CRISPR-Cas9 Allows Photographic Imaging Of Genes In Live Cells
A Science Enthusiast
With biotechnology, we have been able to use marker genes like green fluorescent protein (GFP) to follow the activities of individual cells, but that is a far cry from interior mechanisms within a cell. Since the dawn of the field, technology has advanced so that fluorescent microscopy has taken the step to using fluorescent proteins to be able to cause only particular cellular components to glow.
This, in turn, has allowed scientists to follow the actions of individual macromolecules and their activities within the cell. But there is one part of the cell that still remains largely aloof. The part containing the highly coiled genome of the cell.
How To Picture DNA
Past methods used to image DNA and showcase its three-dimensional structure has run into difficulties. The chromatin, that is the packaged architecture that contains the DNA in the genome wrapped around histone proteins in complex arrangements, and its 3D shape is incredibly important for geneticists wanting to understand more about how certain genes are regulated and controlled.
But most imaging methods that are able to reach the level of detail required are also invasive enough that they can’t be performed on live cells. And, in order to make a full picture, the process often requires millions of individual cells to be imaged. All in all, an inefficient process and one that isn’t helpful, since DNA denatures and begins to lose its structure upon cell death.
Gene editing methods of the past and still the present, such as TALENs and ZFNs, have allowed some amount of sequencing of genes while in their 3D form, to know the physical locations of each gene segment within the coiled structure. But these methods could only view repeating segments of the genome, as the repetition made them easier to pick out and use gene editing to be noticed.
CRISPRing A Solution
Targeting the non-repeating parts could still be done, but it required such a large array of the prior mentioned protein tools that the effort wasn’t feasible. However, as i’m sure you’re well aware, we have a new gene editing tool available now. One that can finally fix this problem once and for all.
Though even with CRISPR-Cas9, there are complications. Previous studies have shown that single guide RNAs (sgRNA) can be used in conjunction with Cas9 to have much more flexibility in targeting gene locations. These Cas9 complexes can also be made to fluoresce when they reach their target genes, showing off where the location is, even with different colors for different Cas9s. Even with this though, there are challenges in targeting non-repetitive sequences.
A repeating sequence is easy to pick out with a fluorescing Cas9 next to it, but non-repeating gene sequences are easier to lose the fluorescing signal in. One way to get around this is to use 26 sgRNAs to target the same region of DNA, but this has a fair failure rate due to them not all finding the same sequence thanks to their amount.
Finding A Signal
Now, researchers at the University of Virginia and the University of California – Berkeley have developed a method that allows the use of CRISPR-Cas9 to image several different gene locations at the same time within a live cell.
To accomplish this, they engineered an sgRNA that has 16 special motifs as a part of its sequence. A motif is a sequence of nucleotides (or amino acids, for proteins) that is very common or have a special significance, making it easy to target and follow. There are also structural motifs that involve a particular 3-dimensional arrangement of amino acids that is created to be followed.
The motifs used were those from the bacteriophage MS2, the first fully sequenced genome of an organism. In fact, the first gene ever sequenced was the MS2 coat protein called MCP. This well known sequence was chosen because of its significance and easy ability to follow it. The motifs were then tagged with MCP genes that also had an added fluorescence.
Using a large number of MS2 gene motifs allowed them to be followed easier, lowering the “signal to noise” ratio that was blocking out locating the Cas9 complex in previous attempts. It also meant that less such complexes were needed, even to target multiple gene positions on the genome (the locus or loci of the gene).
Imaging For Regulation
The scientists were able to target and detect the locations of non-repetitive sequences with as few as four sgRNA sequences. Extended RNA sequences that last longer were later used and actually allowed imaging of the gene locations and their movement throughout the entire lifecycle of the cell.
This allowed the researchers to see which genes were actively being transcribed and which genes were inactive during different points of activity within the cell, helping make clear the functions of different genes and when they are needed.
The new method as devised finally permits the imaging of a cell’s genome with its full 3D structure, including the actions of different genes, without requiring extensive preparations of materials.
Finally, a new step in cell imaging may let genetic function and epigenetic researchers more closely study the genes of various organisms and push forward the scientific knowledge of genetics by huge strides.
All thanks, as usual, to CRISPR-Cas9.