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Location: Home > Custom Services > Immunology Services > CRISPR-CAS Technology

CRISPR-CAS Technology

Date: 2019-12-29 Author: Leading Biology Click: 1705

Q: What are genome editing and CRISPR-Cas9?

Genome editing (also known as gene editing) is a set of techniques that enable scientists to alter the DNA of an organism. These techniques allow genetic material to be added, removed, or altered at specific locations in the genome. Several genome editing methods have been developed. The most recent one is called CRISPR-Cas9, which is short aggregation regular interval short backtracking repeat and CRISPR-related protein 9. The CRISPR-Cas9 system has caused a stir in the scientific community because it is faster, cheaper, more accurate and more efficient than other existing genome editing methods.

 

CRISPR-Cas9 is adapted from a genome editing system that naturally exists in bacteria. Bacteria capture DNA fragments from invading viruses and use them to create fragments of DNA called CRISPR arrays. THE CRISPR array allows bacteria to "remember" viruses (or closely related viruses). If the virus attacks again, bacteria produce RNA fragments from the CRISPR array to target the virus's DNA. Bacteria then use Cas9 or similar enzymes to isolate DNA, which disables the virus.

 


The CRISPR-Cas9 system works in a similar way in the lab. Using a small piece of RNA, the researchers had a short "guide" sequence that binds to specific DNA target sequences in the genome. RNA is also bound to Cas9 enzymes. Like bacteria, modified RNA is used to identify DNA sequences, and Cas9 enzymes cut DNA at the target location. Although Cas9 is the most commonly used enzyme, other enzymes (such as Cpf1) can also be used. Once THE DNA is cut, the researchers use the cell's own DNA repair mechanism to add or remove fragments of genetic material, or to alter DNA by replacing existing fragments with customized DNA sequences.

 

Genome editing is of great interest in the prevention and treatment of human diseases. Currently, most research on genome editing is done to understand diseases using cells and animal models. Scientists are still working to determine whether this approach is safe and effective for use in people. It is being explored in research on a wide variety of diseases, including single-gene disorders such as cystic fibrosis, hemophilia, and sickle cell disease. It also holds promise for the treatment and prevention of more complex diseases, such as cancer, heart disease, mental illness, and human immunodeficiency virus (HIV) infection.

 

Ethical issues arise when genome editing is done using technologies such as CRISPR-Cas9. Most of the changes introduced by genome editing are limited to somatic cells, not egg and sperm cells. These changes affect only certain organizations and do not pass from one generation to the next. However, changes to the genes of an egg or sperm cell (reproductive cell) or an embryonic gene can be passed on to future generations. The editing of reproductive cells and embryonic genomes presents many ethical challenges, including whether this technique is allowed to enhance normal human characteristics, such as height or intelligence. Due to ethical and safety concerns, reproductive cell and embryonic genome editing is currently illegal in many countries.

Advantages of CRISPR Genome Engineering

Arguably, the most important advantage of CRISPR/Cas9 over other genome editing technologies is its simplicity and efficiency.
Since CRISPR/Cas9 can be applied directly to embryos, CRISPR/Cas9 reduces the time required to modify the target gene compared to gene targeting techniques based on embryonic stem cells. Improved bioinformatics tools - identifying the most appropriate sequence to design guide RNA - and optimizing experimental conditions to enable very powerful programs to ensure the successful introduction of the required mutations.

Limitations of the CRISPR/Cas9 System

The molecular mechanism used to insert DNA fragments (e.g. cDNA) is mediated by a DNA repair mechanism activated by a double-stranded fracture introduced by Cas9. Because the scope of the DNA repair system is not to integrate DNA fragments into the genome, targeted alleles often carry additional modifications, such as deletions, partial or all integration of the target vector, or even duplications 6,7,8.


Target spot plague standard ES cell-based program for secondary unwanted mutation events, researchers have learned how to avoid producing mice carrying passenger mutations. To identify the correct recombination events in ES cells, most laboratories use a combination of positive-negative selection procedures and validation procedures designed to detect other mutations in the target site.


On the other hand, when the CRISPR/Cas9 procedure is performed directly on the embryo, it is not possible to select the desired event, which greatly limits the possibility of identifying the required allele. In addition, mosaics observed in founder mice using the CRISPR/Cas9 method make it challenging to identify unwanted genomic modifications at the target site.

CRISPR Performance in the Field

CRISPR/Cas9 in embryos is very effective for the production of simple alleles, such as forming knock-off and tapping mutations 1,6, but does not use more complex modification techniques, relying on homologous recombination in larger regions, such as the introduction of pairs of lox P sites or cDNA.


Although Taconic Biosciences and others have successfully introduced complex modifications to the embryo using CRISPR/Cas9 in the mouse genome, the complexity of genome editing and validation procedures for these projects can lead to increased time urgency and cost, reducing or even offsetting the inherent benefits of the technology.

Future Applications

To take full advantage of the potential of CRISPR/Cas9 to modify the mouse genome, an intriguing choice is to take a step back and use it to genetically engineer ES cells, not embryos.


The main advantage of using CRISPR/Cas9 in ES cells compared to traditional gene-targeted methods is that THE frequency of the same-source recombination events caused by DNA damage caused by Cas9 increases by many orders of magnitude. As a result, there is no need to identify ES cell clones that carry modified alleles, simplifying the process of generating target vectors, ES cell screening, and validation.


By separating the clone population of cells, mosaicism can be avoided and in-depth quality control procedures are performed to verify that modified alleles do not carry any passenger mutations. Since there is no selection marker box in the target ES cell, the kimmara extracted from the validated clone can be used directly for rapid colony expansion to speed up the production of mouse models.

Conclusions

CRISPR/Cas9 genomic engineering technology provides researchers with a valuable tool to accelerate the generation of mouse models for biomedical invivy research. The intense pace of CRISPR development, coupled with its versatility and ease of use, has left its mark in the field of molecular genetics. Its combination with established technologies will greatly increase the chances of generating new and valuable genetically engineered mouse models for based and transformational research.

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