Gene editing has rapidly developed over the past decade to become something of a scientific and biomedical revolution. The ability to precisely manipulate the genomes of cells or organisms is a powerful biotechnological tool that has huge potential for treating genetic diseases, creating more resistant crops and improving the efficiency of biofuels, to name a few applications.

The technique involves making permanent modifications at a specific site on the genome – whether this be by removing, adding or replacing sections of DNA – which allows for the engineering of desired traits into organisms and systems of interest at will.

There are three main types of gene editing that have been used since the technology first came into play in the 1970s: zinc finger nucleotides (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR-Cas. These techniques offer a significant improvement over previous attempts to modify genomic DNA because of their higher specificity to the target, and each will be explored in more detail in this article.

What is gene editing and how does it work?

The general principle of gene editing is to make a cut in DNA, called a double-stranded break (DSB), at a target site using an enzyme, which activates the cell’s machinery to repair the DSB and incorporate the required changes into the genome. This can be done by one of two repair mechanisms – homology directed repair (HDR) or non-homologous end joining (NHEJ). While HDR is an accurate process that uses long stretches of homologous DNA as a template to mend lesions, NHEJ works by directly joining DNA ends and is naturally error-prone, often resulting in a mix of different insertions and deletions of DNA nucleotides.

Introducing a DSB allows either a knock-out or knock-in within a defined gene, resulting in a loss of function (where the gene is deleted) or a change in its sequence, respectively. NHEJ repair can cause this loss of gene function by generating null mutations, while edits can be incorporated either through targeted HDR of the DSB or by replacing specific sequences.

This process must be tightly controlled to ensure no edits occur elsewhere in the genome other than at the intended target site, which could have unknown and potentially dangerous consequences. It is still too risky to edit the genomes of human embryos for implantation, for example.

Zinc finger nucleases

The first breakthrough in gene editing came in the 1990s with ZFNs, a class of engineered endonuclease enzymes that specifically recognise and cleave DNA. Based on a type of transcription factor found in eukaryotes called a zinc finger protein, ZFNs consist of a DNA binding domain fused to a FokI endonuclease, which acts as the DNA cleavage domain. The zinc fingers within the binding domain are used to determine where the DNA should be cleaved, before FokI makes the cut.

Because FokI cuts just one DNA strand, ZFNs must be used in pairs to generate DSBs. The ZFN dimer recognises nucleotide sequences that are in proximity to each other on the target site, and the simultaneous recognition and binding of both ZFNs is important for improving specificity and limiting off-target effects.

ZFNs have been successfully used to target genes in model research organisms, such as fruit flies and zebrafish, by injecting them into embryos, as well as to create gene knockouts or optimised cell lines that produce more proteins or antibodies. But they have several disadvantages: they are difficult and time-consuming to design and engineer, and a new ZFN must be constructed for every new DNA target.

Transcription activator-like effector nucleases

More than a decade after ZFNs (which during that time were the only programmable site-specific nuclease available), and thanks to the discovery of proteins secreted by the plant bacterium Xanthomonas called transcription activator-like effectors (TALEs),.a new family of gene editing proteins called TALENs was created.  TALENs are like ZFNs in that they possess a DNA binding and cleavage domain, again using a fused FokI endonuclease to introduce DSBs.

TALENs, however, recognise single nucleotides rather than longer sequences, making them more specific than ZFNs.

A single TALE recognises and binds a single DNA nucleotide without affecting the binding specificity of other TALEs around it. This simplicity makes TALENs a more favourable method for gene editing compared to ZFNs because they are easier to engineer and have greater flexibility, producing faster and cheaper results. To target a longer sequence, a series of TALENs can be linked in an array. Though this is also achievable with ZFNs, it presents a greater technical challenge as zinc finger motifs can affect neighbouring ones.

TALENs have been used to reach key milestones in gene editing, including the commercialisation of the first genome-edited crop – a soybean that contains fewer trans fats – and the first successful cancer therapy for humans.

CRIPSR-Cas

Despite the success of TALENs, the method was shortly overtaken by the most powerful and efficient gene editing tool yet: the CRISPR-Cas system. Developed in 2009, it makes use of a type of adaptive immunity that some bacteria employ to kill invading viruses, CRISPR (short for clustered regularly interspaced short palindromic repeats) paired with a Cas endonuclease.

For gene editing, a short RNA molecule called single guide RNA (sgRNA) is synthesised to match a corresponding DNA sequence of choice. This sgRNA guides Cas – typically a type called Cas9 – to the intended part of the genome where it recognises a defined DNA sequence known as PAM. Cas9 then cleaves the target DNA at a site adjacent to the PAM to make the desired edit.

The ease of creating a sgRNA rather than an entire protein to recognise a target site makes CRISPR-Cas9 cheap, easy to employ and efficient, with estimates its efficiency is six times that of both ZFNs and TALENs. Compared to these methods, CRISPR-Cas9 is also the most flexible and scalable and can be achieved with standard molecular biology techniques. Adaptations to the system have been developed, such as the use of different Cas enzymes, to further optimise the technology or to edit genes in specific contexts.

CRISPR-Cas9 has been used for a multitude of applications, ranging from making specific gene alterations to creating transgenic organisms. It has successfully treated diseases in animals, such as muscular dystrophy in mice, and used to treat blindness and blood disorders in humans as part of clinical trials. Numerous clinical trials are underway to develop therapies for various diseases including cancer and cystic fibrosis.

When to use each technique

There are four factors to consider when selecting which platform to use for gene editing: specificity, selection of the target site, efficiency, and ease of design. Out of the three, CRISPR-Cas9 is generally the most efficient and straightforward to use – it is less labour intensive yet targeting of the intended genomic sequence is highly specific and predictable. The system also allows for multiple genes to be edited simultaneously by introducing multiple sgRNAs.

However, ZFNs or TALENs may be more favourable in certain contexts – for example in applications involving heterochromatin, a condensed form of DNA. Here, TALENs is a more efficient approach for accessing the DNA than CRISPR-Cas9 because of Cas9’s limited ability to locate the target site.

Comparison of ZFNs, TALENs and CRISPR

 ZFNsTALENsCRISPR-Cas9
Design methodEngineer a protein for each targetEngineer a protein for each target~20-nucleotide gRNA
Ease of designChallengingEasyEasy
Targeting mechanismProtein-DNA interactionProtein-DNA interactionRNA-DNA interaction
DeliveryTwo ZFNs around the targetTwo TALENs around the targetsgRNA and Cas9
Off-target effectsHighLowVariable
EfficiencyVariableModerateHigh
CostHigh (resources and time)High (time)Low

Message Us!

* These fields are required.

© Copyright 2022. All Rights Reserved.

X