CRISPR/Cas taught reversible edits

The new system attacks the "letters" of RNA

Ekaterina Ryumina, "The Attic"

A group of scientists from the USA led by Feng Zhang, one of the pioneers of CRISPR/Cas editing (he was the first to show how the system works in human cells), expanded the range of CRISPR/Cas capabilities by teaching it to correct RNA sequences. A few years ago, they managed to force the system to attack adenine nucleotides, now cytosine ones are next in line. Such editing allows you to change the activity of the proteins encoded by them, not the genes.

The CRISPR/Cas system is usually used for genome editing, i.e. DNA. However, it can also be adapted to RNA editing by taking instead of the "ninth" CRISPR-associated protein – Cas13, which binds specifically to RNA. In 2017, Feng Zhang's group developed the REPAIR (RNA editing for programmable A to I(G) re-placement) system, a CRISPR/Cas13 system in which the ADAR2 domain is "sewn" to the "toothless" Cas13 (dCas13, an inactivated protein that can only recognize the sequence, but not cut it). This domain is used for editing nitrogenous bases, that is, replacing one "letter" in the text of a nucleic acid with another. The REPAIR system specialized in the conversion of adenine nucleotide (A) into inosine (analogues of guanine, G), but was powerless against other types of nucleotides.

Now Feng Zhang's group has taken up the editing of cytosine nucleotides. The authors of the work (Abudayyeh et al., A cytosine deaminase for programmable single-base RNA editing) obtained the necessary protein deaminase from the already used ADAR2 by controlled mutagenesis. The resulting deaminase contained 16 mutations and worked exclusively with double-stranded RNA. Just like in the REPAIR system, it was attached to dCas13. The new system is able to replace cytosine nucleotides (C) with uracyl (Y) and therefore was named RESCUE (R NA E dieting for S pecific C to UE xchange).

The next step was to test the effectiveness of the system on human cell culture. The targets for editing were places on the RNA, the change of which should lead to the activity of proteins: depending on the amino acids located in certain parts of the protein molecule, a "black mark" is more or less likely to be attached to the protein, leading to its cleavage. Therefore, by changing the RNA sequence, it is possible to change the amino acid composition and affect the stability of the protein in the cell and, consequently, its activity. In these experiments, the percentage of edited RNAs ranged from 5% to 42%.

The resulting ADAR2 retained the ability to deaminate adenosine with the formation of inositol, therefore, under certain conditions, the RESCUE system can turn both G into C and A into I, but the efficiency of this process is low – 15% and 5%, respectively. In addition, there is also inappropriate editing, which was partially reduced by once again "conjuring" ADAR2 with the help of controlled mutagenesis.

RESCUE expands the tools of molecular biologists, because it allows you to make reversible changes in the work of the cell: not to "break" the whole genome, but to pointwise influence the activity of its individual products. The reversibility and short duration of such edits makes Zhang's new tool attractive for experiments on manipulating the expression profile of the human genome for medical purposes.

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