Shredder instead of scissors
CRISPR cascade crumbled DNA in human cells
Daria Spasskaya, N+1
Geneticists have adapted the Cascade/Cas3 CRISPR system of the first type for use in human cells, in which not one, but many proteins are used to recognize and cut DNA. As reported in an article in Molecular Cell Dolan et al., Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas, due to the ability of the Cas3 protein to make numerous incisions in extended sections of DNA, this system can be used to create large deletions in the human genome.
CRISPR systems, which have been discovered as a component of bacterial "immunity" against viruses, are very diverse in nature, and include both systems with one effector protein and several. The functions of recognizing a given sequence and cutting DNA in systems of the second type are performed by a single protein, which makes them especially attractive for biotechnology. This includes a system that includes the Cas9 nuclease, which is currently actively used to edit the genomes of a wide variety of organisms. However, such systems make up no more than 10 percent of the known CRISPR systems.
The most common CRISPR systems of the first class and the first type in nature include several effectors. The functions of sequence recognition here are played by the Cascade multiunit complex, and the cut in the DNA is made by the helicase-nuclease Cas3. The Cascade/Cas3 complex is intensively studied in bacteria, but it has not yet been used in eukaryotic cells.
Researchers from Cornell University and the University of Michigan decided to test how the most studied Cascade from the thermophilic bacterium Thermobifida fusca will behave in human embryonic cells (these cells were chosen as a "healthy" cell model). To deliver the complex to cells, scientists expressed all seven Cascade subunits in bacteria with a nuclear localization signal sewn to them, then mixed them with guide RNA and Cas3, and delivered them to cells using electroporation. In order for the complex to work at 37 degrees, a mutation was introduced into one of the subunits.
Diagram of the Cascade/Cas3 operon in the bacterial genome (figures from the article in Molecular Cell).
The efficiency of editing was tested on the example of reporter genes – green and red fluorescent proteins. It turned out that, despite the complexity of the assembly of the complex, it works well in human cells, accurately recognizes the specified sequences and makes breaks in the overlying DNA sequence. The complex introduced a gap in about ten percent of the cells, the maximum editing efficiency for the selected guide RNAs was 13 percent. However, in the case of another gene in the HAP1 cancer cell line, the editing efficiency was already 30-60 percent.
One of the goals of the authors of the work was to clarify the mechanism of the Cas3 protein. Unlike Cas9, it does not make a single break at the recognition site, but "arrives" at the right place on Cascade, then detaches and travels along the DNA far enough. It turned out that in eukaryotic cells, as a result of the work of the complex, quite large deletions are formed with a size of several hundred to tens of thousands of base pairs.
Apparently, Cas3, passing through DNA, introduces several single- or double-stranded breaks, thus cutting a sufficiently long DNA sequence into pieces.
Of course, if we talk about practical application in human cells, Cascade/Cas3 is not suitable for precise editing, the authors of the work note. However, more than 90 percent of the human genome consists of non-coding sequences, and in order to study their functions, it is often necessary to remove sufficiently long sections of chromosomes. In this case, Cascade could replenish the genetic toolkit for eukaryotes.
In addition to the most studied Cas9 protein, scientists proposed using other proteins of CRISPR systems of the second class – Cas12a for editing the human genome (Cpf1), Cas13a, which can turn off genes at the mRNA level, and the recently discovered "small" analogue of Cas9 – CasX.
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