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What is CRISPR Technology and how is it used?

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CRISPR is a technology that enables gene editing, and as such, it has the potential to transform society. Finding a specific DNA sequence inside a cell is simple with CRISPR. After that, the section of DNA is usually modified in CRISPR gene editing. The CRISPR system has been modified to perform additional tasks as well, including turning genes on or off without changing their sequence.

Before the CRISPR technique was introduced in 2012, it was possible to change the genomes of some plants and animals, but it took years and hundreds of thousands of dollars. CRISPR has made it simple and affordable. In the future, many of the plants and animals in our farms, gardens, and homes may have to be modified with CRISPR, which is already widely employed in scientific studies. CRISPR food is already being consumed by some humans. Additionally, CRISPR technology has the potential to revolutionize medicine by allowing us to both treat and prevent a wide range of diseases. We might even choose to utilize it to alter the genes of our offspring. Although a Chinese attempt to do this has been criticized as premature and unethical, other people believe it could eventually be advantageous for kids. Other uses for CRISPR include gene drives and the control of evolution, as well as the fingerprinting of cells and recording of internal events.

CRISPR Technique

 

What is CRISPR technology?

CRISPR is a powerful tool for genome editing that enables researchers to quickly alter DNA sequences and alter how genes function. It offers a wide range of possible uses, including the correction of inheritable diseases, the treatment and prevention of illness, and the enhancement of crop growth and adaptability.

Cas9, also known as “CRISPR-associated protein 9,” is an enzyme that recognizes and cleaves particular DNA strands that are complementary to CRISPR sequences by using the CRISPR sequences as a guide. The CRISPR-Cas9 technique can be used to modify the genes of organisms by combining Cas9 enzymes with CRISPR sequences. The production of biotechnological products, the treatment of diseases, and basic biological research are just a few of the many uses for this editing technique.

Cas12a

The nuclease Cas12a, formerly known as Cpf1, was identified in the Francisella novicida bacterium’s CRISPR/Cpf1 system in 2015. Its initial name, derived from a TIGRFAMs protein family definition created in 2012, refers to the frequency with which the Prevotella Francisella lineages include this CRISPR-Cas subtype. In comparison to Cas9, Cas12a caused a “staggered” cut in the double-stranded DNA, as opposed to Cas9’s “blunt” cut. It also relied on a “T-rich” PAM (giving Cas9 alternative, targeting sites), and it only needed a CRISPR RNA (crRNA) for successful targeting. In contrast, Cas9 needs both a transactivating crRNA and a crRNA (tracrRNA). These variations could provide Cas12a an edge over Cas9.

Cas13

The Leptotrichia shahii bacterium’s nuclease Cas13a, formerly known as C2c2, was described in 2016. Cas13 is an RNA-guided RNA endonuclease, which means it can only cut single-stranded RNA and not DNA. Cas13 attaches to and cleaves an ssRNA target after being directed there by its crRNA.

Components

  • CRISPR/Cas systems are divided into two categories, Class 1 and Class 2.
  • While Class 2 systems only use one Cas protein, Class 1 systems use many Cas proteins to cleave foreign DNA. 
  • Types I, III, and IV are contained in Class 1, and Types II, V, and VI are contained in Class 2. 
  • The additional Cas genes present and a gene that is often found only within that type serve to define the type. 
  • 19 further subtypes divided the categories.

How CRISPR works?

Genome editing includes changing the DNA sequences that makeup genomes, so altering the signals and instructions that are encoded inside them. The intended modifications can be introduced by inserting a cut or break in the DNA and deceiving a cell’s normal DNA repair systems. CRISPR-Cas9 offers a method for doing this.
Two important studies that demonstrated how the bacterial CRISPR-Cas9 might be used to destroy any DNA, not simply that of viruses, were published in the journals Science and PNAS in 2012. The natural CRISPR system might be modified into a straightforward, programmable genome-editing tool in this way.

Scientists may simply alter the sequence of the crRNA, which binds to a complementary sequence in the target DNA, to instruct Cas9 to snip a particular section of DNA, according to the findings. The technique was further streamlined by Martin Jinek and his team by combining crRNA and tracrRNA to form a single “guide RNA.” Thus, a guide RNA and the Cas9 protein are the only two components needed for genome editing.

