Approaches to Genome Editing
|28.10.2020||Posted by tactical33 under Advertising & Marketing|
Genome editing is a method of making specific changes to the DNA of a cell or organism. An enzyme scissors the DNA at a specific sequence, and when this is repaired by the cell, a change or ‘edit’ is made to the sequence. This method lets scientists change the DNA of many organisms, including plants, bacteria, and animals. Gene editing can result in changes in physical traits, like eye color, and disease risk. Scientists use different technologies to do genome editing.
Genome editing technologies help scientists to make changes to DNA, leading to changes in physical traits. These genome editing technologies act like scissors, cutting the DNA at a specific spot. After that, scientists can remove, add, or replace the DNA where it was cut.
The first genome editing technologies were developed in the late 1900s. Recently, there are four foundational technologies-Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nuclease (TALEN), Engineered Meganucleases/Homing Endonucleases (HE), and CRISPR/Cas Nuclease.
Mechanisms of Genome Editing
Genome editing is to efficiently induce targeted DNA double-strand breaks (DSBs). These DSBs then drive the activation of cellular DNA repair pathways and facilitate the introduction of site-specific genomic modifications. This process is most often used to achieve gene knockoutvia random base gene insertions and/or gene deletions that can be introduced by nonhomologous end joining (NHEJ). Alternatively, in the presence of a donor template with homology to the targeted chromosomal site, gene integration, or base correction via homology-directed repair (HDR) can occur.
Targeted Nucleases for Genome Editing
It has been recognized for several years that the ability to create unique DSBs would afford the capability to replace one genomic segment for another both in vitro and in vivo. The concept of using customized sequence-specific engineered endonucleases to perform genome editing was made possible on a large scale by the advent of ZFNs.
Figure 1. The structure of Zinc Finger Nuclease. (Gaj, 2016)
ZFNs, which are fusions between a custom-designed Cys2-His2 zinc-finger protein and the cleavage domain of the FokI restriction endonuclease. ZFNs function as dimers, with each monomer recognizing a specific “half site” sequence-typically 9 to 18 base pairs (bps) of DNA via the zinc-finger DNA-binding domain.
Two methods for constructing ZFNs have been widely used. The first one is oligomerized pool engineering (OPEN) involves creating a pool of zinc finger arrays (ZFAs), then assessing them for use. The second one is a new ZFN construction technique termed context-dependent assembly (CoDA).
Xanthomonas is able to express a newly discovered class of DNA-binding proteins, the transcription activator-like effectors (TALEs). These proteins encompass many conserved repeats of 34 amino acids with two residues that change in sequence at positions 12 and 13. TALENs are modular in form and function, comprised of an amino-terminal TALE DNA-binding domain fused to a carboxy-terminal FokI cleavage domain.
Using TALEN capabilities to target nuclear-encoded genes, TALEN proteins harboring mitochondrial localization signals were used to specifically cleave mutant mitochondrial genomes, potentially paving the way for their use in a gene therapy strategy toward mitochondrial disease. TALEN technology has also been used to produce transgenic animals.
Some TALEN construction methods for which reagents are publicly available to have been employed. The simplest but most labor-intensive step involves traditional plasmid cloning by restriction digest and ligation of each TALE repeat. TALENs also can be constructed from plasmid libraries containing three or four TALE repeats. Recently, the developed Golden Gate strategy is used.
Meganucleases or homing endonucleases (HEs) are endonuclease proteins that are found in a number of prokaryotes, archaea, and unicellular eukaryotes. These proteins can be encoded as free-standing genes, within an intron or as self-splicing inteins, and function in nature to support horizontal gene transfer of their coding sequences.
Using HEs with modified DNA-binding specificities, cell culture experiments specifically targeted the human Recombination activating gene 1 (RAG1), Xeroderma pigmentosum, complementation group C (XPC), and Monoamine oxidase B (MAO-B) genes.
The most recently described methods for genome engineering are based on the CRISPR and CRISPR associated (Cas) systems. They function to protect their host from the horizontal introduction of exogenous genetic material delivered by phages or plasmids.
CRISPR/Cas systems have three types (I, II, and III), which are relied on different accessory RNAs and proteins. Among the three types, type II CRISPR/Cas systems have been identified for use in genome engineering. Types I and III are composed of a number of Cas proteins, whereas the Cas9 protein is the only protein constituent of type II CRISPR/Cas systems.
1. Gaj, T.; et al. (2016). Genome-editing technologies: principles and applications. Cold Spring Harbor perspectives in biology. 8(12), a023754.