CRISPR-cas9 and Ethics, Update One: The Mechanism

The first phase of constructing my literature review was to obtain a solid foundation of knowledge regarding the technology of clustered regularly interspaced palindromic repeats (CRISPR) cas9 itself.  This information is necessary in order to accurately assess ethical concerns moving forward.  Throughout my reading, I realized there are two levels to understanding CRISPR-cas9.  The first is how it operates as a biological mechanism in nature, and the second is how this mechanism has been adapted into a functional tool.

There are six different CRISPR systems, but type II is the most useful for study in terms of gene-editing due to its simplicity.  Whereas other types will contain multi-cas complexes, type II only utilizes the cas9 endonuclease.  CRISPR are segments of DNA and “cas9” is short for “CRISPR associated”, in reference to the enzyme which can cut DNA.  CRISPR segments are characterized by repeating nucleotide sequences and spacers in-between those sequences.  The system is anti-viral, and the spacers within the DNA are taken from viruses which have previously attacked the organism, allowing for the virus to be recognized and targeted in the future.  In the event of a re-attack, the portion of the DNA segment matching the virus will be transcribed into crRNA, which acts as a guide RNA.  Cas9 then binds the crRNA with tracrRNA, forming a crRNA-effector complex.  The two RNAs then guide cas9 to the target site, where it performs a double-stranded break (DSB).  The CRISPR region of DNA also contains protospacer adjacent motifs (PAMs).  These motifs are located next to the target sequence to be transcribed, and act as a safety mechanism so that cas9 does not attack the DNA of the organism rather than the virus.

In order to adapt this system into applicable technology, the natural repair mechanisms need to be tricked.  Genes can either be removed or inserted using this method and nearly any site following a PAM sequence can be targeted.   A guide RNA sequence of twenty nucleotide base pairs must be designed in order to achieve this.  For an insertion, an additional donor DNA template will be inserted following the DSB performed by cas9.  The donor template must match the ends of the cleaved DNA and contains the change desired.

Overall, I have had to dedicate more hours than I anticipated to the initial stages of this project.  The complexities of CRISPR itself, especially in higher-level literature, proved to be very dense.  There are some aspects of the process that are not even fully explained, such as how invading DNA is incorporated into the CRISPR sequence.  However, I am satisfied with the information I obtained as result and I believe it will benefit the resulting work.  Moving forward, I will now begin to focus on the faults and risks of the mechanism and begin to analyze overarching scientific ethics and their connections to this specific technology.