRISPR array, the CRISPR Cas system distinguishes between the self and foreign DNA through protospacer adjacent motif (PAM) (Figure 1). PAM is a short DNA sequence containing 2 to 6 base pairs immediately followed by the DNA sequence that is going to be targeted by the CRISPR-Cas 9 proteins, which is a part of the foreign genome. The invader’s genome will not be recognized by the CRISPR-associated proteins (Cas9) if it is not followed by the PAM sequence. Thus, the PAM sequence prevents autoimmunity and eliminates the risk of the CRISPR array being destroyed by the CRISPR-associated proteins.
Guide RNA The CRISPR arrays in the bacterial genome are transcribed into guide RNA that make complex with Cas 9 proteins and roam in the bacterial cell to target the invading DNA on subsequent infection. The guide RNA consists of two parts; CRISPR RNA (crRNA) that contains 17 to 20 base pairs which are complementary target DNA, and a tracr RNA which acts as a binding scaffold for Cas nucleases.
The Cas9 proteins identify the foreign DNA with the help of guide RNA, cut it into different segments and one of the segments is adjusted in size called a spacer sequence, is integrated into the bacterial genome in the CRISPR array as a memory site.
Figure 1: The CRISPR-Cas9 system. CRISPR are genomic sequences present in bacterial DNA. Cas9 is one of the related enzymes (an endonuclease) that produce a straight cut in dsDNA.
Infectious Diseases Applications
CRISPR Cas 9 system has been widely employed in genome editing in mammalian cells by developing a guide RNA. This can also be used to knock out infectious or mutated genes from the host cells. For this a single guide RNA (sgRNA) is generated, the sequence of which is complementary to the target gene or genome. This guide RNA assists Cas-9 nuclease to target a specific target gene inside the cell. The double-stranded DNA breaks made in the host genome are then repaired via a non-homologous end joining (NHEJ) repair pathway (Figure 2).
Figure 2: Working of CRISPR Cas9 system for gene knockout.
Most of the existing antivirals are found to be ineffective against emerging viral diseases i.e., MERS (middle east respiratory syndrome virus), SARS (severe acute respiratory syndrome), and COVID-19. The development of an antiviral drug is a time taking procedure. The only treatment available so far for these fatal infectious diseases is supportive and symptomatic, but lack of specific treatment will result in a high mortality rate.
Recently huge interest has been shown by the scientist to use the CRISPR Cas9 system to eliminate specific viral genomes from the host cell DNA through genome editing tools to treat the disease. In a successful clinical trial, Edward M. Kennedy used CRISPR Cas9 technology to remove the E6E7 gene of the herpes virus to treat cervical cancer. the covalently closed circular DNA of the Hepatitis B virus has also been knocked out in an animal study via CRISPR Cas9 that results in an effective cure from the disease.
Epstein-Barr virus and Cytomegaloviruses have also been removed successfully in in-vitro studies. In addition to the removal of the viral genes from the host cells, T lymphocytes have also been engineered via CRISPR Cas9 technology to target specific diseases and eliminate them indirectly from the body. T cells have been modified to cure incurable B-cell lymphomas and leukemias by establishing specific Cas9 proteins.
Other than this CD4+ T lymphocytes are also engineered through the CRISPR-Cas9 system to treat human immune deficiency virus infection that can provide a new insight for the cure of highly fatal and refractory diseases. CRISPR Cas9 system with this genome editing technology is likely to become one of the best cures in the future for emerging and existing infectious diseases.
For RNA viruses, Cas13 targeted genome editing is more successful as compared to Cas9 nucleases. Dengue virus NS3 gene has been knocked out through Cas13a protein in vivo testing. SARS-CoV-2 and human influenza virus have been successfully destroyed in human lung epithelial cells by the development of Cas13d assisted prophylactic antiviral CRISPR in human cells (PAC-MAN) (Figure 3).
Figure 3: Mechanism of systematic elimination of SARS-COV via CRISPR/Cas9
The most economical way to control and prevent disease is vaccination. In addition to genome editing, CRISPR Cas technology can also be used to develop recombinant vaccines against emerging infectious diseases. The development of vaccines via CRISPR would be more efficient and less time taking as compared to conventional vaccines.
The CRISPR Cas9 along with Cre-lox genome editing was used by scientists for simultaneous knockout of the lethal genes as well as integration of the desired antigen into a vector infectious laryngotracheitis virus to develop a multivalent recombinant vaccine. Another herpes virus vector recombinant avian influenza virus, the African swine fever virus vaccine has been developed by using homology repair CRISPR Cas9 system. The Cas9 knock-in technology is also being used by scientists to develop mouse models that express human angiotensin-converting enzyme, that will help in understanding the pathogenesis and transmissions of the SARS-CoV virus, also provides future insights to evaluate therapeutic agents and vaccines against COVID-19. (Figure 4).
