An Introduction to Understanding the CRISPR/Cas9 System
The technology that allows researchers to edit and influence gene expression
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CRISPR/Cas9 is a gene editing tool that can manipulate gene expression in plants, humans and animals. CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, are short sections of bacterial DNA containing repetitive base sequences. CRISPR plays a crucial role in the immune response of bacteria against foreign DNA. When a bacterium detects viral DNA, it produces two strands of short RNA called guideRNA, which then go on to form a complex with an endonuclease enzyme called Cas9 (CRISPR associated protein 9). This complex targets and cuts out the viral DNA rendering the virus disabled. The Cas9 nuclease will not bind to the DNA if it the target sequence is not followed by the Protospacer Adjacent Motif, or PAM, which helps the enzyme distinguish between the bacterial DNA and the viral DNA target. The CRISPR/Cas9 system then has the ability to store this viral data so that it will be able to recognize and eliminate future viral threats.
In vivo excision of HIV-1 proviral DNA by sgRNAs/saCas9 in solid tissues/organs can be achieved via AAV delivery, a significant step toward human clinical trials, according to Chaoran Yin C et al. (2017).
Researchers Jennifer Doudna of the University of California, Berkeley and Emmanuelle Charpentier from Umea University in Sweden discovered a way to exploit these abilities to control gene expression by manufacturing guideRNA with a specific sequence and feeding it to the Cas9 enzyme. The enzyme will search for anything with that specific code and cut it up, yielding a window to implant a desired DNA sequence. Once the sequence is planted, the strands are sealed and the target sequence will be expressed. This process can be done in a cell’s nucleus, in stem cells, embryos and even extracellularly in a test tube. CRISPR/Cas9 is able to edge out other gene editing techniques like TALEN and Zinc finger nucleases because it is the only technique that is highly efficient and precise, extremely customizable and can target multiple genes at once.
The versatility of the CRISPR/Cas9 system and its ability to locate and alter specific genes can yield advancements in drug discovery, basic medical research, agriculture and even the possibility of preventing genetic diseases, heart disease, and blood conditions in humans. One example is the possibility of creating a Malaria-resistant Mosquito, in which scientists were able to alter multiple genes in a type of mosquito that allow it to become resistant to Plasmodium falciparum, the parasite that causes Malaria.
Scientists are also utilizing CRISPR/Cas9 to make advancements in the fight against HIV. In a study led by researchers at the University of Pittsburgh and the Lewis Katz School of Medicine at Temple University, the CRISPR/Cas9 system has shown promise in effectively locating and shutting down HIV infected human immune cells in mice. The researchers look to take the next step toward human application by testing their findings in primates.
CRISPR/Cas9 also will play a multi-fold role in agriculture by making it possible to edit crops to make them more nutritious, better tasting, disease resistant, and less susceptible to drought. The benefits of CRISPR/Cas9 gene editing are seemingly endless across multiple areas of study.
To further support scientists in the advancement of CRISPR/Cas9 research, EpiGentek offers products like EpiQuik CRISPR/SaCas9 (S. aureus) Assay ELISA Kit (Colorimetric) to measure CRISPR-associated protein 9 (Cas9 and dCas9: S. aureus) amounts with use of purified SaCas9 nuclease and whole cell extracts isolated from tissues and cultured cells of various species, and the CRISPR/Cas9 Monoclonal Antibody [7A9] to aid in genomic editing studies.
References:
Bomgardner, M. “CRISPR: A New Toolbox for Better Crops.” CEN RSS, 12 June 2017
Buguliskis J. “CRISPR Eliminates HIV in Live Animals.” GEN, 2 May 2017
Gantz, V et al. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi PNAS 2015 112 (49) E6736-E6743; published ahead of print November 23, 2015, doi:10.1073/pnas.1521077112
Chaoran Yin C et al., In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models, Molecular Therapy, Volume 25, Issue 5, 2017, Pages 1168-1186, ISSN 1525-0016.
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