Altering the DNA or gene sequences present into the genome of an organism is called as a gene editing. The alteration may be addition or deletion. CRISPR-CAS9 is the system used as a gene editing tool in recent days.

In simple words, we can describe the gene editing as editing the messages of the DNA. In gene editing, we are changing the sequences thereby changing the message-driven by that particular DNA sequence.

In this article, we will discuss the gene editing or genome editing and related tools. The content of the article is,

  • History of gene editing
  • What is a need of the gene editing?
  • What is gene editing?
  • How gene editing is done?
  • Importance of gene editing
  • What are the different types of gene editing systems?
  • CRISPR-CAS9 gene editing system
  • CRISPR-CAS9 in Bacteria
  • CRISPR-CAS9 in genetic engineering
  • Applications of CRISPR-CAS9
  • Limitation of CRISPR-CAS9
  • Ethical issues in gene transfer technique

“When the gene or the sequence of DNA deleted, inserted, replaced or modified with the help of the bacterial nuclease enzyme, it is called as a gene editing, also called as genetic engineering”.

History of Gene editing:

Some prefer to say it as genome editing but both the terms can be used, we can use any of it. The main point of editing DNA is to remove the abnormal or faulty DNA sequence. The story for gene editing came into the light in the late ’70s when Herb Boyer and Stanley Cohen discovered the antibiotic-resistant genetically engineered bacteria. Until 2012, the idea for gene editing was just a fairy tale.

In 2012, the CRISPR gene editing system was discovered which changed the era of genetics. A group of scientists from the University of California discovered a so-called bacterial immune system which can be edit genes at the targeted location and named it as a CRISPR.

However, the idea was evolved in the year 1982 when the human insulin-producing bacteria were discovered. This synthetic insulin becomes so successful that it was commercially available after FDA approval.

After the successful implementation of the synthetic insulin, A roadmap was created for the genetically engineered Flvr shower tomato, bringer and some of the species of cotton. This is the short history and the milestone in gene editing technology.

What is gene editing and CRISPR-CAS9?

Image credit: www.pioneeringminds.com. The guided RNA/DNA Heteroduplex.

What is a need of the gene editing?

Genetic diseases are lethal and nearly incurable. After the discovery of DNA into 1953, the picture of inheritance of disease becomes more clear. The chromosome carries the DNA and inherited from one generation to another. More detail on the DNA packaging, read the article: DNA packaging in eukaryotes.

In this manner, the genetic material inherited from parents to their offspring and so the mutation is. Any alterations occurred into the genome inherited into the next generation which causes severe disease condition.

Gene editing can help to eliminate this mutated portion of the genome.

What is gene/genome editing?

“ Manipulating the genetic composition of an organism artificially is called as a gene editing”.

Editing genes of bacteria, yeast and mice are nowadays quite easy, however, not possible for the human genome. Actually, it is not impossible but there are some ethical issues associated with a human embryo. We will discuss ethical issues in the later part of the article.

The process of gene editing is divided into the following steps:

  • Identification of target site into the genome
  • Designing a healthy copy of that particular targeted site
  • Guided molecules find the target site
  • An enzymatic reaction for cutting the faulty DNA
  • Insertion of new DNA
  • Repairing the site of action

The targeted site into the genome or in a gene is a place which is faulty or mutated and produces faulty/mutated protein. The specially designed “Guided molecules” (which are nucleic acids) binds to the specific target site into the genome with the help of the enzyme.

The enzyme is a nuclease, a bacterial nuclease also called as molecular scissor or engineered nuclease which helps in cutting the DNA, After binding of a guided molecule to the target site.

Once the guided molecule binds to the site of action the nuclease cuts that portion of the DNA from the genome and removes it. A nick is created at this place is later filled by the cells natural DNA repair mechanism.

The cells natural DNA repair mechanism finds any change or break into the genome and fill it with the complementary bases during the process of the replication. We can also insert the piece of DNA of our interest on that particular site.

By means of nuclease governed gene editing, we can delete or insert the DNA sequence of our interest. This is the simple process of gene editing. The pictorial representation of gene editing is shown in the figure below.

What is gene editing and CRISPR-CAS9?

