CRISPR and Its Applications

Introduction

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), is a family of short sequences of DNA fragments found in the immune system of bacteria(50%) and archaea(90%) known as CRISPR. These sequences are used to protect the immune system of prokaryotes against the viral infection by invading and cutting the viral DNA. These specific short sequences are actually derived from the part of the viral genome i.e. bacteriophage that infects the prokaryotes and now became the part of the prokaryotic genome which it uses as an antiviral property in the defence system. Now, scientists are able to alter the DNA of various organisms by using CRISPR technology as a GENOME EDITING TOOL. By altering gene sequences and gene functions, CRISPR technology may allow to treat various disease including cancer and to improve agricultural crops. In 1987, Yoshizumi Ishino firstly discovered clustered repeats by cloning ‘iap’ gene along with CRISPR sequences but he did not know about the function of these specific sequences. [1]

CRISPR Cas-9

CRISPR  is the special part of genomic DNA which have two specific features: spacers and repeats of nucleotide. Spacers are derived from the viruses that previously infected the bacterial genome and now act as a bank of memory to recognize and cut the similar viral DNA in pieces if it attacks in future. Repeated sequences are building blocks of genomic DNA and these are regularly spread over the entire length of the genome while spacers are interspersed between these repeated sequences.

CRISPR RNA

When spacer becomes part of the DNA genome from the previous viral infection, some part of CRISPR will be transcribed into CRISPR RNA (crRNA) when the virus again attacks it. In 2014, Jennifer Doudna published in a review article that transcribed RNA is complementary to the nucleotide sequence of CRISPR which act as a template strand. CRISPR RNA consists of distinct features i.e. nucleotide sequence and a spacer portion.

Cas-9

The CRISPR system was studied from Streptococcus Pyogenes and scientists discovered that Cas-9 protein is the main key feature of the defence system of bacteria that work against viral infection. The function of Cas-9 protein is to cut or degrade foreign DNA. The endonucleases of Cas 9 protein has two key components which play part in the degradation of foreign DNA i.e. CRISPR RNA (crRNA) and the other one is trans-activating CRISPR RNA (tracrRNA). Jennifer Doudna and Emmanuelle Charpentier compressed these two components into a single guide RNA molecule. When Cas-9 bind with this guide RNA, it could easily recognize and cut the specific sites of genomic DNA. Protospacer adjacent motifs PAMs are short nucleotide sequences which act as tags and specify the location for the activity of Cas-9 protein. It will act as a safety and more targeted mechanism for CRISPR technology.

 

Genome Editing Tool

CRISPR Cas-9 is used as a gene-editing tool to cut and modify nucleotide sequences within DNA of any organism using genetic engineering techniques. In this method, Cas-9 nucleases along with the guide RNA are transferred into the desired cell in which this complex show their activity by cutting the specific nucleotide sequence and replace it with new genes by using in vivo technique. There are four major components of genome engineering which edits the genome i.e.

• crRNA it forms a complex with guide RNA which finds the right sequence in the host genome along with tracrRNA.
• tracrRNA it forms an active complex along with guide RNA.
• sgRNA it is a group of single guided RNAs which have tracrRNA and one crRNA.
• CAS 9 it is an enzyme of the bacterial immune system which is able to cut and modify the host genome at specific locations.
• Repair template it consists of a DNA molecule which is used to repair the DNA of the host genome after cutting by Cas-9 enzyme.

Genome editing functions of CRISPR complex are utilized in many organisms and species which are giving major goals in various medicines, disease diagnosis and agricultural areas. [2]

Applications of CRISPR

1.    Biomedicine

CRISPR technology would be successfully used as therapeutic medicine to many diseases and it can also treat genetic mutations in the human genome. It has the potential to treat cancer, sickle cell anaemia, cystic fibrosis, thalassemia, heart diseases and haemophilia etc. because CRISPR can knock out disrupted genes and replace them with healthy genes. In 2016, CRISPR was used for lung cancer treatment. FDA also approved clinical trials of CRISPR technology for cancer treatment.

2.    Disease models

CRISPR technique allows rapid and efficient production of organism model for the study of various diseases which give accurate information about how Cas-9 proteins functions and modify when it interacts with the host genome. The first transgenic model created from CRISPR was Drosophila melanogaster. By using such in vivo methods of CRISPR, it could also treat down syndrome and related diseases. Genome editing was also done in Saccharomyces cerevisiae, Arabidopsis spp., Danio rerio, and E.coli.

3.    Treat infections

We know that Cas-9 enzyme is the part of the immune system of bacteria thus it can cut foreign invading virus and will be used as anti-viral and also anti-bacterial mechanisms. In recent studies, CRISPR technology was observed to control the multiplication of herpesviruses. It is used in the form of anti-herpesvirus CRISPR which can eradicate Epstein Bar Virus (EBV) from tumour cells.

4.    Biofuel production

CRISPR is used to produce biofuel from algae which will be eco-friendly and safe without causing any damage to environment as well as humans. Recently, synthetic genomics incorporation produces biofuel by using CRISPR from aquatic animals.

5.    Food production

The shelf life of fruits and vegetables would be increased by using CRISPR. It can also remove allergens from food. Tokushima University produces seedless tomatoes by using genome editing CRISPR technology.

6.    Fight against malaria

Mosquitos which spread malaria in humans could be genetically modified by altering their genome using CRISPR. Gene drive is used in this technology which states that transgenic mosquitos will transfer their resistance against malaria to next generation and hence it will not spread the disease.

7.    Revive extinct species

Extinct species could be revived by using CRISPR. Researchers from Harvard University announced that mammoth embryo could be revived by combing mammoth genes (from fossils) and elephant genes and then placed it in the womb.

8.    In agriculture

Cas 9 along with Cas12a also been used to produce a variety of plant species. CRISPR technology also provides genetically modified crops which are quality based crops. Resistance genes are incorporated into plants to fight against weeds and insects which will increase the crop production. Scientists from Mosanto also revealed that virus resistant and drought-tolerant crops could also be produced by using CRISPR.

9.    In IVF treatments

Gene editing could be done during in vitro fertilization to improve chances of pregnancy and to decrease chances of miscarriages. Chinese scientists recently used CRISPR in three human embryos to correct their mutations in genes.

10.  CRISPR as a diagnostic tool

In 2017, several scientists revealed that CRISPR along with nucleases would be beneficial as a diagnostic tool of nucleic acid for screening purposes. CRISPR is coupled with SPRINT (sherlock-based profiling of in-vitro transcription) to detect metabolites in the human body. It can also be used to detect toxic particles from the environment. [3][4]

By: Maryam Baig

REFERENCES

1. Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213).
2. Kleinstiver, B. P., Prew, M. S., Tsai, S. Q., Topkar, V. V., Nguyen, N. T., Zheng, Z., ... & Aryee, M. J. (2015). Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature, 523(7561), 481-485.
3. Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262-1278.
4. Uppada, V., Gokara, M., & Rasineni, G. K. (2018). Diagnosis and therapy with CRISPR advanced CRISPR based tools for point of care diagnostics and early therapies. Gene, 656, 22-29.