It is of no doubt that the recent advancements of the CRISPR/Cas9 genome-editing technique has provided a huge boost to the genetic research field. Evolutionary, as a defensive mechanism found in some bacteria and archaea against invasive viruses and exploited by the Church group in Harvard and Zhang group in MIT, such a genome-editing technique is gradually becoming the standard protocol for performing genetic engineering in a wide range of organisms, from cells and animals to crops and livestock. A more recent (and radical) investigation even applied this technique on infertile human embryos, with another application pending to apply it on normal human embryos.
In short, the Cas9 protein, with the assistance of two RNA molecules (crRNA/tracrRNA, although in practice these two RNA molecules are replaced by a single chimeric sgRNA), has the ability to “home-in” on its target site in the genome and either make double-strand cuts or introduce new DNA fragments. Basically, the sgRNA is the only element that needs to be re-designed in each experiment, while all the other reagents, including the Cas9 protein itself, have already been widely commercialized and can be purchased off-the-shelf. The expenses for each independent experiment design is also minimal: approximately $30 for the cost of the sgRNA to be designed and synthesized.
Albeit with all these above-mentioned advantages, the Zhang group still thought there were spaces to maneuver. In April, they reported the discovery of a smaller version from the bacterial strain Staphylococcus aureus, which may provide better penetrability into cells due to its relatively smaller size. Another major breakthrough from their lab was just released in last week’s Cell, where they reported the discovery of Cpf1, an enzyme that shares the same genome-editing abilities as Cas9. More importantly, after exhaustively screening more than 16 bacteria strains that harbour Cpf1, two strains were isolated with their Cpf1 that possess the abilities to function on human DNA. The most striking feature of Cpf1 is, unlike its counterpart Cas9 that makes a clean cut on double stranded DNA and leaves “blunt” ends after its job is done, Cpf1 creates “sticky” ends that leaves one strand longer than the other (5’- overhang). For people who are familiar with molecular biology, a “blunt” end is much more difficult to work with than a “sticky” end. For example, one of the advantages of the CRISPR system is its ability to insert exogenous DNA fragments at the cutting site. However, the existence of a “blunt” end means that only 50% of the inserted fragments are expected to be functional since the double strand can be ligated in two different ways. The creation of “sticky” ends at the cutting sites eliminates such problems since the fragments would have only one viable direction to be inserted in, which greatly enhances the integration efficiency. Moreover, the homology guide sequence (seed sequence) are spatially separated from the cleavage site in the case of Cpf1 which prevents the destruction of the seed sequence after insertion (as in the case of Cas9) and allows re-insertion of additional sequences using the same seed site.
At one week old, this new genome-editing technique is still too early to be put into action. It is nevertheless important and worthwhile to begin evaluating its potentials, limits, and possible applications in the current context of the biotechnology industry. Insightful bio-entrepreneurs should act fast, before the new scissors are taken by their competitors.
References:
Zetsche et al., Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System, Cell (2015), http://dx.doi.org/10.1016/j.cell.2015.09.038
Image courtesy of pixabay.com
Learn more about PreScouter at www.prescouter.com.
