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The genome of an organism has always fascinated researchers.
The discovery of restriction endonucleases, enabled scientists to make targeted manipulations (or knockouts) in any gene sequence of any organism, by the technique popularly known as genome engineering.
But today is an era of Gene Editing dominated by the CRISPR/Cas9 tool due to its ease of design and handling.
The article introduces you to the basics and applications of gene editing, base editing, and CRISPR technology.
This article covers zinc finger nucleases, TALENs, the CRISPR system, and the latest techniques like BASE EDITING andPRIME EDITING.

What is Gene Editing?

GENE EDITING or GENOME EDITING with programmable nucleases is a rapidly growing field in genome engineering.
It involves transformative technologies that allow researchers to make precise changes in the DNA of a living organism for numerous applications such as biomedical research, biotechnology, agriculture, human gene therapy, and patient treatment.
GENOME EDITING is a sophisticated type of genetic engineering that can add, remove, or alter genetic material at specific locations within an organism’s genome.
In GENOME EDITING, there are TWO BASIC PROCESSES.

Genome editing techniques like CRISPR, TALENs, and ZFPs are now in widespread use to alter specific sequences in the genomes of cultured cells.

GENOME Editing Vs Genetic Engineering

Gene Editing and Genetic Engineering both make changes in the genetic material of organisms, but they differ in their methods and precision.
The current genetic engineering technologies precisely modify the DNA to mimic the naturally occurring differences in DNA sequence.
They randomly insert genetic material into a host genome whereas gene editing targets site-specific locations.

What is Gene Editing used for today? | Applications of Gene Editing

Gene editing is a powerful tool that has a wide range of applications across various fields. Some of the key areas of application are Medicine, Agriculture, Environmental Protection, Biofuels, and Basic Research.
Scientists use Gene Editing to develop treatments for genetic disorders such as muscular dystrophy, cystic fibrosis, and sickle cell anaemia.

CRISPR-based therapies have shown promising results in clinical trials to treat these conditions.
In agriculture, gene editing helps create disease, pest, and environmental stress-resistant plants.
It also enhances yield and the nutritional value of crops.
Genome editing techniques can introduce precisely targeted modifications in any crop.
It is used to manage harmful species and control disease vectors like mosquitoes that spread Malaria and Dengue. This can help protect ecosystems and reduce the transmission of diseases in humans and crops.
Gene-edited microbes break down agricultural and industrial waste into biofuels.
This not only provides a renewable energy source but also helps in Waste Management and Environmental Protection.
Scientists are using Gene Editing to modify microorganisms like algae and bacteria to produce biofuels at higher rates and with greater yields.
Gene Editing technology can reduce the need for chemical inputs to produce biofuels and make the production process more sustainable and environmentally friendly.
Scientists use gene editing to understand the fundamental aspects of biology and genetics including- gene function studies, disease models, genetic pathways, drug development, and evolutionary biology.
This can lead to discoveries and innovations in biology, agriculture, and medicine such as inserting a new gene to make organisms produce useful medicines; treating genetic diseases;
creating tailor-made organisms to study human diseases; and producing replacements for damaged or diseased tissues and organs.

Chronology of gene editing tools and techniques | History of Gene Editing

The evolution of genome engineering started about seven decades ago with the discovery of the DNA double helix.
Research in this field has been active since the 1970s.
During these years, researchers uncovered different techniques to perform gene editing.
The journey of gene editing starts with Meganucleases and Zinc Finger Nucleases (or ZFN), walks through Transcription Activator-Like Effector Nucleases (or TALENs) and CRISPR-Cas-9 to reach Base Editing and Prime Editing. 
Meganucleases discovered in the 1990s, were among the first tools used for gene editing.
They are natural enzymes that recognize and cut specific DNA sequences.
However, their use was limited due to the difficulty in targeting new sequences.

Precise gene editing techniques began with the introduction of zinc finger nucleases (ZFNs) again in the 1990s.
These are artificially produced restriction enzymes for custom site-specific genome editing.

TALENs, or Transcription Activator-Like Effector Nucleases, came in 2011, are a molecular tool derived from a plant pathogen. These are capable of single-nucleotide recognition, thereby increasing targeting capabilities and specificity compared to zinc finger nucleases.
TALENS are cheaper, safer, more efficient, and capable of targeting a specified region in the genome.
In 1987, an important tool ‘Clustered regularly interspaced short palindromic repeats (CRISPR) for gene engineering was discovered in Escherichia coli by Ishino and colleagues.

The CRISPER technology uses a guide RNA and the ‘CRISPER associated protein 9’ or Cas9 endonuclease, hence it’s name is CRISPER/Cas9.
The Base Editing developed in 2016 unlike previous techniques, avoids cleavage of nucleic acid backbones and directly modifies target nucleobases. It offers a more precise way to correct point mutations.
The latest in the armamentarium is Prime Editing which came in year 2019.
Compared to base editing it is a more versatile molecular tool because it can precisely perform targeted small insertions, deletions, and base exchanges while limiting unwanted effects without causing double-strand breaks.

