Scientists and scientific publications often discuss CRISPR as a tool that, once inserted successfully into a cell, just gets to work on modifying the host cell genome. Its efficiency and accuracy may vary, but it is often thought of in conversation as a highly effective gene modifying device. That as it turns out is only partially true. There are plenty of roadblocks and hindrances that work together to prevent CRISPR from even doing its most basic of functions.
The Building Blockage of DNA
An apparent representation of this is the existence of chromatin. That being the specific form that DNA (and RNA) takes when it is folded up and packaged together in a wound up fiber, the most complex levels of which are recognized as individual chromosomes. Since the introduction of CRISPR Cas9 as a gene editing tool, several studies have found that packaged chromatin unsurprisingly blocks access to the particular DNA sequences that are being targeted. If the genes are wound up in a complicated overlapping fiber-like structure, then the capability to alter the desired length of DNA is prevented almost entirely.
Up until now, a large amount of DNA editing targets have been on genes likely to be under active transcription, meaning that the cell itself is in the process of producing proteins and RNA from the sequence. So it was unlikely to be in a turned off and bound up chromatin state, therefore allowing CRISPR Cas9 to be successful in its goals. But as genetic engineering advances and more subtle targets are being considered, the issue of chromatin interference is becoming ever more prevalent in CRISPR experiments. Direct testing of this using introduced chromatin-forming enzymes have shown that, depending on the circumstances, Cas9 can be suppressed in its activity or outright blocked.
This leaves only one alternative option: unwind and reverse the chromatin state via other introduced genetic elements. Even if done just transiently, that should be enough so long as the correct length of sequence is exposed for the CRISPR complex to do its work. And the best choice to target in this regard is the process of chromatin remodeling, whereby transcription machinery proteins access localized parts of the genome when they need it. If this can be purposefully harnessed, then the entire disruptive problem can be avoided.
Dual Tactics: Inhibition and Promotion
Researchers from Arizona State University decided to attempt to address this issue. In prior publications, they had used cell lines with a transgene for luciferase that is bioluminescent and had already been inserted into a packaged chromatin region. Therefore, if they manage to get around the chromatin to edit the region to activate the transgene, achieving this would be visible. Previous knockdown and silencing of a polycomb group protein responsible for gene expression had shown an increase in efficiency of editing the transgene, but the research group wanted to do even better than just that.
In the current experiment, they focused on two separate approaches for opening up chromatin: one based around inhibiting the enhancer for a histone methylation and transcriptional repression protein and another using a fusion protein trying to activate transcription through direct DNA binding. They wanted to see if inhibition of chromatin formation or promotion of chromatin unwinding would work better for the purposes of allowing CRISPR Cas9 access. After conducting the process, they then measured transcription levels of the desired sequence and used that data to figure out CRISPR efficiencies.
Overall, they found that taking out the inhibition protein only had a partial improvement and did not reach full gene expression levels. On the other hand, using the transcriptional activator known as Gal4P65 and having it be targeted to the luciferase transgene had a multitude of desired effects. Levels of a silencing mark that shows an epigenetic change causing chromatin folding was reduced generally and expression of the luciferase was increased tremendously. Testing with other Gal4-AAP fusion proteins, even ones that didn’t activate the luciferase marker, were still effective in improving editing efficiencies. That means that fusion proteins aimed at promoting transcription in chromatin regions is the best option for getting around this barrier.
Opening The Black Box
It is highly beneficial that things worked out this way, as activator fusion proteins can be targeted to a specific gene area without affecting other surrounding genes. The other method with knockout of methylation genes could have untoward side effects. The scientists suggest trying out the efficiencies when using other gene editing tools, such as zinc finger nucleases, TALENS, and also dead Cas9 binding complexes. Another improvement option is to fuse the activation associated peptides (AAPs) and other molecules that modulate chromatin expression onto the Cas9 complex itself. This would create an all in one device that can potentially modify the chromatin and conduct the gene editing all together with less complications.
But a lot of further testing is needed to prove these other suggestions work just as well. For now, all we can say is that it is indeed possible to open up chromatin and have CRISPR Cas9 edit those sequences with the same full efficiencies as with unwound DNA itself. With further developments in this area, greater portions of genomes will be accessible and create a multitude of additional options for making useful and beneficial changes to any number of organisms.
Photo CCs: Chromatin and the PAD4 enzyme from the Wang Lab at Penn State