Figure 4: Applications of CRISPR/Cas9 in drug development, construction animal models, genome screening, and understanding signaling pathways
The optimal clinical care and targeted therapies require a comprehensive understanding of the mechanisms through which a pathogen (virus, bacteria, parasites, and fungi) interacts with host body cells. CRISPR Cas9 technology is widely employed to discover various virulent genes and associated proteins that are responsible for pathogenesis and onset of clinical signs in a host. Knocking out of these virulent genes from the host cells will ultimately result in the recovery from the disease.
Winter et al., has used developed a single guide RNA library to demonstrate an important virulence factor- alpha-hemolysins that plays important role in the pathogenesis of Staphylococcus aureus. Three essential genes TSPAN14, SYS1, and ARFRP1 were identified through a single guide RNA that regulates a metalloproteinase and disintegrin enzyme that reduces the level of alpha-hemolysin on the surface of a cell, thus minimizing the cytotoxicity.
Genetic screening of West Nile Fever Virus through CRISPR single guide RNA library identified 7 important genes EMC3, DERL2, UBE2G2, HRD1, SEL1L, EMC2, and UBE2J1, when inactivated through CRISPR technology provided protection against neuronal cells death induced by West Nile Fever Virus. Identification of these genes provides an important insight as a target for new drug development.
CRISPR cas9 technology has also been applied to study the genes of Trypanosoma cruzi and Toxoplasma gondi, the causative agents of Chagas disease and toxoplasmosis respectively. Toxoplasma gondi gene that encodes GP72 protein was inactivated through CRISPR single guide RNA that resulted in inhibition of flagellar attachment of the parasites to the host body cells. These examples explain the breadth of the CRISPR Cas 9 to evaluate interactions of the pathogen and host body thus providing important targets to the development of novel drugs for their effective treatment.
CRISPR Cas9 system has also been applied in the field of diagnostics by researchers to develop novel and modern tools with higher specificity and sensitivity. The isothermal amplification, nucleic acid-based amplification methods have been combined with CRISPR Cas9 to accurately identify closely related strains and serotypes of Zika virus in the macaque model.
The antibacterial resistance genes in bacteria have also been identified by combining the CRISPR Cas9 system with optical DNA mapping. The specific single-guide RNA is designed for this purpose that cleaves the plasmid containing antibiotic-resistant genes specific sites, the resulting DNA segments are labeled with a fluorescence dye netrospin and YOYO-1, that produce a unique pattern of omission intensity for each DNA segment, like a bar code. Using this technique scientists were able to identify various plasmid DNA that produces various antibiotic-resistant genes including ESBLs (extended-spectrum beta-lactamases) and carbapenemases.
CRISPR Cas9 together within DNA (FISH) fluorescence in situ hybridization has been used to identify MRSA (methicillin-resistant staphylococcus aureus). The mecA gene of MRSA was identified with the help of a single guide RNA and Cas9 complex together with a fluorescence-labeled specific probe. This technique can identify MRSA even at a very low concentration of 10cfy/ml concentration very efficiently and can differentiate between various isolates of staphylococcus aureus with and without antibiotic-resistant mecA gene.
Genetic blood diseases
Sickle cell disease also called sickle cell anemia is an inheritable disease that arises due to point mutation at chromosome 11. That changes the reading frame and results in the formation of another amino acid. The glutamic acid is replaced by valine in the B-globin gene at the 6th codon. This point mutation results in the synthesis of haemoglobin-S, which is a deoxygenated hemoglobin. This is a sticky molecule that leads towards the formation of the polymers of hemoglobin-S. these polymers further aggregate and bundles or stacks are produced. The formation of these stacks changes the shape of RBCs and blood cells. The disease has been characterized long ago and the only recommended treatment is bone marrow transplantation. CRISPR technology can be utilized to treat this condition through genetic engineering or gene editing.
Two approaches have been devised for the treatment of sickle cell disease by using the CRISPR-Cas9 system. In both these strategies, the progenitor cells of hemopoietic stem cells are modified with CRISPR-Cas9 technology and then re-implanted in the patient’s blood circulation.
- Repairing genes that produce defective haemoglobin-S, causing them to form standard hemoglobin.
The B-globin gene is broken up by using the CRISPR-Cas9 system. The donor guide templates responsible for the production of standard hemoglobin are also developed to rectify the mutation during the cell repairs, and then the broken pieces of DNA join by NHEJ (non-homologous end joining). These modified cells are re-implanted into the patient’s blood circulation to produce normal haemoglobin-F. The method has been tested in in vitro trials and found to be very efficient and reproducible.
