CRISPR/Cas9: An Introduction and a Brief Glimpse of Recent Literature

Ridwan Hossain

Sophomore

School of Life Sciences

Independent University, Bangladesh

November 9th, 2017

CRISPR/Cas9 is a new revolutionary gene editing tool that is causing quite a stir in the world of science. It is opening up possibilities to cure cancer, HIV and other major diseases which are incurable by other methods till now. To learn how CRISPR works, we need to understand what genomes, genes, and gene editing are. Every living organism ranging from the most sophisticated ones like humans to the simplest but deadliest of organism like viruses contains specific instructional codes called genes. The complete set of genes carried by an organism is its genome. With the exception of some viruses, genomes are made up of DNA.  These instructional codes decide the characteristics of the organisms; genes code for their physical structures, and determine the biochemical processes that they utilize to make use of nutrients and produce energy. Your eye color, how a virus will infect an organism, how a plant will carry out photosynthesis, the fur color of a dog, these are all decided by each of their respective genetic codes. Changing these genetic codes will alter different characteristics of an organism. We have discovered and developed multiple methods to modify genetic codes and this is what gene editing refers to. CRISPR/Cas9 is a promising new tool that can carry out very specific gene editing in less time than the other methods we have traditionally used. Now, what exactly is CRISPR/Cas9?

Viruses are a type of microorganism that survive by feeding on other organisms. Certain viruses, known as bacteriophages, only infect another type of microorganism known as bacteria. Bacteriophages bind to the surface of bacteria and inject their genetic information inside the bacteria. The bacteriophage takes over the building machinery of the bacterium, and uses it to make hundreds of copies of itself (that is, its offspring). Bacteria have evolved several mechanisms to defend themselves from bacteriophages. The CRISPR/Cas system, found in many bacteria, allows them to recognize and cut foreign pieces of genetic material (such as viral DNA), and if they survive, store this information into their own genome using the CRISPR system. Now that it has information about this particular phage, if it is attacked again, it will use this stored information to specifically recognize and rapidly destroy the phage. We have adapted a particular bacteria’s CRISPR/Cas system (namely the CRISPR/Cas9 system of Streptococcus pyogenes) to target genes for editing. So how does this system really work?

An animated Cas9 targeting a gene. McGovern Institute for Brain Research at MIT

We do not need the initial step of recognizing DNA as foreign for our gene-editing system. The CRISPR/Cas9 system works based on stored sequences in the CRISPR genetic region, an enzyme called Cas9 for cutting DNA, and a guiding RNA. The guiding RNA is transcribed from the stored sequence in the CRISPR region. We have the technology to target any part of the genome (in any organism) by making the appropriate guiding RNA. The guiding RNA carries the Cas9 enzyme to the gene we want to cut. Cutting a gene can lead to its deactivation, and also allow us to introduce mutations of our choice by providing an appropriate repair template for the cell’s repair machinery. Let us look at a couple of recent studies and potential applications of the system.

An increasingly large number of studies are using CRISPR/Cas9 to edit genes for a variety of purposes. One recent study used thesystem to target the nervous system in adult living mice. The target gene in this study was an X-chromosome gene known as MECP2. The normal functioning of nerve cells depends on the protein encoded by MECP2. A mutated version of this gene causes a syndrome called Rett syndrome. Rett syndrome is a disease that causes problems in development of a victim’s brain and causes major disabilities and symptoms similar to autism spectrum disorders. In this study, they were able to use CRISPR/Cas9 to specifically target this gene and thereby reduce the production of its products, which resulted in Rett syndrome. This seems counter-intuitive and scary, but what they did is basically show that it is possible to target and alter a gene in an adult living organism. The next step would be to figure out how to fix a defective gene through CRISPR-mediated targeting.

But deactivating genes can be quite beneficial on its own, as demonstrated by a study where they targeted HIV genes with CRISPR/Cas9. HIV, which is the virus that ultimately causes AIDS, still eludes a complete cure. According to WHO there were 36.7 million people living with HIV by the end of 2015. HIV is particularly hard to find a cure for because it reproduces really fast and its genome goes through rapid changes. So if we come up with a way to destroy HIV virus they just change into something different and our cure does not work anymore. Additionally, the virus incorporates itself into the host genome, and can stay dormant, making viral clearance impossible. Maybe CRISPR can change that. Current methods target different steps of the HIV lifecycle simultaneously as multiple resistance mutations are less likely to occur. CRISPR could do even better. A study was recently conducted to see how CRISPR could stop HIV. A CRISPR/Cas9 system with ten different types of guiding RNAs, each with its specific genome sequence, was used to target different areas of the HIV genome infecting cells in laboratory cell culture. Results showed that this process was highly effective in the inhibition of HIV-1 (a certain lineage of HIV) genome. CRISPR was working by inserting inactivating mutations into different parts of the HIV genome. An important advantage to this is that HIV viruses that had been directly incorporated into the host genome were directly deactivated, unlike what current drugs do (which only work on actively replicating viruses). This effectively means viral clearance, and if developed further, could completely cure AIDS.

CRISPR/Cas9 holds the cure for genetic and viral diseases and so much more. The technology is, in fact, still being developed for even greater efficiency. CRISPR could be used to get rid of disorders that children are born with such as Down Syndrome. More excitingly, and perhaps scarily, we can decide the characteristics of our offspring and even modify and change ourselves with complete freedom. It is hard to say what will be possible with it in the near future but it will eventually change the way we live and see our world. Are we ready to accept all the changes though?

Ridwan is a sophomore at IUB whose dream is to be a renowned mad scientist. He will be a Nobel laureate.