Genome Editing Techniques – Gateway to the era of Molecular Medicine

Anish Pyne, Indian Institute of Technology Kharagpur

With the growing prowess of personalized medicines and molecular drugs, a group of methods took the central stage allowing the introduction of highly specific changes within the genome by either adding or deleting, or substituting any desired sequence, thus modifying the natural characteristics of a gene. And this was how we were introduced to the world of genome editing technologies. Over the past decade, genome editing has evolved from initial attempts like homing endonucleases, ZFNs (zinc-finger nucleases), and TALENs (Transcription activator-like effector nucleases) which faced several challenges like minimized specificity and excessive off-target effects to the ground-breaking discovery of CRISPR/Cas9 which shows great specificity and exactness in its activity. Such techniques work on the principle where a targeted change can give way to correct genetic diseases and manipulate conditions like cancer. Since these techniques directly impact the domain of gene therapy, there is constant growth in this field is opening limitless possibilities for therapeutic interventions.

Genome editing techniques range from conventional systems like homologous recombination and chemical systems like pseudo-complimentary peptide nucleic acids (pcPNA) to protein-based nuclease systems like ZFNs and TALENs and RNA based systems like CRISPR/Cas. 

A number of the notable techniques are discussed below in detail: –

• Conventional Techniques: These include homologous recombination related to gene intervention. The techniques are based on physiological processes involving a double-stranded repair system. However, the technique as of now could not successfully gain widespread usage, thanks to the emergence of newer techniques.

• The Artificial Restriction DNA Cutter (ARCUT): This technique uses pseudo-complementary peptide nucleic acid (pcPNA), which specifies the cleavage site within the chromosome or the telomeric region. Once pcPNA specifies the location, excision is meted out by cerium (CE) and EDTA (chemical mixture), which performs the splicing function. Furthermore, the technology uses a DNA ligase which will later attach any desirable DNA within the spliced site.

• Homing Endonucleases (HEs): These nucleases occur naturally, with a size of approximately 14 bp (base pairs) and they’re capable of splicing slightly larger DNA sequences. The overall concept involves a DNA segment where a site is removed by the endonucleases, thus leading to the formation of two segments of the DNA fragment. Recently, the introduction of recombinant adeno-associated viruses (rAAVs) has made them efficient vehicles for transporting genetic tools of genome engineering into the cell and hence made the work of homing endonucleases easier.

• Protein-based nuclease systems: These systems incorporate nuclease proteins for genome editing. Among them, Zinc finger nucleases (ZFNs) are a category of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations. Each Zinc Finger Nuclease (ZFN) consists of two functional domains: (i) A DNA-binding domain comprised of a sequence of two-finger modules, each recognizing a novel hexamer (6 bp) sequence of DNA and (ii) a DNA-cleaving domain comprised of the nuclease domain of FokI endonuclease. When the DNA-binding and DNA-cleaving domains are fused, a highly-specific pair of ‘molecular genomic scissors’ are created. TALENs on the opposite hand is just like ZFNs and comprise a nonspecific FokI nuclease domain fused to a customizable DNA- binding domain. This DNA-binding domain consists of highly conserved repeats derived from transcription activator-like effectors (TALEs). The difference between TALENs and ZFNs is that the former can target 3 nucleotides in one go, while the latter can only target one nucleotide, thus making TALENs slightly more site-specific with fewer off-target effects.

• RNA-DNA systems: These systems include the various sorts of CRISPR methods. CRISPR has two components, including SPR, sometimes termed as spacers, which are hallmarked by varying and differing nucleotide sequences, and possibly every one of them represents a past exposure to foreign antigen and also the CRI which represents the genetic memory for a bacterium and might be re-activated once encountered with the same foreign antigen. Cas9 encompasses a nuclease function. Whenever CRISPR RNA (crRNA; also termed guide RNA [gRNA]) guides the Cas9 protein regarding a possible antigenic threat, it creates a double-stranded DNA (dsDNA) nicks at the guided selected sites with the assistance of gRNA. This causes a site-specific cleavage and, thus, destruction of the antigen. Moreover, the memory from the antigen is stored as a spacer within CRISPR. Over the previous couple of years, CRISPR/Cas9 technology has gained widespread popularity on account of its simplicity and specificity. 

In the present context, CRISPR/Cas technologies are probably superseding ZFNs and TALENs. However, the CRISPR/Cas methods are also being improvised constantly, and newer additions have further enhanced its functional capabilities with reduced off-target effects. But similar to the other modern technologies, genome editing technology is extremely powerful in terms of its potential to alter the characteristics and properties of any gene. Thus, they must be handled with extreme responsibility because the bioethical concerns need serious attention.

Also read: World Milk Day –Sustainability in the Dairy Sector

Reference:

1. Khan S. H. (2019). Genome-Editing Technologies: Concept, Pros, and Cons of Various Genome-Editing Techniques and Bioethical Concerns for Clinical Application. Molecular therapy. Nucleic acids, 16, 326–334. https://doi.org/10.1016/j.omtn.2019.02.027

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