- Replacement of haemoglobin-S with hemoglobin-F (normal hemoglobin)
Another promising CRISPR-Cas 9 sickle cell therapy is the knockout of hemoglobin F natural repressors. By inducing mutations in the BCL11A gene, we can indirectly promote the expression of the standard hemoglobin molecule. This approach of treatment was suggested when it was found that sickle cell patients having a mutation in the BCL11A gene do not show disease symptoms. In in vitro cell line studies, the expression of BCL11A was suppressed by antisense therapy which ultimately promoted the expression of haemoglobin-F. In antisense therapy, a strand of DNA has been introduced that binds with target mutated mRNA and deactivates it, thus preventing the formation of the respective protein.
Thalassemia is an inheritable disease associated with inefficient erythropoiesis that arises due to mutation in the HBB gene that leads towards the formation of non-existent (β0) or diminished (+β)B-globin. This mutation also results in loss of balance and equilibrium between α- and β-like globin chains of hemoglobin. CTX001 is a CRISPR-Cas9 mediated gene editing treatment devised for thalassemia. The genome of hematopoietic stem cells (HBB gene) is edited for reactivation of the fetal hemoglobin production which suppresses the disease symptoms. The clinical trials for this treatment are in progress in rabbit models, where the HBB gene would be knocked out using the CRISPR-Cas0 technology. Cosenza in a clinical trial determined that correction of β0039 thalassemia mutation via CRISPR-Cas9 system resulted in regulation of proper functioning of β-globin genes. Thus genomic editing via this technology anticipates new treatment strategies for thalassemia with a higher success rate.
Genetic lung diseases
Inherited surfactant protein syndrome and cystic fibrosis are some of the genetic lung diseases that are congenital as well. These diseases have a high mortality rate due to respiratory failure. Cystic fibrosis is a highly fatal disease that arises due to mutation in the CFPR gene. Researchers have developed an embryonic mouse model harboring human SP gene SFTPCI73T mutations to propose the genome editing treatment of this disease. CRISPR mediated fluorescent-labeled system has been established for the timely inoculation of CRISPR-Cas9 components to inactivate the expression of SFTPCI73T mutant through non-homologous end-joining. This genetic modification enhances the life expectancy of the patient by 22.8% by reducing pulmonary pathogenesis. With the help of selection markers, scientists corrected the F508 deletion on the 10th exon of the CFTR gene in iPSCs cell lines. an iPSC cell line is developed from individuals suffering from this condition. The efficacy rate for this treatment was found to be 90%. The genomic repair mediated by the CRISPR Cas9 system restores the function of CFTR on epithelial cells of the lungs.
Cancer is a disease with high mortality rates, with very minimal treatment and cure strategies. However, CRISPR therapy has been devised based upon modification of T lymphocytes making them able to kill cancerous cells. The CRISPR Cas9 devised treatment technique functions in the following way:
- Administration of the synthetic genes donor T lymphocytes through genetic means.
- Removal of the three genes by CRISPR Cas9 system; one that reduces the cancer-killing ability of the cell and two genes that hamper the NY-ESO-1 receptors.
- Growth of NYCE T lymphocytes in large amounts as an end product.
- Administration of the prepared and edited T lymphocytes in the patient.
Another therapeutic application of CRISPR Cas9 technology for cancer is cell transformation which is performed in several steps. In this treatment approach, cancer cells are removed from the patient’s body and cultures in the laboratory. Then death receptor gene of these cells is knocked out through the CRISPR Cas9 system. The gene that converts ganciclovir to cancer-killing drug and other associated ligand molecules are administered to the cancer cells. These cells are then administered back to the respective person from which these were obtained. These cells when observed critically were able to recognize and attack metastasized cancer and tumors followed by apoptosis or self-destruction. This strategy anticipates the improved survival of cancer patients.
The CRISPR Cas9 system has the potential to treat human immunodeficiency virus infection (AIDS). The concept behind the use of this system as a treatment strategy is to reduce the HIV infection as well as target the cellular co-factors (that aid in its replication) to pro-viruses from the cells. Targeting long terminal repeats (LTRs) of HIV suppresses the expression of HIV genes in Jurkat cell lines. CRISPR Cas9 technology can also be used to remove the embedded or integrated genes of HIV from the infected cell genome, thus anticipating the great potential of this technique in AIDS cure. Scientists developed Cas9-guide RNA that was used to target the conserved parts of the LTR U3 region of HIV, which inactivated the expression of the HIV genes as well as inhibited the virus replication in pro-monocytes, microglial cells, and T cell lines. The scientist also has induced mutations in HIV pro-viruses through single guide RNA that resulted in the inactivation of the virus. Thus CRISPR Cas9 system provided future therapeutics for AIDA.
