Jennifer Doudna, the co-inventor of the new gene editing technology, CRISPR-Cas9, explains how it works and its potential uses. The breakthrough for this new technology came from the study of bacteria. The CRISPR technology uses a protein called Cas9. A guide RNA (synthetically made) that matches the sequence of the DNA in question, at a specific location, attaches to the Cas9 protein to form a complex. Once this complex finds matching DNA sequences, it attaches at that site, and the Cas9 makes a precise cut, removing that DNA strand. After a break, the cell can repair the site by integrating a new piece of DNA. This is why the CRISPR technology is so important for genome engineering- it allows us to insert new DNA into an organism at a specific location.
Many new gene editing tools have been developed but CRISPR is the most efficient and easy to use. Previous methods couldn’t control where a gene was cut or where new genes were added. CRISPR, however, through the use of Cas9 and the synthetically made guide RNAs (combination of tracrRNA and spacer RNA), is able to cut a genome in an exact location. The CRISPR technique has been used on mice embryos to produce new mice models. The CRISPR method has also been used on crops to disable genes and recently has been used to target genes related to human diseases. The CRISPR method, however, is not perfect. According to some tests, there is a possibility the guide RNA can target DNA that differs from the intended DNA by up to five bases (A, T, G, C). In some cases, the guide RNA targets the wrong DNA, potentially causing unnecessary breaks.
- Caplan, A. L., B. Parent, M. Shen, and C. Plunkett. “No Time to Waste–the Ethical Challenges Created by CRISPR.” EMBO Reports 16.11 (2015): 1421-426. Web.
There are many ethical concerns with the CRISPR technology. One concern is the ability to edit human embryos. Many people argue this is unsafe and crosses ethical lines. The next big concern is related to GMOs. Organisms have been genetically modified for years, yet many people still have major concerns. With CRISPR technology, it will be much easier and cheaper to produce these potentially unsafe genetically modified organisms. Because the process is so simple, in the wrong hands, CRISPR could also be used to create harmful viruses and bioweapons.
This article first introduces previous gene editing methods such as zinc fingers and explains the problems associated with those methods. It then goes on to describe how the CRISPR (clustered regularly interspaced short palindromic repeat) system has been used in Drosophila (flies) and the varying outcomes (links to studies are also included). Many tests have been done on flies using a multitude of techniques (inserting plasmids, inserting mRNA, etc). They all showed positive changes in the Drosophila DNA because of the CRISPR method. These studies are important for expanding the CRISPR technology. The article also discusses limitations to the CRISPR system such as the sgRNA (simple guide RNA) cleaving efficiency, where the complex isn’t able to cut the DNA. This could occur because of the structure of the Cas9/RNA complex or the chromatin structure. Any disfiguration in these structures could decrease the complex’s performance.
This article goes into more detail about how the CRISPR technology works and discusses Cas9’s ability to attach to its true target. It is quite common for Cas9 to attach to the wrong site, but very uncommon for cleaving to occur. Scientists performed a study to determine how Cas9 avoids cleaving at incorrect sites (the complex quickly moves on when the RNA and DNA aren’t an exact match, not leaving enough time for a cut) and how it can quickly find its true target in a sea of DNA. Another study of Cas9 shows it surprisingly persisting in heterochromatin regions. In clustered DNA, the protein is slowed but still persists.