Glossary used into the article:

CRISPRClustered Regularly Interspaced Short Palindromic Repeats
CAS9CRISPR associated protein 9
gRNA Guided RNA
crRNA CRISPR RNA
DSBDouble-stranded break
ZFNZinc finger nuclease
TALENTranscriptional Activator-Like Effector Nuclease
NHEJNon-homologous end joining
HDR Homology direct repair
PAMPhoto-spacer adjacent motif

Importance of gene editing:

  • It is used to edit the genome of any organism however, it is most applicable in the bacteria, yeast, mice and other model organisms.
  • It is used to study the gene expression of the organism.
  • The technique can remove mutated genes from the genome of an organism, also, it can be used to create new variation.
  • One can change the characteristics of the organism.
  • Economically important genetically modified organisms can be created by gene editing technology.

What are the different types of gene editing systems?

The molecular scissor, genetically modified nuclease enzyme plays an important role in gene editing technique. Therefore, depending on the types of nuclease there are several gene editing techniques used since long in genetic engineering. Three major techniques are enlisted here:

  1. ZFNs (Zinc finger nuclease)
  2. TALEN (Transcriptional Activator-Like Effector based Nuclease)
  3. CRISPR-CAS9 (clustered regularly interspaced short palindromic repeats)

ZFNs and TALENs:

The difference between the ZFNs and TALENs are only because of the use of different molecules.

The ZFN technique was introduced in early 1991 by Palvetich and Pabo. The Zinc finger proteins are an artificially engineered protein binds to the specific location into the targeted site. The ZNFs bind to the 3 to 4 nucleotide away from the cutting site.

Two copies of the ZNFs are required for the gene editing. Once it binds to the target sequence, the bacterial nuclease Fok1 cuts the DNA sequences. Fok1 is the bacterial nuclease encoded by the DNA binding domains.

Read more,

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  2. DNA digital data storage
  3. DNA fingerprinting

Once the nuclease cuts the DNA, the portion can be deleted or our DNA of interest can be inserted into the site. Here the Fok1 recognise the ZFNs at both the side of the DNA sequence as well as cuts DNA at the exact location.

The TALEN works similar to ZFNs However, it can cut the larger sequence efficiently than ZNFs. In TALEN the Fok1 cuts the double-stranded DNA for gene editing.

The graphical representation of both the techniques are given into the figure below,

What is gene editing and CRISPR-CAS9?

Zinc finger nuclease, Transcriptional activator-like effector based nuclease and meganuclease are the traditional techniques which are not applicable in the recent day due to several limitations.

  • These techniques are time-consuming and costly. More time and cost are required to engineer artificial proteins such as TALEN and ZFNs.
  • The specificity of gene editing is also poor.
  • The end result, the rate of gene edition is also low.
  • The technique is more sensitive and accuracy is less.

In the year 2012, a new method of gene editing was demonstrated by the group of scientists from the University of California named it CRISPR-CAS9. The CRISPR-CAS9 is most advanced and futuristic technique. It is fast, reliable, cost-effective, efficient and accurate.

CRISPR-CAS9 gene editing system:

CRISPR- Clustered Regularly Interspaced Short Palindromic Repeats present into the bacteria and some archaea. It is the elements of the bacterial natural defence mechanism. The CRISPR derived RNA helps the bacteria to protect it from the invading viruses and phages. The nuclease, CAS9 protein cuts the DNA strand of the foreign pathogens and destroys them.

The CRISPR gene sequences were discovered into the year 1980 in E.Coli but their function was not known at that time. Later on, in the year 2007, Barrangou et al., reported their role in the adaptive immune system of the bacteria.

CRISPR-CAS9 in Bacteria:

During the viral infection, the virus injects its DNA into the Bacteria through the bacterial cell wall. The viral DNA integrated into the genome of the host and transcribed and translated. The new viral protein forms from the bacterial genome.

To avoid viral infection, bacteria developed a defence mechanism called as a CRISPR-CAS9 based defence mechanism.  

During the viral invasion, the bacteria took some of the DNA sequences as a reference and inserted it into the palindromic repeats that generate CRISPR. CRISPR is the combination of bacterial palindromic repeat sequences and the viral spacer DNA.

The spacer DNA transcribed into the RNA called as crRNA which guide to bind at the specific location into the viral genome. The DNA-RNA heteroduplex is created at the site of the invasion.

Along with the crRNA, the bacteria also transcribed several short tracrRNA (trans-activating crRNA), partially complementary to the crRNA. The tracrRNA helps for the maturation of the crRNA from the multiple pre-crRNA.

The junction of crRNA and tracrRNA is called as a gRNA or guided RNA which guides the nuclease for cutting the target sequence.

