The other day on Bioscription, we discussed the South Korean Institute for Basic Science (IBS) and their work with CRISPR-Cpf1 in soybeans. Today, the same organization has released a study on a new form of CRISPR-Cas9 that they isolated from Campylobacter jejuni. This resulted in the acronym CjCas9 for it.
A Problem Of Size
A known limitation of CRISPR systems, at least the earliest ones used like the Cas9 from Streptococcus pyogenes, was that their protein structure was fairly large. For that one, it measured at 1,368 amino acids in length. This made it too large for many plasmid or viral vectors to properly transport it into desired cells in order to do its work.
Scientists got around this in the early stages by delivering the Cas9 in two separate viral packages, but this resulted in it not being as fully active as it could have been. It’s easy to see how this is not the most optimal usage of the technology.
After that, a new Cas9 was obtained from Staphylococcus aureus, the common horrible medical infection bacteria. This version was only 1,053 amino acids in length, just barely short enough to be packaged in a single viral vector. This is a better solution, but it still limits the usage of CRISPR as no additional protein material can be added to guide its workings.
Until now, that is. CRISPR-CjCas9 fills in at only 984 amino acids in length and an additional guide RNA and even a fluorescent protein. Not Green Fluorescent Protein (GFP) sadly, the beloved phosphorescence of genetics classes worldwide, it’s a bit too long at 238 amino acids.
A Need For PAM
It was also given a Protospacer Adjacent Motif (PAM) sequence as well in order for it to function. These sequences are essentially a target board that allows the CRISPR mechanism to recognize the genes connected to the PAM sequence as targets to be cut. Otherwise, Cas9 will not be capable of cutting the desired genome.
The targeting system in this case involves taking the target gene that is desired to be cut out and sticking the PAM sequence on the end, then bundling it with the CRISPR-Cas9 sequence. This allows the mechanism to recognize the target gene, even though its target within the actual cellular genome doesn’t have a PAM sequence on the end.
For their test of the new Cas9, the researchers bundled it with two guide RNAs related to age-related macular degeneration (AMD). The first of these codes for a common target that, if inactivated, would improve the condition, a gene known as Vascular endothelial growth factor A (VEGF A) and the other is just a transcription factor that activates the former sequence called HIF-1a.
The latter being a transcription factor is important. They have long been considered to be “undruggable” targets. That is, proteins and molecules that do not favorably bind to the target drug and thus cannot be altered or induced by medical means. With this new CRISPR however and this guide RNA version, the Cas9 can be guided to target that transcription factor in the genome and inactivate it.
The capability of CRISPR being able to target previously untargetable sequences has always been one of the primary reasons to praise its discovery, but it was always difficult to get it to work properly due to the system’s size. Now that isn’t an issue anymore.
The Capability Is Finally Here
With this improved size capability, the hope is that more conditions can be treated by Cas9 that previously were unavailable by not being able to include guide RNAs to aim at the target genes. This also means that practically all undruggable targets in medicine are open to be focused on and modified to alter medical conditions.
Such a simple improvement to CRISPR-Cas9 as finding a smaller version in another bacteria may make all the difference in the fight against genetic disorders.
Photo CCs: Macular Degeneration from Wikimedia Commons