Gene Editing and CRISPR
An overview to applications of Gene Editing and CRISPR
Gene editing has revolutionised biomedical research, provided scientists with multiple significant breakthroughs, by allowing genes to be altered, removed, or added. This technique has permitted researchers to edit genomes not only accurately, but cheaply and easily. There is a wide range of applications gene editing can impact positively, namely new medicines, agricultural products, and genetically modified organisms, or as a means of controlling pathogens and pests. More significant to human life are the speculative developments toward clinically treating inherited diseases and somatic mutations including cancer.
CRISPR-Cas9 is amongst the most popular methods of gene editing, this is because of its incredible ease of use in addition to its low cost and editing effectiveness. However, older methods such as Zinc Finger Nucleases (ZFN) and Transcription activator-like effector nucleases (TALENs) are still utilised. There are many reasons as to why these older methodologies are still used today, one of which is for applications where high specificity is crucial, since they can be more precise and have a lower risk of off-target effects compared to CRISPR-Cas9. Additionally, these older techniques can be designed to target longer DNA sequences, resulting in greater flexibility.
For more information on how CRISPR/Cas9 gene editing works, please take a look at “Why CRISPR Matters” and for a more scientific breakdown of the technology behind it, please visit “How does CRISPR work?”.
Comparisons of Most Common Gene Editing Methods
| ZFNs | TALENs | CRISPR-Cas9 | |
|---|---|---|---|
| Length of recognised DNA target | 9–18 bp | 30–40 bp | 22 bp + PAM sequence |
| Mechanism of target DNA recognition | DNA–protein interaction | DNA–protein interaction | DNA–RNA interaction via Watson-Crick base pairing |
| Mechanism of DNA cleavage and repair | Double-strand break induced by FokI | Double-strand break induced by FokI | Single- or double-strand break induced by Cas9 |
| Design | Challenging. Available libraries of zinc finger motifs with pre-defined target specificity. | Easy. TALE motifs with target specificities are well defined. | Easy. SgRNA design based on complementarity with the target DNA. |
| Cloning | Requires engineering linkages between zinc finger motifs. | TALE do not require linkages. Cloning of separate TALE motifs can be done using Golden Gate assembly5. | Expression vectors for Cas9 are available. SgRNA can be delivered to cells as a DNA expression vector or directly as an RNA molecule or pre-loaded Cas9-RNA complex. |
Zinc Finger Nucleases (ZFNs)
ZFNs, or Zinc Finger Nucleases, are a type of engineered nuclease that can create specific, targeted breaks in DNA molecules. They are composed of two functional domains: the DNA-binding domain (DBD) and the nuclease domain. The DBD is made up of zinc finger proteins, which can be engineered to recognise and bind to specific DNA sequences. The nuclease domain then cuts the DNA at the site specified by the DBD.
ZFNs have been used in a variety of applications, including gene editing and gene therapy. By creating targeted breaks in the DNA, ZFNs can stimulate the cell's natural DNA repair mechanisms, leading to precise changes in the genetic code. This technology has the potential to correct genetic mutations that cause diseases, as well as to create novel traits in organisms for various purposes.
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Transcription Activator-Like Effector Nucleases (TALENs)
TALENs, or Transcription Activator-Like Effector Nucleases, are another type of engineered nuclease that can create targeted breaks in DNA. Like ZFNs, TALENs are composed of two functional domains: the DNA-binding domain and the nuclease domain. However, TALENs use a different DNA-binding domain that is based on transcription activator-like effectors (TALEs) derived from bacteria.
TALEs are naturally occurring proteins that are capable of binding to specific DNA sequences. They contain a repeating domain that can be engineered to recognise and bind to specific DNA nucleotides. By fusing TALEs with a nuclease domain, TALENs can be designed to cut DNA at specific sites within the genome. The TALEN technology has been used in a variety of applications, including gene therapy, gene editing, and synthetic biology.
2BScientific has partnered with ACRO Biosystems, Progen, Cellecta, Innoprot, Cell Biologics, Creative Biolabs, IQ Biosciences, Life Science Group, MoBiTec, Origene, and Cusabio to provide customers with a wide range of gene editing products.
To get access to our CRISPR associated and genomic analysis Products and Services, please contact us at Sales@2BScientific.com so that you can identify and select the best products for your experiments. 2BScientific is passionate about ensuring that customers get the precise, required product and have a kind and knowledgeable team ready to answer any technical queries.