CRISPR-Cas9

The Future Of CRISPR-Cas9

Genome editing, which is also called  gene editing, includes technologies that have made it possible for scientists to alter an organism’s DNA. By the use of gene editing, genetic material can be added, replaced, or removed at particular locations in the genome. Several approaches to gene editing have been developed. One of them is known as CRISPR-Cas9. In comparison to other methods, CRISPR-Cas9 is faster, efficient, cheaper, and more accurate.

Introduction to CRISPR-Cas9

The term CRISPR/Cas9 stands for Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9. The name itself speaks to features of cas9 identified during its discovery, but the expanse of study has gone much beyond that. Discovery of CRISPR and Cas9 was made by the research results of two scientists at different places.

In 1993, Francisco Mojica was the first researcher to characterize the CRISPR locus. Based on his findings, he hypothesized that CRISPR is an adaptive immune system (Mojica et al., 2005). In that same time, similar reports were published by another group of researchers (Pourcel et al., 2005).

Another scientist, Bolotin, was doing research on the bacteria Streptococcus thermophilus, which revealed an unusual CRISPR locus when sequenced (Bolotin et al., 2005). Findings state that the CRISPR array lacked known cas genes but it contained a novel cas gene with some nuclease activity predicted with an encoded large protein.This novel cas gene is now termed as Cas9.

Zhang, who had previous experience of working with other genome editing technologies like TALENs, was first to successfully work with CRISPR-Cas9 for gene editing in eukaryotic cells (Cong et al., 2013).  Two major applications of the technology were suggested as follows:

  1. Could target a number of genomic loci if programmed
  2. Could drive homology-directed repair

What does that mean?

In non-scientific terms, it means that CRISPR-Cas9 could work as (a) a targeted editing effect to produce specific and desired outcomes from changes within a DNA or genome. Think of this as a potential to eradicate a disorder that is embedded within the genome, or create something within an organism like a fruit that could change taste or aesthetic properties. And (b) repair damage that’s been done through an editing process that may wholly replace damage. More info can be found here.

CRISPR-Cas9 Graphic For Gene Editing

How does CRISPR work?

Bacteria feature  naturally occurring systems for genome editing and CRISPR-Cas9 technology was actually adopted from observing these systems. Bacteria use snippets of DNA captured from invading viruses to create CRISPR arrays. These arrays are DNA segments, which help bacteria remember the viruses. If attacked again by viruses, bacteria recalls the CRISPR arrays to produce RNA segments and target the DNA of viruses. Later, Cas9 or some relative enzyme breaks DNA apart to disable the virus. More information on this can be found on the National Institute of Health’s website.

CRISPR-Cas9 technology is meant to replicate this system in the lab. A small piece of RNA is created by researchers in the lab, with a short “guide” sequence that binds in a genome to a specific target sequence of DNA. Binding of Cas9 to RNA also takes place. The modified RNA recognizes the DNA sequence and the DNA is cleaved at the targeted points by Cas9 enzyme. Cas9 is used mostly but other enzymes like Cpf1, can also be used here.

After cutting down the DNA into pieces, repair machinery in DNA is used for addition or deletion of genetic material, or to alter the placement of existing segment in a DNA with another customized DNA sequence.

A new CRISPR editing system involves the delivery of an enzyme to a particular gene. This alters a gene by changing a C to a T or an A to a G, instead of cutting or putting anything inside or outside.

CRISPR’s Part In The Future Of Genetic Medicine

The technology has been already adapted for genome editing in a variety of organisms including mice, fungi, frogs, yeast, tobacco, monkeys, dogs, fungi, rabbits, rice, mosquitoes, and humans. This has allowed scientists to study or design models of different diseases with human cell lines and mouse models.

The concept of gene therapy has the potential to treat disease caused by a mutation in a single gene. One example often used in this case is sickle cell anemia. The methodology used for correction of any defective gene is called gene therapy, whereas CRISPR is seen as one of the most powerful and potential options to perform gene therapy. The potential of gene therapy using this technology has been demonstrated using mouse models, but practices for human gene therapy are still untested.

Few other major potentials are listed below:

  • Gene drives, which ensure the transfer of a genetic trait to offspring.
  • Creation of antimicrobials using new ways to fight antibiotic resistance.
  • Targeted manipulation to improve crop yields by making the alteration in nutritious value.
  • Human design building, from the genetic level.

Applications of this technology have such a huge potential diversity that there still remains much more to be considered.

Closeup of DNA and Gene Editing

The Future of CRISPR-Cas-9

This technology has provided the means for genome alteration, regulation, and visualization with more accessibility and adaptability. The technology has enabled biological and biotechnological research with increased range of their applications. The pace of research has been accelerated and made it possible to find out previously undiscovered genes that may contribute to disease. As well, some see the potential for CRISPR science to find sequences that may prevent disease when implemented.

Multiple clinical practices and trials are in progress or near their beginning, which ensures that the research and development in Cas-9 biotechnology are being fast-tracked simply due to the technology’s potential.

This will decide the future in terms of usage of somatic cell editing either in-vivo or ex-vivo. Research on a variety of diseases is in progress to determine the safety and efficiency of genome editing in humans. As it promises for the prevention, cure or treatment of complex diseases, for example, HIV infection, cancer, mental illness, and heart diseases.   

Beside all these clinical approaches, the CRISPR technology has derived applications to be implemented on the agricultural sector and one of them is the production of a whole new plant from a single cell. It has already lead the production sector to overcome the market. This is one of the reasons behind the modifications made by the U.S. Department of Agriculture in regards to their regulations.

CRISPR-Cas9 Ethical Future Concerns

The CRISPR technology seems to have the scope and potential to improve the life on earth, but some of the ethical concerns are highly sensitive. When a technology like genome editing tool is considered for variation in the genetics of human, this is more limited to somatic cells and highly rejected for its application to make changes to genes in a sperm cell or an egg or an embryo. As this can transfer the modifications and changes to the next generation, concerns surface about what that means for evolutionary life forces and kinds.

Use of this technology with germ-line cell and embryo bring up various ethical challenges including the permissibility to use it for enhancement of normal human traits. Looking at these ethical concerns related to germ-line and embryo, the technology has been regulated or shut down already in several countries.

Future of CRISPR References

Mojica FJ., Diez-Villasenor C., Garcia-Martinez J.,  and Soria E. (2005). Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 60:174-82. doi: 10.1007/s00239-004-004603

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Pourcel C., Salvignol G., and Vergnaud G. (2005). CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology. 151:653-63. doi: 10.1099/mic.0.27437-0

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Bolotin A., Quinguis B., Sorokin A., and Ehrlich SD. (2005). Clustered regularly interspaced short palindrome repeats(CRISPRs) have spacers of extrachromosomal origin. Microbiology. 151:2551-61. doi: 10.1099/mic.0.28048-0

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Cong L., Ran FA., Cox D., Lin S., Barretto R., Habib N., Hsu PD., Wu X., Jiang W., Marraffini LA., and Zhang F (2013). Multiplex genome engineering using CRISPR/Cas systems. Science. 339:819-23. doi: 10.1126/science.1231143

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