CRISPR/Cas13 instead of RNA interference

The new CRISPR/Cas13 system helped turn off genes in human cells

Daria Spasskaya, N+1

The newly discovered Cas13a protein – a component of the CRISPR/Cas system that is capable of purposefully destroying RNA molecules – was forced to work in human cells. Scientists from The Massachusetts Institute of Technology has shown that using CRISPR/Cas13, it is possible to effectively destroy the mRNA of selected genes, thereby "turning off" their activity. The inactive form of Cas13a, which can only bind but not cleave RNA, was adapted by scientists to track the localization of RNA molecules inside the cell. The study is published in Nature (Abudayyeh et al., RNA targeting with CRISPR–Cas13).

Often, to assess the effect of a particular protein on some intracellular process, researchers need to turn off the gene that encodes it. This can be done in two ways – to irreversibly "break" the gene, that is, to introduce a mutation into the DNA that will disrupt its function, or to suppress its activity at the level of transcription, that is, synthesis of matrix RNA.

In particular, the DNA sequence can be changed using the CRISPR/Cas9 system. This system consists of the Cas9 protein, which cleaves DNA, and a short guide RNA that "tells" the protein where to make the cut. In the process of healing the incision in the cell DNA mutation is introduced into the gene sequence.

However, the efficiency of this process is not as high as we would like, besides, the gene under study may be so important for vital activity that its irreversible breakdown is incompatible with life. In such cases, gene transcription can be temporarily suppressed by RNA interference. This process is based on small RNA molecules complementary to the site inside the matrix RNA (they are called small interfering RNAs). A special protein complex recognizes the matrix RNA associated with small RNA and cleaves it.

The effectiveness of suppressing gene activity using RNA interference is usually less than one hundred percent, which makes it possible to work even with vital targets. However, this method also has disadvantages: firstly, some genes cannot be "silenced" even by RNA interference, and secondly, small RNAs do not bind very accurately to their target, which leads to the non-targeted destruction of mRNA.

Last year we wrote about the discovery by an international team, which included Russian scientists, of a new protein from the Cas family, which is capable of directionally splitting single-stranded RNA, not DNA. The protein was named C2c2, or Cas13a. In a paper published in the journal Science, the researchers managed to purposefully destroy the bacteriophage RNA in E. coli cells, as well as direct the degradation of the fluorescent protein mRNA. In addition, the new protein has been successfully used for in vitro detection of nucleic acids in a method called SHERLOCK.

In the new work, scientists have shown that Cas13a from the bacterium Leptotrichia wadei can be used in human cells as well as in plant cells (a protein from another species, Leptotrichia shahii, was tested in E. coli cells), and that the CRISPR/Cas13 system can effectively replace RNA interference.

To begin with, the authors optimized the nucleotide composition of the Cas13 gene so that the protein was effectively synthesized in eukaryotic cells, and "sewed" a sequence to it that ensures delivery to the nucleus. The efficiency of RNA cleavage was tested on two human cell lines and rice cells. The efficiency of shutting down the reporter gene Gluc encoding luciferase everywhere turned out to be quite high and amounted to about 75 percent.

Comparison of the efficiency and accuracy of the CRISPR/Cas13a system and the RNA interference system. The graph on the left shows the number of non-target (off-target) targets for samples used for RNA interference (shRNA) and for CRISPR-off genes (LwaCas13a). The graph on the right reflects the effectiveness of gene shutdown by different samples. Drawings from become in Nature.

After that, the authors tested the effectiveness of the system for suppressing the activity of several human own genes on fibroblasts and melanoma cells. For different genes, the shutdown efficiency ranged from 30 to 85 percent in several experiments, which on average corresponded to the effectiveness of control samples for RNA interference.

Using Cas13, the authors managed to significantly reduce the number of transcripts for genes that could not be suppressed using RNA interference (MALAT1 and XIST). An important advantage of CRISPR/Cas13 over interference was the binding sensitivity: compared with small interfering RNAs, for which many non-target targets were detected, there was no non-target binding at all for Cas13.

The introduction of mutations into the catalytic domain of the protein led to the fact that Cas13a stopped cutting RNA, and there was no decrease in the number of target transcripts. The ability of inactive mutant dCas13a to specifically bind RNA molecules has been used as a method for monitoring matrix RNAs in the cytoplasm. To do this, the dCas13a protein had to be modified in such a way that, in the absence of a target, it was concentrated in the nucleus and suppressed its own transcription. Using the example of the mRNA of the actin gene (ACTB), the authors showed that with the help of dCas13a, it is possible to detect the accumulation of these RNAs in the cytoplasm as part of stress granules – clusters that form in unfavorable conditions for the cell. In the presence of a guide RNA for the actin gene, dCas13a bound the mRNA of this gene and passed into the cytoplasm, and in the presence of a control RNA that did not direct it anywhere, it was detected in the nucleus.

Modified Cas13a can be used for mRNA monitoring. In order to localize the protein unrelated to the target in the nucleus, a DNA binding domain and a repressor domain (c) were sewn to it. In the presence of a guide RNA for the ACTB gene, the protein binds to the mRNA of the gene (d, f). The images obtained by fluorescence microscopy of Cas13a fused with a green fluorescent protein show that the mRNA of the ACTB gene tends to accumulate in the cytoplasm as part of stress granules. NT guide is a control guide RNA that does not correspond to any gene.

According to Konstantin Severinov, who participated in the discovery of a new protein, and an interview with whom we published on this occasion, the practical application of a new Cas protein with RNase activity in cells was hardly possible. However, his colleagues quickly showed that this was not the case. Time will tell whether Cas13 will become as popular and indispensable a laboratory tool as RNA interference and its "brother" protein Cas9.

Previously, scientists managed to get the Cas9 protein to bind to RNA and split it by artificially sewing RNase to it. Using a variant of this protein called RCas9, the authors were able to destroy clusters of microsatellite RNA in cells taken from patients with myodystrophy. 

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