Treatment of the mitochondrial disorder
Mitochondrial diseases and inheritable disorders arise due to mutations in mitochondrial DNA. Up to the present, there is no specific treatment for their cure. However different gene-editing strategies have been devised that include MitoTALENs (mitochondrial-targeted endonucleases ) and zinc finger DNA cutting enzyme.
CRISPR Cas9 system is one of the most advanced techniques to propose the cure and treatment of mitochondrial disorders. Scientists have targeted the mitochondrial DNA, removed the foreign ssDNA (single-stranded) as well as lowered the number of mitochondrial DNA by using CRISPR technology. The cytidine deamination had also been performed in double-stranded mtDNA by associating CRISPR with a bacterial cytidine deaminase enzyme. But the treatment of such diseases is challenging because the size of cas9 protein is large and it’s quite difficult to take it inside the mitochondria. The delivery of guide RNA is also not very successful. However, a few guide RNA and Cas9 bases approaches are found to be effective to some extent to reduce the percentage of mitochondrial DNA mutations. Further research is also needed to explore and enhance the potential of the CRISPR-Cas0 system in treating mitochondrial disorders.
- Bakhrebah, M. A., Nassar, M. S., Alsuabeyl, M. S., Zaher, W. A., & Meo, S. A. (2018). CRISPR technology: new paradigm to target the infectious disease pathogens. Eur Rev Med Pharmacol Sci, 22(11), 3448-3452.
- Ding, R., Long, J., Yuan, M., Jin, Y., Yang, H., Chen, M., … & Duan, G. (2021). CRISPR/Cas System: A Potential Technology for the Prevention and Control of COVID-19 and Emerging Infectious Diseases. Frontiers in cellular and infection microbiology, 11.
- Strich, J. R., & Chertow, D. S. (2018). CRISPR-Cas Biology and Infectious Diseases Applications. Journal of Clinical Microbiology.
- White, M. K., Hu, W., & Khalili, K. (2015). The CRISPR/Cas9 genome editing methodology as a weapon against human viruses. Discovery medicine, 19(105), 255.
- Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262-1278.
- Reis, A., Hornblower, B., Robb, B., & Tzertzinis, G. (2014). CRISPR/Cas9 and targeted genome editing: a new era in molecular biology. NEB expressions, 1, 3-6.
- Liu, C., Zhang, L., Liu, H., & Cheng, K. (2017). Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. Journal of controlled release, 266, 17-26.
- Abbott, T. R., Dhamdhere, G., Liu, Y., Lin, X., Goudy, L., Zeng, L., … & Qi, L. S. (2020). VADR: Validation and annotation of virus sequence submissions to GenBank.
- Atasoy, M. O., Rohaim, M. A., & Munir, M. (2019). Simultaneous deletion of virulence factors and insertion of antigens into the infectious laryngotracheitis virus using NHEJ-CRISPR/Cas9 and cre–lox system for construction of a stable vaccine vector. Vaccines, 7(4), 207.
- Behan, F. M., Iorio, F., Picco, G., Gonçalves, E., Beaver, C. M., Migliardi, G., … & Garnett, M. J. (2019). Prioritization of cancer therapeutic targets using CRISPR–Cas9 screens. Nature, 568(7753), 511-516.
- Demirci, S., Leonard, A., Haro-Mora, J. J., Uchida, N., & Tisdale, J. F. (2019). CRISPR/Cas9 for sickle cell disease: applications, future possibilities, and challenges. Cell Biology and Translational Medicine, Volume 5, 37-52.
- Bak, R. O., Dever, D. P., & Porteus, M. H. (2018). CRISPR/Cas9 genome editing in human hematopoietic stem cells. Nature protocols, 13(2), 358-376.
- Yang, Y., Kang, X., Hu, S., Chen, B., Xie, Y., Song, B., … & Sun, X. (2021). CRISPR/Cas9-mediated β-globin gene knockout in rabbits recapitulates human β-thalassemia. Journal of Biological Chemistry, 296.
- Rogan, M. P., Stoltz, D. A., & Hornick, D. B. (2011). Cystic fibrosis transmembrane conductance regulator intracellular processing, trafficking, and opportunities for mutation-specific treatment. Chest, 139(6), 1480-1490.
- Zhan, T., Rindtorff, N., Betge, J., Ebert, M. P., & Boutros, M. (2019, April). CRISPR/Cas9 for cancer research and therapy. In Seminars in cancer biology(Vol. 55, pp. 106-119). Academic Press.
- Liao, H. K., Gu, Y., Diaz, A., Marlett, J., Takahashi, Y., Li, M., … & Belmonte, J. C. I. (2015). Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells. Nature communications, 6(1), 1-10.
- Al Khatib, T.E. Shutt, Advances towards therapeutic approaches for mtDNA disease, Adv. Exp. Med. Biol. 1158 (2019) 217–246, https://doi.org/10.1007/ 978-981-13-8367-0_12.