From there, one may determine what the complementary crRNA sequence would be. According to the authors, “operationally, you build a stretch of 20 base pairs that match a gene that you want to change.” The target gene must be the sole location in the genome where the nucleotide sequence is present, according to Church.
The RNA and the protein [Cas9] will then cut the DNA at that location, and ideally, nowhere else, Church explained.
Edits to the genome can then be done after the DNA is cut when the cell’s natural repair processes begin to put the DNA back together. For this, there are two useful strategies:

  1. Gluing the two cuts together is one mending technique. This technique, called “non-homologous end joining,” frequently causes mistakes where nucleotides are unintentionally added or removed, leading to alterations that may disrupt a gene.
  2. The gap is filled up with a series of nucleotides to repair the break. The cell does this by using a brief strand of DNA as a template. By providing the DNA template of their choice, scientists can add any gene they desire or fix a mutation.

Mechanism of CRISPR

The CRISPR/Cas-9 genome editing process can be broadly divided into three steps

  • Recognition 
  • Cleavage 
  • Repair 

The intended sgRNA controls Cas-9 and, through its complementary base pair in the 5 crRNA, recognizes the target sequence in the relevant gene. Without sgRNA, the Cas-9 protein remains dormant. Double-stranded breaks (DSBs) are produced by the Cas-9 nuclease at a location of three base pairs upstream of PAM. A short (2–5 base-pair length), conserved DNA sequence called the PAM sequence is located downstream of the cut site. The size of the PAM sequence changes depending on the type of bacterial species. The Cas-9 protein, which is the most commonly employed nuclease in the genome-editing tool, recognizes the PAM sequence at 5-NGG-3 (N can be any nucleotide base). When Cas-9 finds a target site with the right PAM, it causes local DNA melting, which is followed by the synthesis of an RNA-DNA hybrid. However, the process by which the Cas-9 enzyme melts the target DNA sequence is still not precisely understood. To cut DNA, the Cas-9 protein is then activated. Target DNA is broken up into its complementary and non-complementary strands by the HNH and RuvC domains, respectively, resulting in DSBs that are mostly blunt-ended. Finally, the host cellular machinery fixes the DSB.

Mechanisms for Double-Stranded Break Repair The two mechanisms used to repair the DSBs produced by the Cas-9 protein in the CRISPR/Cas-9 process are non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways. In the absence of external homologous DNA, NHEJ enhances the repair of DSBs by connecting DNA fragments through an enzymatic mechanism. NHEJ is active throughout the whole cell cycle. Although it is the most prevalent and effective cellular repair process, it is prone to mistakes and can produce minor random insertions or deletions (indels) at the cleavage site, which can result in frameshift mutations or premature stop codons. HDR requires the use of a homologous DNA template and is extremely precise. The late S and G2 phases of the cell cycle are when it is most active. A significant number of donor (exogenous) DNA templates bearing an interesting sequence are necessary for HDR in CRISPR gene editing. By inserting a donor DNA template with sequence homology at the anticipated DSB site, HDR carries out the precise gene insertion or replacement.

Identification of CRISPR

Bacteria and archaea both have a lot of CRISPRs, and they exhibit some sequence similarities. Their direct repeats and repeated spacers stand out as their most distinctive features. Due to the low possibility of a false positive match due to the high number of repeats, CRISPRs are easily distinguishable in lengthy DNA sequences.

Since CRISPR loci don’t generally assemble due to their repetitive nature or through strain heterogeneity, which confounds assembly methods, analyzing CRISPRs in metagenomic data is more difficult. Polymerase chain reaction (PCR) can be used to amplify CRISPR arrays and analyze spacer content in environments with a large number of reference genomes. However, this method only provides data for CRISPRs that are specifically targeted and for organisms that are sufficiently represented in public databases to allow for the development of trustworthy polymerase chain reaction (PCR) primers. Long CRISPR arrays can be created by computationally assembling amplicons comprising two or three spacers that were amplified using degenerate repeat-specific primers directly from environmental materials.