The CAS9 recognise this gRNA and binds to the location of the heteroduplex. Once it placed properly, two unrelated domains of a nuclease (CAS9 nuclease) named as RuvC- like domain and HNH domains lysed the dsDNA of the virus.

What is gene editing and CRISPR-CAS9?

CRISPR-CAS9 defence system in the bacteria.

During the nuclease attack, the CRISPR altered the DNA sequences of the pathogen which helps the bacteria from the viral invasion. The CAS9 work as a dimeric molecular scissor for cutting DNA at two different points on two different strands.

The RuvC domain cuts the non- homologous domain while the HNH cuts the homologous domain (homologous to gRNA).

The foreign pathogen or the virus destroyed by the combined effort of CRISPR and CAS9 protein. One question arises after this explanation that, “does it cut its own DNA?” the answer is “Yes”.

The viral DNA sequence is present into the CRISPR as well. Therefore, it is might possible that bacteria CRISPR-CAS9 recognises these spacer sequences as a foreign DNA and edit it. Actually, it does not happen, thanks to the motif called as a PAM.

The photo spacer-associated motif (PAM) helps the CAS9 to recognise the gRNA. The PAM binds to the foreign DNA immediately after the formation of RNA-DNA heteroduplex. It binds at the 3’OH end after the gRNA.

This will give a signal to CAS9 for cleaving the DNA. CAS9 recognise the PAM-gRNA complex on dsDNA and performs the catalytic reaction. PAM does never bind to bacteria’s own DNA. It is a kind of built-in safety mechanism developed by the bacteria itself (How smart they are).

PAM protects bacterial DNA from their own nuclease activity.

The CRISPR are the DNA sequences made up of the spacers and the repeats. The spacers are present between the short palindromic repeats throughout the CRISPR.  Different spacer sequences are taken from the different pathogen which helps the bacteria during the pathogenic attack in future.

Bacteria use the information of these sequences to destroys the pathogens. The bacterial CRISPR-CAS9 system has the potential for gene editing other than their own genome.

CRISPR-CAS9 in genetic engineering:

Guided RNA and the dimeric CAS9 protein are the only requirements in the gene editing technique.

Once the nuclease cuts the DNA at the specific location, it creates the double-stranded cut into the DNA. Now, the natural DNA repair mechanism of the cell repairs this gap in one of the two ways. The DNA repair mechanism involved in the CRISPR-CAS9 gene editing is double-stranded break repair.

Non-homologous end joining: in this type of DNA repair mechanism, the gap is directly filled by the joining of two opposite non-homologous strands directly. Here, in NHEJ several important pieces of genetic information might be lost because the entire fragment of DNA is lost.

This DNA fragment will never be present in the next cell division, will create a mutation into the genome. If some of the important gene for the organism is knocked out, the condition becomes lethal for the organism.

Homologous direct repair: the gaps are not filled directly in homologous repair. The Homologous direct repair follows the mechanism of recombination. Here the nucleotides are inserted into the site of the cut based on the information of the previous replication.

For doing this, the cell uses single-stranded DNA as a template for the addition of new nucleotides. Now, this is the time we can insert a single-stranded DNA of our interest which synthesised naturally in consecutive cell division by the cell itself.  

Knocking out any faulty DNA can be possible by non-homologous end joining and insertion of new DNA into the genome can be possible by homologous direct repair.

In the molecular genetics or in genetic engineering the CRISPR-CAS9 is used as a gene editing tool. Based on the activity of the CAS9, three different gene editing systems are postulated. All three types of systems are enlisted below:

  1. CAS9 with only cleavage activity
  2. CAS9 nuclease full activity
  3. CAS without the cleavage activity.

In the first system, cas9D10A mutant is developed from the CAS9 encoding gene. This mutant can not cleave both strands of the DNA. instead, cas9D10A can recognise the gRNA and cleaves only a single strand of the target DNA.

What is the benefit of this mutant? Well, it increases the precision of the gene editing. It allows only high-fidelity homologous direct repair of the single-stranded gap reduces the deletion-insertion mutation chance into the gnome.

In short, cas9D10A variant does not allow the non-homologous end joining.

The second system is the normal function of CRISPR-CAS9. None of the sequences is mutated for CAS9 proteins. We had already discussed the CRISPR-CAS9 system.

The third system depends on dCas9. The dCas9 is called as nuclease deficient CAS9. Some mutations are artificially introduced into the RuvC and HNH domains lead to suppressing the function of nuclease.

