CRISPR-Cas9 is a genome editing technology that is based on the immune system of bacteria and archaea.
The whole technology consists of two blocks:
Fig.1 CRISPR region: repeating regions alternate with distinct spacers
Once the foreign genetic elements have been integrated into CRISPR, they need to be translated into a suitable form to work with Cas. To do this, CRISPR sequences are transcribed (turned into RNA molecules) and cut into short RNA fragments. This form of RNA is called guide or guide (gRNA, gRNA) and contains unique sequences that are complementary to targets.
In short, sgRNA is a kind of “sight” or “guide rail” for the “scissors” Cas . Their combination allows targeted cutting of certain parts of the genome.
After the gRNA sequence binds to the target, the Cas9 nuclease begins its work – it introduces a double-strand break.
Currently, most research on genomic DNA editing uses Cas9 nuclease in combination with a single guide RNA (single guide RNA, sgRNA). It consists of variable crRNA (depends on the target sequence) and non-variable tracrRNA (which binds to Cas9).
But not all purposes require double-strand breaks. In order for Cas9 to cut only one strand or even become inactive, point mutations are introduced into it and then forms are possible: nickase ( nCas9 ) – cuts only one strand of DNA and “dead” (dead, dCas9) – binds to the sequence and does not cut anything. But such an inactive protein can be used to repress entire sets of genes or as a platform for creating complex regulatory complexes. For example, if an activating domain is attached to it, then the expression of target genes is activated. And by adding fluorescent labels, you can visualize different regions of chromosomes.
sgRNAs allow dCas9 activators to increase the expression of any gene in an organism’s genome. For this, dCas9 activation systems use transcription activators.
It is important to say here that overexpression is the increased activity of a gene, in which this gene produces an excess amount of the protein encoded in it.
Fig.2 Schematic representation of gene expression and overexpression
Until 2015, all studies of Cas9 activators were carried out in cell culture, and in vivo activation was not shown in any multicellular animal, so Chinese scientists decided for the first time to show how it all works already in Drosophila. They used dCas9-VPR (VPR transcription activator) and several sgRNAs on target genes. And it turned out that such a complex reliably works to activate the transcription factors Twist and Snail in cells, both individually and together. The authors even adapted the dCas9-VPR system for Gal4-UAS activation and showed that this approach can activate target genes in vivo at levels sufficient to generate dominant phenotypes1.
Or, for example, the use of a gene activator based on dCas9 made it possible to increase the expression of the laminin gene in the muscles of mice with type 1A congenital muscular dystrophy (MDC1A). To do this, Canadian scientists altered the expression of the Lama1 gene using dCas9, VP64 transcription activators, and a guide RNA for the Lama1 promoter . It turned out that the introduction of such a design to newborn mice generally prevents muscle destruction. At the same time, even if dCas9 is administered to adult mice with all the signs of the disease, they stop further development of the disease and improve mobility2.
And a group of scientists from Duke University created two lines of transgenic mice to regulate target genes using dCas9 in vivo . One line of Rosa26 mice: LSL-dCas9-p300 for gene activation, and the other Rosa26: LSL-dCas9-KRAB for gene repression. These mice allow researchers to change gene expression levels and observe how these changes affect the tissues and physiology of an entire living organism3.
In all the examples described above, the researchers used different transcription activators in their work. These transcription activators have protein domains or even entire proteins associated with dCas9 or sgRNA that help recruit important cofactors as well as RNA polymerase for target gene transcription. And we need RNA polymerase to get a protein from a gene, that is, for transcription.
One of the first known transcriptional activators, VP64, consists of four tandem copies of VP16. VP64 works as a strong transcriptional activator only when connected to a DNA-binding domain. In this construct, the VP64 transcription activator is attached to the C-terminus of dCas9. dCas9-VP64 is considered a “first generation” CRISPR activator and shows moderate levels of gene activation.
For higher levels of expression, VP64-p65-Rta, or dCas9-VPR, a modification of dCas9-VP64, has been developed. It turns out that all three transcription factors target the same gene, and this use of three transcription factors leads to an increase in the expression of target genes. In addition to increasing the expression of a particular gene, we can also use several sgRNAs and thus act on several genes at once in the same cell4.
Interestingly, one study in yeast used different sgRNAs to target different parts of a gene. And it turned out that dCas9-VPR can act as an activator or repressor, depending on its binding site in the genome. sgRNAs targeted to the promoter (a launching pad for transcription) allow dCas9-VPR to increase expression, while those targeted to the coding region of the gene lead to a decrease in expression5.
The dCas9-VPR system can also be actively used in Drosophila melanogaster cells , using it as a model organism.
SAM uses MS2, p65, and HSF1 proteins, and with their help, the dCas9-SAM system recruits various transcription factors that work synergistically to activate the gene of interest.
dCas9-SAM uses a modified sgRNA that has binding sites for the MS2 protein. Other transcription factors can bind to the MS2 protein without destroying the dCas9-sgRNA complex.
Fig. 5 Synergistic activation mediator (SAM)
When targeting individual genes, SAM shows the highest levels of gene activation compared to other CRISPR activators, making it a popular technique for gene activation experiments. However, in cases of multiplex gene regulation (simultaneous activation of several genes), SAM activation levels do not differ from other popular activation methods (VPR and SunTag)6.
The SunTag activator system uses the dCas9 protein modified to bind to SunTag. Instead of using one copy of VP64 for each dCas9, SunTag uses an array of repetitive peptides to bind to multiple copies of VP64. By having multiple copies of VP64 at each locus of interest, this allows more transcription factors to be recruited for each target gene.
Fig. 6 SunTag
SunTag performs better than first generation activators, but shows a lower activation rate than SAM.