Gene editing with CRISPRs

GloFish have coral DNA spliced into their own DNA. Photo source.

Things aren’t made the way they used to be. When you shop at the grocery store, chances are you’ll buy some genetically modified fruit or vegetable. For a long time, selective breeding has been used to produce crops and livestock with favorable traits. In the 1970’s, scientists developed the first methods for directly engineering the genome, or genetic material, of an organism. Since then, many of these genetically modified organisms (GMOs) have become widely available for commercial use. These include transgenic bacteria that produce insulin (Humulin), corn that makes its own pesticide, and fluorescent GloFish. GMOs pose both benefits and risks that need to be fully assessed before they are produced and distributed. For example, genetically modified Atlantic salmon, which are pending FDA approval in the USA, are able to grow to full size in half the time. However, a recent study demonstrated that genetically modified salmon can hybridize with and outcompete wild stocks, which could prove disastrous for native populations.

In the laboratory, the information gained from studying genetically modified organisms has proven invaluable. Several methods have been developed in the last two decades to make genome engineering easier and more efficient including ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), and CRISPRs (clustered regularly interspaced, short palindromic repeats). The latest technology available is the CRISPR-Cas9 system, which was developed from a bacterial immune system. In 2012, this system was modified to edit virtually any gene in any organism and, since then, the system has been developed by numerous laboratories. An ongoing legal battle has ensued between these groups to identify the original inventor and each has started up their own company (Editas, CRISPR Therapeutics, Intellia Therapeutics). CRISPRs have created quite a frenzy in the molecular biology world. In just the past two years, over a thousand papers have been published related to CRISPRs.

CRISPR-Cas9 targeting a sequence of DNA. Image courtesy of Michael Peters.

CRISPR-Cas9 targeting a sequence of DNA. Image courtesy of Michael Peters.

How do CRISPRs work?

CRISPRs, or Clustered Regularly Interspaced Short Palindromic Repeats, were discovered in bacteria as a mode of acquired immunity to remove DNA introduced by viruses and other infectious agents. In many viruses, just like in humans, DNA is the molecule that is used to store biological information in the form of genes. These encoded genes are first transcribed into RNA, which is the message to produce the proteins that are needed for the virus to replicate and infect other cells. The CRISPR system is used by bacteria to remove viral DNA in a few short steps (check out this for more info). First, the bacterial cell samples a very small piece of viral DNA, which is like taking a fingerprint from the virus. The bacteria uses this piece to then make an RNA molecule that acts as a guide for a protein called Cas9. Together, they target and cut up the viral DNA, functionally removing the viral DNA from the bacteria.

Researchers have harnessed the DNA targeting ability of the CRISPR system to modify the genomes of zebrafish, fruit flies, human cells, and mice, among others. A target sequence can be chosen almost anywhere in the genome. After introduction into the organism, the guide RNA and Cas9 complex makes a cut at the target site in the DNA. Mutations (changes in DNA) are then introduced as small deletions or insertions of genetic material (aka indels). These indels can scramble the encoded message and render the gene nonfunctional.  This approach, called “Reverse Genetics,” allows scientists to remove a gene and find out what it was doing. It differs from “Forward Genetics,” which attempts to identify the gene(s) that cause a trait or defect that is already known. For example, in a forward genetics screen for mutants, fruit flies that grew legs on their head were found to have a mutation in a gene that was thereafter named antennapedia.

The left fruit fly has normally developed antennae whereas the fly on the right has a mutation at the AntP gene which makes the fly grow legs in place of antennae. The understanding of this phenomenon was elucidated through forward genetics. Photo source.

The left fruit fly has normally developed antennae whereas the fly on the right has a mutation at the AntP gene which makes the fly grow legs in place of antennae. The understanding of this phenomenon was elucidated through forward genetics. Photo source.

What are the potential applications?

Mutations are the basis for evolutionary change and these “mistakes” have produced all of the diverse lifeforms found on our planet. Humans have about 24,000 genes that code for proteins and mutations in individual genes can cause 10,000 different genetic disorders that affect millions of people. Removing a gene from an animal can have drastic effects on development. CRISPRs have been used to produce mutant models to study genetic diseases such as liver cancer, thalassemia, and cleft palate. Gene therapy is the ultimate goal for genome engineering but, unlike their predecessor, zinc finger nucleases (ZFNs), CRISPRs have not yet been used in clinical trials. ZFNs were recently used to edit a gene in cells that were taken from and transplanted back into HIV-1 patients with promising results (viral RNA was reduced in 4 patients!).

However, ZFNs have had a decade head start and CRISPRs are not far behind. The CRISPR system has already been used to cut out and replace the damaged beta-globin gene with the correct sequence in cells taken from thalassemia patients. By fusing parts of other proteins to Cas9, the CRISPR system can be modified to carry out other jobs, like turning genes on. Since the technology is advancing at a rapid rate, it is important to consider that manipulating the human genome could have unintended consequences, especially since most of it is still uncharacterized. Several of the gene editing bigwigs met in January to discuss the potential dangers of genome engineering and they published an article in one of the top scientific journals, Science, strongly discouraging germline genome modification of humans. Remarkable achievements have been made with this technology in just two years. How will CRISPRs be used in the coming years? Keep reading FTDM to find out!

Find out what else has been done with CRISPRs

Michael Peters received a BS in Biology at the University of New Hampshire in 2012. Currently, he is pursuing his PhD in Evolution, Ecology, and Marine Biology at Northeastern University. He studies the mutations that led to the evolutionary loss of red blood cells in Antarctic icefish. His work utilizes CRISPRs to reproduce these mutations in pet-store variety zebrafish. When not in the lab, he enjoys fishing, running, and traveling to new places.

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