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How to Use CRISPR/Cas9

Shrewsbury, MA

From the smallest microorganisms to the biggest creature on the planet, DNA (deoxyribonucleic acid) serves as the building blocks for all organisms. Found in nearly all cells, DNA is a complex, long-chained molecule that contains the genetic blueprint for building and maintaining all living organisms. DNA is made up of four nucleotide bases that are strung together in precise, yet unique sequences that make every organism different from another.


However, scientists have found new ways to edit specific genes of organisms to give them new and improved traits. Scientists can make drought-resistant crops, mosquitoes that don’t spread malaria, and so much more. Before we learn how this works, we need to know where it comes from.


One of the best gene-editing tools scientists use is called CRISPR. CRISPR itself is a naturally occurring process that was first discovered as the immune system for bacteria. CRISPR has two main parts. The first one are Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), which are short repetitive sequences of DNA. The second component are proteins called Cas (“CRISPR-Associated” proteins).


gene editing, CRISPR/Cas9, PAM sequence
Bacteria's Immune System with Cas Proteins

When a virus enters a bacteria, Cas proteins copy the DNA of the virus and insert it into the DNA of the bacteria. Then, the virus’ DNA is copied into RNA. RNA is similar to DNA, but it is used as a messaging device that copies DNA codes and transports them throughout the cell. In the bacteria, the RNA of the viral DNA will bind to moving Cas9 proteins that defend the bacteria from the next time the same virus DNA tries to invade the cell. Once the Cas9 protein sees that the virus has the same DNA that matches its RNA, it will destroy the virus, protecting the bacteria.


Having said that, what is to stop the Cas9 protein from destroying the DNA sequence that matches its RNA? Well, when Cas proteins copy the viral DNA, they also copy a PAM (protospacer adjacent motif) sequence which is a small portion of the DNA that follows the viral DNA. Cas9 proteins will only destroy the object that has the matching DNA sequence that is followed by the PAM sequences. Since the bacteria’s DNA doesn’t have the PAM sequence, Cas9 will not destroy the bacteria’s genome sequence.


However, CRISPR/Cas9 is not limited to bacteria. In 2012, scientists found a way to take the editing technique of CRISPR/Cas9 and modify targeted regions of gene sequences. But how did they do it? Scientists modify DNA by designing a guide RNA (gRNA) that matches the specific gene they want to target in the DNA sequence. They make sure to match a PAM sequence that matches the DNA. Then, they attach the RNA to Cas9 proteins and deliver it into the cell. The modified Cas9 protein will find the match to its RNA on the DNA strand and cut that part out as its PAM sequence matches the DNA.


gene editing, gene therapy, homologous repair, PAM sequence, CRISPR/Cas9, DNA
Gene Editing with Cas Proteins and RNA

After the DNA is cut, the cell will start a repair process called “nonhomologous end joining” where the cell tries to erase the cut DNA and join the ends back together. However, this repair process can cause mutations that can make the gene sequence unusable. So, scientists will add a template DNA to start homologous recombination instead, completing the gene editing process.



Homologous recombination allows the DNA to insert a new genome sequence into the DNA strand using the template DNA they created. Scientists can craft the template DNA to do whatever they want. The ability to repair the DNA sequence allows scientists to cure genetic diseases or give the organism new traits.


Scientists and doctors have been experimenting with CRISPR and determining the extent of its applications. In 2016, scientists used CRISPR to eliminate HIV from over 50% of all cells in rats. CRISPR trials have been going on to cure other disorders, and many more will occur in the future. Additionally, gene editing can be used on plants and insects as well. We can make larger fruit, or make parasites harmless.


The major limitation of using CRISPR is that it cannot be passed down. It dies with the organism. However, scientists are still trying to use CRISPR to edit gametes (sex cells) and potentially engineer embryos. However, that raises some controversies as scientists still have much to do to fully understand the long-term effects of gene editing.


The future of gene editing will change the way doctors treat their patients. By using technologies such as CRISPR/Cas9, doctors may be able to eliminate certain diseases and genetic disorders. Not only that, genetic engineering can potentially lead to a breakthrough in curing cancer. The possibilities are endless. What has long been thought of as science fiction just became a reality.


Learn more about the future of medicine from other articles at STEM-E!

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