The nuclease deficient CAS9 can only bind to the target sequence but can not cut it. This facilitates gene silencing or activation of a particular gene with the help of many effector domains.  

What is gene editing and CRISPR-CAS9?

Read further,

Application of CRISPR-CAS9:

The technique is reliable, cost-effective and fast.

Creation of large deletion, rearrangement and inversion are possible by CRISPR-CAS9.

A point mutation can be induced with the help of single gRNA.

The gRNA can be easily synthesised and design that is not possible in case of ZFN and TALEN.

Gene silencing is achieved by the dCas9 system. Any mutated gene or gene of our interest can be suppressed by CRISPR-CAS9.

CRISPR-CAS9 is applicable in the creation of genetically modified organisms (also called as genetically engineered organism). Many different genetically engineered plant species can be developed by CRISPR-CAS9.

Furthermore, gene expression studies in mice, sea archaea, zebrafish and fungus are possible because of the CRISPR-CAS9. Knockout mice can be generated by CRISPR-CAS9 which helps in the study of the role of the particular gene.

Microscopic examination of specific gene loci is facilitated by CRISPR-CAS9.

What is gene editing and CRISPR-CAS9?


Do you know

We can visualise any gene with the help of CRISPR-CAS9?

The naked CAS9 (CAS9 without nuclease activity) can only bind to the specific gene loci. By introducing the mutation D10A into the RuvC domain and H840A into the HNH domain of CAS9 the activity of nuclease suppresses, however, it allows CAS9 binding to the gene of interest. The gene of interest does visualise microscopically.

 

EGFPs, Enhanced Green Fluorescent Protein fused with the  dCAS9 was first used to visualise the repetitive sequences into the genome.

 


Limitations of CRISPR-CAS9:

CRISPR-CAS9 is the best tool in recent years. Nonetheless, only a single limitation makes it restrictive. The “off-target effect” introduces mutation other than the target site. CRISPR-CAS9 cleaves off-target DNA too and reduces the efficiency of the gene editing.

In some organisms such as zebrafish and sea archaea >70% efficiency of gene editing was reported (WY Hwang et al., 2013). In contrary, only 3% to 4% efficiency was reported into human pluripotent cells (P Mali. et al., 2013).

Efficiency deviation in different cell types is the only limitation of the CRISPR-CAS9 tool.  

What are the ethical issues in gene editing?

Ethics are the moral principle of the individual. We have to take care of the moral principles of the organism while performing any scientific experiments on them. The prior concern is further required before performing any experiment on any organism.

Safety is the first ethical concern of any experiment. So the gene transfer technique must be safe for use in routine diagnostics.

It should be available for all. If it is too costly, only rich people can afford it which means it creates discrimination based on wealth. The gene transfer technique must be cheaper enough to available for all.

The technique must be restricted for trait dependent disease diagnosis, not for personal health care. One can use it for increasing athletic capabilities and beauty treatments. Therefore, the use of the technique is restricted for some meaningful purpose.

The gene editing technique should not have any lethal side effects.

The risk level of the technique should be minimum otherwise it will adversely affect the organism as well as the environment.

What is gene editing and CRISPR-CAS9?

Gene editing should be avoided into the germline cells or into the developing embryo because it can create any new deleterious, lethal or harmful mutations into the population.

Creating, manipulating or destroying a human embryo is not a legal activity and prohibited by USA, UK and other governments. After all, social, religious or moral sentiments of any individuals should not be affected.  

The off-target effect is a big challenge in the CRISPR-CAS9 gene editing technique. Many homologous DNA sequences are present in the genome of an organism. The gRNA can bind to any of this homologous DNA sequence.

This results in the induction of the unknown mutation into the genome. The deleterious effect of new mutation may be harmful to the cell or the organism.

Read more on PCR,

  1. A Complete Guide of the Polymerase Chain Reaction
  2. The Function of dNTPs in PCR reaction
  3. Role of DMSO in PCR: DMSO a PCR enhancer
  4. Function of taq DNA polymerase in PCR
  5. PCR primer design guidelines
  6. Role of MgCl2 in PCR reaction

Conclusion:

Though the gene transfer technique is revolutionary and emerging genetic technique, experiments on humans and other animals should be avoided. No doubt, CRISPR-CAS9 like gene editing tools will be available in medical science in the nearby future but manipulating genetic composition of an organism can create serious problems for mankind.