RNA expression modulating or editing method via regulation of Cas13 protein
View Patent ↗The present invention relates to a RNA expression modulating or editing method through chemical or optogenetic regulation of Cas13 protein activity. Specifically, in order to regulate the activity of Cas13 in the CRISPR-Cas13 system, a fragment of the Cas13 protein was generated to enable recombination, and a chemogenetically or optogenetically bindable protein was linked to each fragment. And, it was confirmed that the Cas13 protein can be activated by treatment of a low molecular weight compound or irradiation of light. Accordingly, the method of the present invention can be effectively used for disease treatment by regulating RNA expression or editing RNA mutations related to disease.
1 . A method for regulating the activity of Cas13 protein, comprising a step of treating a first fragment containing the N-terminal segment of Cas13 protein and a first regulatory protein; and a second fragment containing a second regulatory protein and the C-terminal segment of Cas 13 protein with a compound;
wherein the N-terminal segment of the Cas13 protein is a segment from the N-terminus of the Cas 13 protein to the 272nd, 286th, 351st, 480th or 928th amino acid of the Cas13 protein, and wherein the C-terminal segment of the Cas13 protein is a segment from the 273rd, 287th, 352nd, 481st or 929th amino acid of the Cas13 protein to the C-terminus of the Cas13 protein.
2 . The method for regulating the activity of Cas13 protein according to claim 1 , wherein the compound treatment induces dimerization of the first regulatory protein and the second regulatory protein.
3 . The method for regulating the activity of Cas13 protein according to claim 1 , wherein a linker is inserted between the N-terminal segment of Cas13 protein and the first regulatory protein, and between the second regulatory protein and the C-terminal segment of Cas13 protein.
4 . The method for regulating the activity of Cas13 protein according to claim 3 , wherein the linker includes glycine (G) and serine(S).
5 . The method for regulating the activity of Cas13 protein according to claim 1 , wherein the Cas13 protein is at least one selected from the group consisting of Cas13a, Cas13b, Cas13c and Cas13d.
6 . The method for regulating the activity of Cas13 protein according to claim 5 , wherein the Cas13 protein is PspCas13b protein.
7 . The method for regulating the activity of Cas13 protein according to claim 1 , wherein the compound is rapamycin.
8 . The method for regulating the activity of Cas13 protein according to claim 1 , wherein the combination of the first regulatory protein and the second regulatory protein is FKBP-FRB.
9 . A method for modulating the expression of a target RNA comprising the following steps:
1) preparing a vector comprising a construct containing a sequence encoding the N-terminal segment of Cas13 protein and a sequence encoding a first regulatory protein; and a vector comprising a construct containing a second fragment comprising a sequence encoding a second regulatory protein and a sequence encoding the C-terminal segment of Cas13 protein;
2) injecting the vector above and crRNA (CRISPR RNA) capable of hybridizing to the target RNA sequence into mammalian cells; and
3) treating the cells with a compound;
wherein the N-terminal segment of the Cas13 protein is a segment from the N-terminus of the Cas13 protein to the 272nd, 286th, 351st, 480th or 928th amino acid of the Cas13 protein, and
wherein the C-terminal segment of the Cas13 protein is a segment from the 273rd, 287th, 352nd, 481st or 929th amino acid of the Cas13 protein to the C-terminus of the Cas13 protein.
10 . The method for modulating the expression of a target RNA according to claim 9 , wherein the crRNA includes a guide sequence and a direct repeat sequence capable of hybridizing to the target RNA sequence.
11 . The method for modulating the expression of a target RNA according to claim 9 , wherein the vector is a virus or a plasmid.
12 . The method for modulating the expression of a target RNA according to claim 11 , wherein the virus is adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, modified vaccinia virus ankara (MVA), herpes simplex virus or baculovirus.