Zinc Finger Nuclease (ZFN)

Zinc fingers were discovered in 1985.
ZFNs are engineered DNA-binding proteins.
Efficient genome editing relies on a site-specific DNA double-strand break (DSB).
Zinc finger (ZF) nucleases (ZFNs) were developed to generate a site-specific DSB at the desired genomic region.
Each zinc finger is a domain stabilized by a zinc ion and a hydrophobic core, allowing for specific DNA recognition.
They facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations which are then repaired by the cell’s natural repair mechanisms, leading to targeted changes in the genome.

ZFNs have two main parts: Zinc Finger DNA-Binding Domain or ZF protein and Fok1 Nuclease Domain.
Each zinc finger protein (ZF protein) can recognize a sequence of 3-4 base pairs, hence targeting longer sequences needs multiple zinc fingers.
The first part recognizes and binds to specific DNA sequences and the second part is responsible for cutting the DNA.
This requires dimerization or pairing with another FokI domain to become active, ensuring that DNA is only cut when two ZFNs bind to adjacent sites on the DNA.
When two ZFNs bind to their target DNA sequences, the Fok1 domains create a double-strand (ds) break.

The cell then repairs this break using one of two methods:

1. Non-Homologous End Joining (NHEJ) and
2. Homology-Directed Repair (HDR).

The NHEJ can introduce small insertions or deletions, potentially disrupting the target gene.
The HDR uses a repair template to make precise changes to the DNA sequence.
Applications of ZFNs are gene therapy, crop modification, and gene study.
ZFNs are one of the earlier tools developed for Genome Editing, alongside other technologies like TALENs and CRISPR/Cas9.

TALENS | Transcription Activator-Like (TAL) Effector Nucleases

TALENs, or transcription activator-like (TAL) effector nucleases, are precise and efficient gene editing tools. Unlike CRISPR-Cas9 technology, TALENs can target any desired sequence within a genome, with no PAM site restrictions.
TALENs consist of a DNA binding domain linked to a specific DNA cleavage domain, usually a nuclease like Fok1, to cut specific DNA sequences.
TALEN gene editing technology is useful when no suitable CRISPR PAM sites are available for specific designs.
It may also be more efficient than CRISPR/Cas9 in editing hard-to-edit genomic regions such as heterochromatin.
TALENs exhibit superior HDR efficiency due to closer proximity to the insertion site. 
The key advantages of TALENs are customizable binding and no PAM site requirement.
Hence, TAL effectors can be engineered to bind almost any DNA sequence, making TALENs highly versatile and allowing for more flexible targeting.
TALENs are used in plants, animals, and human cells, for tasks like gene knockout, gene insertion, and gene editing.

CRISPR/Cas-9 System

CRISPR Gene Editing has evolved as one of humanity’s most powerful technologies.
CRISPR-Cas9 system has emerged as the most flexible and user-friendly tool for Genome Editing.
It eliminates the need for making new proteins to recognize each new target site.
It is less expensive and relatively easy to design and use when compared to older technologies such as zinc finger nucleases. This is one reason why CRISPR is a game-changing technology.

In the first decade of the 21^st Century, the scientific community understood its ability to recognize specific genome sequences and cut them with the ‘CRISPR-associated protein 9’ or ‘Cas-9’ protein. ‘Cas-9’, works with CRISPR and has DNA-cutting abilities.  It inserts small pieces of edited DNA at areas of choice along another  DNA strand.  In this way, CRISPR-Cas9 is a customizable tool in gene editing.

CRISPR is naturally used by bacteria as their immune system to kill invading viruses.
It has now been adapted for use in the gene lab.
CRISPR system has two basic components- a Cas9 protein and a guide RNA or gRNA. Cas9 protein acts like the wrench, and the specific RNA guides known as CRISPRs, act as a set of different socket heads. RNA guides direct the Cas9 protein to the correct gene, or area on the DNA strand, that controls a particular trait. This allows researchers to study our genes in a specific and targeted way in real time.
The guide RNA is a small strand of RNA programmed to look for a specific DNA sequence. Cas-9 enzyme is a strong cutting device that can cut through a double strand of DNA. The guide RNA and Cas-9 remain connected.
When put inside a cell, the CRISPR system locates the target DNA.
A CRISPR-seeking device recognizes and binds to this target DNA.

Then the enzyme Cas9 acts quickly and cuts the double-stranded DNA at two places, removing the desired specific piece to create an edited DNA.
Scientists can now insert these new edited sections of DNA into the cell.
The cell incorporates the new DNA into the gap automatically when it repairs the broken DNA.
CRISPR can also cause deletions in the genome and can insert new DNA sequences.
Many researchers have observed that CRISPR is six times more efficient than ZFNs or TALENs in creating targeted mutations in the genome.
As a result, the large genomics projects that once took many years and huge funds can now be completed in a small fraction of time and cost.