Applications of CRISPR

Basic Science

As of now, the CRISPR/Cas9 gene editing technology seems to function in almost every organism, from Caenorhabditis worms to monkeys, and in every cell type: kidney, heart, and those, like T-cells, that researchers had previously found challenging to modify.

Agriculture

Wheat and rice, two crucial crops for agriculture, were the focus of gene editing. Early on, it became clear that this technique might be used to modify the DNA of these crop species especially to enhance features like disease and drought resistance. Conventional breeding relies on large back-crossing procedures and current natural genetic variation to produce desired features. To create plants resistant to the powdery mildew disease, Wang et al. (2014) targeted and successfully knocked out the genes of the mildew-resistance locus (MLO) in wheat using both the TALEN and CRISPR/Cas9 technologies. 

Disease Modeling

Due to the difficulty in producing genetically engineered animals that faithfully recreate human pathophysiology, disease modeling using animals has been a barrier to the research of many types of human diseases. The procedure was either impossible to complete or inefficient and imprecise. R, ats, monkeys, and other transgenic animals have been created thanks to CRISPR-Cas9, which makes them better than mice at simulating human diseases and enabling more accurate drug testing.
Since mice lack the complex cognitive abilities of primates, neuroscientists have long expected transgenic monkeys the study brain illnesses like autism, schizophrenia, and Alzheimer’s disease that cannot be properly recapitulated in mice. The single-cell monkey embryo’s DNA was modified using CRISPR-Cas9, resulting in the disruption of a gene that is now present in all of the primate’s cells. This will make it easier to research the gene’s role in autism and identify potential new treatments.

Gene Therapy

In mice, a mutation linked to human tyrosinemia was corrected using CRISPR-Cas9. It was the first time CRISPR/Cas9 has been applied to correct a disease-causing mutation in an adult animal, and it was a significant step toward applying the technology to human Gene Therapy.

During infection, the HIV-1 (Human Immunodeficiency Virus) virus can incorporate its genome into the DNA of the host cell. If left untreated, HIV-1 primarily affects CD4+ T cells and results in AIDS (acquired immunodeficiency syndrome). Long terminal repeat (LTR) sequences on the viral genome are used by integrases to insert the viral DNA into the genome of the host cell. Anti-retroviral medication can control AIDS (targeting the reverse-transcription step in the HIV replication process). The condition cannot be treated, however, because this medication does not affect the host cell’s latent pool of HIV-1 DNA. 

Issues

The CRISPR-Cas9 technology causes unintended off-target (effects outside of the intended editing sites) effects in mice, according to a Stanford University study.

  • The worry that the CRISPR system is being hurried into clinical use is still present. This anxiety has been further heightened by three recent reports.
  • Studies have shown that cells modified by CRISPR-Cas9 may cause cancer.
  • This may raise the probability of mutations elsewhere in the genome in those cells.
  • Although CRISPR-Cas9 technology has been utilized successfully to treat several diseases, many things are still unclear, such as how to choose which diseases or features are suitable for gene editing.
  • Ethical issues and concerns also exist over the manipulation of human embryos for personal gain.

Risk

  • It may specifically target genes.
  • Viral infections can transmit to individuals.
  • It can change specific groups of people

Ethical Issues

While CRISPR has the potential to treat some diseases, studies have found that it may also cause mutations which might eventually cause other diseases.

Any genetic alterations done to embryos, eggs, or sperm cells will be passed down to all future generations. One of the main ethical issues with this kind of gene editing is that any changes will have a knock-on impact and be passed down from generation to generation. The effects of DNA editing may eventually be visible throughout the entire human population.

The use of CRISPR/Cas9 technology has enormous potential for both good and bad. It poses some issues with the basic definition of what it is to be a human in some aspects. Its use would have far-reaching effects in ways that had never been seen before.

CRISPR treating Cancer

When we could utilize CRISPR to attack cancerous tissue and healthy cells from harm, it only enters cancer cells. Because of the CRISPR-huge Cas9’s size and poor penetration, existing targeting techniques used to administer chemotherapy to cancer cells are unable to reach enough of the targeted cancer cells.

