The development of recombinant DNA in the 1970s marked a breakthrough in molecular biology: for the first time ever, scientists were able to manipulate DNA molecules. In contrast to the past, where DNA molecules were manipulated and studied outside of the genome, developments in the genome engineering landscape have recently allowed scientists to directly manipulate DNA in their native context. Now that scientists can study how DNA behaves in its natural habitat, so to speak, scientists can uncover the architecture of our genome. This has held, and continues to hold, immense promise for the fields of science and medicine, especially in regards to eliminating non-curable diseases such as cancer.
Recently, the discovery of an ancient defense mechanism found in a wide range of bacteria that eliminates invading genetic material has resulted in an explosion in the genome-editing field. This system, called CRISPR-Cas9 for short, was repurposed by scientists to edit genomes with unprecedented precision, efficiency, and flexibility. The system consists of two components: a Cas9 enzyme and a piece of RNA called guide RNA (gRNA) . The gRNA is a short synthetic RNA composed of a scaffold sequence on one end and a 20-nucleotide sequence designed to target a specific target in the genome. On the other hand, the Cas9 enzyme serves a scissor-like function, cutting up the target DNA so that it can be altered.
How does the CRISPR gene-editing process work? The gRNA binds to its target on the DNA and the scaffold sequence recruits the Cas9 enzyme . This interaction between the two results in an active complex, cleaving the DNA. The DNA is then repaired by endogenous DNA repair machinery present in cells, but these usually result in insertions or deletions of bases, which disrupts the target gene. Depending on which gene is disrupted, there may be beneficial, deleterious, or no effects on the organism under study. Geneticists are currently using CRISPR-Cas9 to functionally characterize our genome, as well as manipulating it to knock-out known cancer causing genes. Many diseases are not caused by a single genetic mutation but rather a combination of a number of aberrant genes. With the CRISPR-Cas9 system, simultaneous targeting of multiple genes can drastically broaden our ability to cure a much wider range of disorders.
CRISPR-Cas9 has become a revolutionary tool in biological research as it is much more precise, faster, and cheaper than previously used genome editing technology . Before CRISPR-Cas9, biologists utilized other genome engineering techniques called ZFNs and TALENs, both of which rely on DNA-binding protein nucleases for their specificity . Both of these techniques proved to be time consuming and expensive as a new nuclease must be designed for each genomic target, and they often have to create several variations before finding one that actually nicks the DNA. In contrast, CRISPR-Cas9 is significantly more versatile as the Cas9 nuclease is constant, and the changing factor, the gRNA, can be designed and produced much more rapidly. Additionally, CRISPR-Cas9 has higher cleavage efficiency than TALENs and ZFNs meaning that CRISPR results in greater successful cleavages than do other genome editing techniques.
Despite these many advantages, CRISPR-Cas9, unlike TALENs and ZFNs, cannot be targeted to anywhere in the genome. It requires the presence of a specific motif called the PAM sequence on the DNA, restricting potential targets for CRISPR-Cas9. This usually does not arise as an issue because of the high occurrence of PAM motifs throughout genomes .
Of course, as with anything in science, controversy is bound to arise, with CRISPR-Cas9 being no different. Immediately after its discovery, ethical concerns were raised regarding the possibility of genome editing in human germline cells, where genetic changes can be passed on to future generations . From this, the issue of “designer babies” (genetically modified for beauty, intelligence, or freedom from diseases) arises with the use of CRISPR-Cas9 in human embryos. Currently, in the scientific community, using CRISPR-Cas9 for somatic cells is largely accepted, however beyond avoiding genetic diseases, the moral and ethical aspects have presented a minefield. Another issue associated with CRISPR-Cas9 is Cas9’s off target activity in editing DNA sequences that bear up to 5 mismatches from the gRNA . This means that when targeting a cancer-causing gene, there is potential for the Cas9 to alter another gene which if disrupted will result in a disease. Kind of like a “catch 22”, but there are a number of ways to increase the specificity of the gRNA.
Recent progress demonstrates CRISPR-Cas9 to be the ultimate molecular tool for genome editing, as it has provided us with unprecedented insights into the genomic architecture of a number of organisms. As long-term implications remain unclear there is a general consensus that there is still a way to go before CRISPR will be ready for safe and effective gene therapy in humans.