Base Editing

Base editing is an advanced type of gene-editing technique.
It allows for precise changes to individual DNA bases without cutting the DNA strands.
The Base editing method is particularly useful for correcting single-point mutations, which are responsible for many genetic diseases.
Base editing uses a modified version of the CRISPR-Cas9 system known as Cas9 nickase. Instead of creating double-strand breaks, the Cas9 nickase only nicks one strand of the DNA.
Guide RNA directs the Cas9 nickase to the specific DNA sequence, where the deaminase enzyme makes the desired base change.

Base editing offers several advantages and differences compared to other gene-editing techniques like traditional CRISPR-Cas9.
It allows for precise single-base changes without creating double-strand breaks (DSBs) in the DNA.
This reduces the risk of unintended insertions or deletions (indels) and chromosomal damage.
It uses a modified CRISPR-Cas9 system combined with a deaminase enzyme to convert one DNA base into another, for example, cytosine to thymine or adenine to guanine).
One notable example of a successful application of base editing is the treatment of progeria.

Prime Editing

Prime editing is a state-of-the-art gene-editing technology.
It is often described as a “search-and-replace” tool for the genome.
Prime editing combines the precision of base editing with the versatility of the traditional CRISPR-Cas9 technique.
It can make targeted insertions, deletions, and all 12 possible base-to-base conversions without creating double-strand breaks (DSBs) in the DNA.
Prime Editing Uses a modified CRISPR-Cas9 nickase and a reverse transcriptase enzyme to directly write new genetic information into the DNA.
It is suitable for a wide range of genetic corrections, including those that require more complex edits than base editing can achieve.

Is it possible to introduce Artificial Intelligence to Gene Editing? | AI Gene Editing

Artificial Intelligence is helping us with everything from identifying abnormal heart rhythms before they appear to detecting skin cancer.
AI gene editing refers to the integration of artificial intelligence (AI) with gene editing technologies like CRISPR and Zinc Finger Nuclease to enhance precision, efficiency, and outcomes in genetic modifications.
A.I. algorithms can analyze vast genomic datasets to identify optimal target sites for gene editing, minimizing negative effects and ensuring more accurate modifications. AI can design more effective guide RNAs or gRNAs and proteins for CRISPR systems, such as the AI-designed protein OpenCRISPR-1.

Now it is becoming possible that artificial intelligence (AI) may enable the production of customizable proteins called ZINC FINGERS to treat diseases by turning genes on and off.
The researchers at NYU Grossman School of Medicine of New York University and the University of Toronto designed a tool that promises to accelerate the development of gene therapies on a large scale.
The convergence of AI and conventional gene editing holds great promise for advancing genetic research and shaping new therapies.

Scenario of Gene Editing in India

Gene Editing is gaining momentum in India across various fields, including healthcare and agriculture.
The Sickle Cell Gene Editing Mission in India focuses on developing gene editing therapies for genetic diseases like sickle cell anaemia and β-thalassemia.
Many government agencies are supporting this mission, including the Council for Scientific and Industrial Research (CSIR) and the Indian Council for Medical Research (ICMR).

Institutions like the Institute of Genomics and Integrative Biology in New Delhi and the Narayana Nethralaya Foundation in Bengaluru are actively involved in preclinical and clinical trials for gene editing therapies.
India has recently relaxed regulations on gene-edited crops, allowing field trials of CRISPR/Cas9-modified rice variants.
This aims to improve crop traits without adding new genetic material.
Sixteen institutions in India engaged in gene editing research for agriculture, focusing on developing crops with improved disease resistance, climate resilience, and nutritional quality.

Indian scientists at the CSIR-Institute of Genomics and Integrative Biology (IGIB) in New Delhi, working with the L.V. Prasad Eye Institute, made a significant breakthrough in gene editing.
They developed an enhanced version of the CRISPR-Cas9 system known as FnCas9.
They claim that the FnCas9 tool is more precise and efficient than the existing CRISPR system.
There is ongoing debate about the ethical and legal implications of these technologies.
While somatic cell editing is progressing, germline gene editing remains banned in India.

Ethical and safety concerns

There are numerous scientific, safety, ethical and policy concerns today in researcher’s minds and governments.
They say that genome editing must be safe before it is used to treat patients.
Serious safety and ethical issues are there in altering the germ line because edits in the germline would be passed down through generations.
Also, a current lack of compelling medical applications does not justify the use of genome editing in embryos.
Many countries and organizations have strict regulations to prevent germline editing for this reason.

In 2017, the National Academies of Science, Engineering, and Medicine (NASEM) recommended that clinical trials using gene editing in embryos should be permitted only within a robust and effective regulatory framework and only when certain criteria are met.
The International Commission concluded in the report, Heritable Human Genome Editing released in September 2020 that clinical use of heritable human gene editing should not be considered until it has been established that it is possible to make precise genomic changes efficiently and reliably without undesired changes in human embryos.

The Dickey-Wicker Amendment prohibits the use of Congressionally appropriated funds for the creation of human embryos for research purposes or for research in which human embryos are destroyed.
If permitted, use should be limited to serious monogenic diseases.
The potential effects of gene drives on individual species and the environment have also raised biosafety, biosecurity and ethical concerns.