Lipid nanoparticles were the answer they came up with. Clinically approved nonviral nucleic acid delivery methods with such potentially huge payloads are lipid nanoparticles (LNPs). The essential element of LNPs that enables effective nucleic acid encapsulation, cellular distribution, and endosomal release is cationic ionizable lipids. To transfer a bigger molecule and enable the delivery of the payload into numerous tissue types, they had to modify the LNPs. The CRISPR system itself targets genes involved in tumor survival. The cancer cell should stop reproducing after these genes are disrupted, and ultimately die and undergo apoptosis. They research ovarian and glioblastoma in mice to examine their innovative LNP delivery technology with CRISPR-Cas9 targeting tumor survival genes. They discovered some very positive information.

The most aggressive type of brain cancer, glioblastoma, has a typical survival time of about 15 months. Because of the way that they penetrate the brain, this cancer has proven to be extremely resistant to treatments with chemotherapy and radiation and cannot be surgically removed. For this reason, the researchers selected glioblastoma specifically. The LNPs with the CRISPR-Cas9 payload were injected into the brains of animals with glioblastoma just once. They discovered that up to 70% of in vivo gene editing led to the death of up to 50% of tumor cells and increased survival by 30%. That would be a remarkable outcome if it applied to people. Although it is not a cure, as is the case with all new anti-cancer therapies, this would greatly increase the continuously rising survival rate of cancer patients.

The researchers further stress that neither the type of cancer nor the malignancy itself is exclusive to this system of LNPs delivering CRISPR-Cas9. Other cell types can use the basic technology once it has been refined. For instance, it might be used to target contagious pathogens. 

In summary, this is a new method of treating cancer that shows incredible possibilities, even for the most dangerous solid tumors, which now have a poor prognosis. But we need to remember that there is a lot of clinical research on the usage of this prospective medication on actual patients in the clinic and the hype. In the best-case scenario, it will be years before these treatments are approved. However, I do believe there is a good chance that this or similar treatment will one day be utilized to treat cancer. It is a novel therapeutic approach that will supplement current therapies and has the potential to considerably increase cancer survival in a variety of cancer types.

FAQs on CRISPR Technique

Question 1: What is the major application of CRISPR technology?

Answer: 

The major application of CRISPR technology is

  • Basic science
  • Agriculture
  • Disease modeling
  • Gene therapy

Question 2: Define CRISPR technology.

Answer: 

CRISPR is a powerful tool for genome editing that enables researchers to quickly alter DNA sequences and alter how genes function.

Question 3: Write in detail about Cas12a.

Answer: 

The nuclease Cas12a, formerly known as Cpf1, was identified in the Francisella novicida bacterium’s CRISPR/Cpf1 system in 2015. Its initial name, derived from a TIGRFAMs protein family definition created in 2012, refers to the frequency with which the Prevotella Francisella lineages include this CRISPR-Cas subtype. In comparison to Cas9, Cas12a caused a “staggered” cut in the double-stranded DNA, as opposed to Cas9’s “blunt” cut. It also relied on a “T rich” PAM (giving Cas9 alternative, targeting sites), and it only needed a CRISPR RNA (crRNA) for successful targeting. In contrast, Cas9 needs both a transactivating crRNA and a crRNA (tracrRNA). These variations could provide Cas12a an edge over Cas9.

Question 4: Write at least 3 issues that are caused by CRISPR technology.

Answer: 

Issues that are caused by CRISPR technology

  1. Studies have shown that cells modified by CRISPR-Cas9 may cause cancer.
  2. Ethical issues and concerns also exist over the manipulation of human embryos for personal gain.\
  3. CRISPR-Cas9 technology has been utilized successfully to treat several diseases, but many things are still unclear, such as how to choose which diseases or features are suitable for gene editing.

Question 5: Write the uses of CRISPR technology.

Answer: 

Uses for CRISPR technology

  1. gene drives and the control of evolution
  2. fingerprinting of cells and recording of internal events.
  3. treat and prevent a wide range of diseases.
  4. many of the plants and animals in our farms, gardens, and homes may have been modified with CRISPR.


Last Updated : 13 Oct, 2022
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