Targeted Genome Editing using Genomic Cruise Missiles


The sequence of the genome has been determined and so has been the association of certain sequences to particular diseases. So, what lies next? Correcting the sequence for diseased (in case of diseases caused by a single gene or monogenic diseases) individuals or the somatic gene therapy.

In the news:
Virginia Tech researchers successfully disrupted the eye colour of Aedes mosquito from black to white using TALENs for genome editing. This is being seen as a big step towards strategies aimed at disrupting transmission of diseases such as dengue. Till now, efforts focussed on addition of genes to the mosquito genome which interfered with the mosquito’s growth, but the expression of the added genes remained a problem. The added gene could just be “selected” out of the populations. This approach would involve subtraction of a gene critical for replication of the pathogen.

Introduction:
Initially, zinc-finger nucleases (ZFNs) were employed to induce genetic alterations. The newly-developed transcription activator-like effector nucleases (TALENs: nicknamed the genomic cruise missiles) enable targeted alteration of nearly any gene in many cell types and organisms. TALENs score above ZFNs in the ease of design, higher rates of cleavage activity and greater targeting range. Like ZFNs, TALENs comprise of a non-specific nuclease (Fokl) fused to a DNA-binding domain.

A comparison of targeted genome editing using ZFNs and TALENs
A comparison of targeted genome editing using ZFNs and TALENs

This domain can be engineered to bind to nearly any sequence. The domain consists of highly conserved repeats derived from transcription activator-like effectors (TALEs), which are proteins secreted by Xanthomonas spp. to alter gene transcription in host plant cells. Genome editing with engineered nucleases was named the 2011 ‘Method of the Year’ and heralded as a major scientific breakthrough in 2012 by the Science magazine, justifying its immense implications in the fields of genetic research and therapy.

Applications:

  1. Agriculture: Newer varieties of commercially important livestock and plants can be produced using genome editing much quicker when compared to the conventional breeding. Plants can be modified at the single-cell level and selected for higher yield, resistance to herbicides or pesticides, or suitability to certain climate. For instance, TALENs were employed to disrupt target sites of naturally occurring TALEs that contribute to pathogenicity and, hence, confer resistance to Xanthomonas in rice.
  2. Disease modelling: TALENs allow direct assessment of the importance of particular sequences in the genome. Such loss-of-function mutations are used to create somatic cell-based disease models. Insertion of genes that encode for fluorescent proteins is used to visualize expression, distribution and interactions of proteins. Genome editing of such precision also allows determining the function of single nucleotide polymorphisms (SNPs).
  3. Therapy: Recent studies have shown that genome editing can correct genetic mutations responsible for sickle cell anaemia or antitrypsin disease in patient specific induced pluripotent stem cells. These studies hint at the possibility of ex-vivo correction of derived cells and reintroducing them back into patients. The patients would undergo minimal immune complications as these would be their own cells. Also, it could be used to disrupt the functioning and hence the product of a gene. This approach is being seen as a therapy for AIDS where ZFNs are used to disrupt expression of CCR5 gene product which is required by certain HIV strains as a co-receptor to infect cells.

Read full paper here:
Joung, J. K., Sander, J. D., TALENs: a widely applicable technology for targeted genome editing, Nature Reviews Molecular Cell Biology 14, 49-55, January 2013, doi:10.1038/nrm3486

and the Virginia Tech breakthrough here:
TALEN-Based Gene Disruption in the Dengue Vector Aedes aegypti (plosone.org)

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