CRISPR-Cas9: Editing and Engineering the Blueprints of Life

Every cell in our body contains a copy of our genome.  From this single blueprint, differential gene expression allows for a variety of structures and functions.  DNA consists of two strands of nucleotides twisted into a double helix and held together by a simple pairing rule.  Genes shape who we are individually and as a species.  Genes also have profound effects on our health.  Some genes can specifically influence our risk for disease.  In order to understand the role of different genes, we need a way to control and manipulate them, and further observe the effects of these changes.

Such control and manipulation is not an easy feat.  Recently, a new method, CRISPR-Cas9, has dramatically improved our ability to edit DNA.

Compared to past gene editing techniques, such as ZFNS or TALENs, CRISPR-Cas9 uses RNA sequences instead of proteins to target the DNA, making this technique more efficient (Rath et al. 2015).  RNA is smaller, making it less challenging to infect cells and associate with DNA.  Synthesizing RNA is also easier and less expensive than synthesizing peptides.

The CRISPR method is based on a natural system used by bacteria to protect themselves from infection by viruses (Rath et al. 2015).  When the bacterium detects the presence of virus DNA, it produces two types of short RNA, one of which contains a sequence that matches that of the virus DNA.  These two RNAs form a complex with a protein called cas-9.  Cas-9 is a nuclease, a type of enzyme that can cut DNA.  When the matching sequence, or guide RNA, finds its target on the virus DNA, the cas-9 cuts the target DNA, disabling the virus.

Researchers studying the system discovered cas-9 and its associated components could be engineered to cut any DNA, not just viral DNA, by changing the guide RNA to match a desired target.  Further, the method can be introduced within the nucleus of a living cell.  Once inside the nucleus, the cas-9 complex locks onto a short sequence of the DNA known as the PAM.  Cas-9 nuclease unzips the DNA and matches it to the target RNA.  If the match is complete, the cas-9 cuts the DNA.  When this happens, the cell tries to repair the cut.  This repair process is prone to error and usually leads to mutation that can disable the gene.  Researchers can then observe and study the effect in the cell and to the organism in order to understand the function of the disabled gene. The mutant gene, or cut segment of DNA, can be replaced with other genes to modify functions in the cell and organism as well.

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The CRISPR-Cas9 system has a slight drawback in that it can only make cuts when adjacent to PAM sequences in the DNA.  The PAM sequence is a 3 amino acid sequence, NGG (Mussolino et al. 2011).  N can be any of the nucleic acids followed by two guanines.  Luckily, this is a common sequence in most DNA.  The other concern, as with all genome editing technology, is off-target effects.  So far, CRISPR-Cas9 seems to have relatively few off-target effects.

            The CRISPR-Cas9 method can be performed not only in test tubes, but also in living organisms.  The method can be used to target many genes, or sequences, at once, allowing for multiple mutations at a time.  Such multiplicity is beneficial when studying diseases that work by targeting many genes at once.  Applications of CRISPR-Cas9 include mutating, silencing, and over-activating genes.

Genome engineering can also control cellular metabolism for enhanced productivity of chemicals, fuels, and even medicines.  In a demonstration of this potential, Li et al. recently published a study describing their development of a CRISPR-Cas9 based method for repetitive genome editing and metabolic engineering of Escherichia coli.  The bacterium was used for integration of a β-carotene synthetic pathway, resulting in overproduction of this beneficial compound.  β-carotene is a safe source of vitamin A and an antioxidant that may reduce the risk of lung cancer for non-smokers and slow cognitive decline.

The study embodies a successful application of metabolic engineering, as Li et al. achieved 100% editing efficiency while introducing three mutations at a time.  The mutations advanced the β-carotene, methylerythritol phosphate, and central metabolic pathways simultaneously.  The short editing cycle of two days also adds to their method’s efficiency.

Other applications of CRISPR-Cas9 include basic research, drug development, agriculture, and hopefully treating human patients with genetic disease.  Therapeutic genome editing comes with both prospects and challenges.  A shadow of fear is cast over the potential benefits of this technology.  Many are weary of an eventual manipulation of genomes for the creation of transgenic humans.  As with all great scientific discoveries, CRISPR-Cas9 will require great responsibility.  Sound ethical and moral principles will help navigate the use of this technology.

Hess, Terry.  CRISPR/Cas9 Mice Model Service.  12 January 2015.  <https://www.flickr.com/photos/130205442@N07/15640213654/> (image credit)

Li, Y., Z. Lin, C. Huang, Y. Zhang, Z. Wang, Y. Tang, T. Chen, and X. Zhao.  2015.  Metabolic engineering of Escherichia coli using CRISPR-Cas9 mediated genome editing.  Metabolic Engineering 31: 13-21.

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Mussolino, C., R. Morbitzer, F. Lütge, N. Dannemann, T. Lahave, and T. Cathomen.  2011. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity.  Nucleic Acids Res 39: 9283-9293. 

Rath, D., L. Amlinger, A. Rath, and M. Lundgren.  2015.  The CRISPR-Cas immune system: Biology, mechanisms and applications.  Biochimie 117: 119-128.