Composition for genome editing using CRISPR/CPF1 system and use thereof
The present invention relates to a composition for genome editing using a CRISPR/Cpf1 system and a use thereof and, more particularly, to a composition for genome editing comprising: a CRISPR RNA (crRNA) including a guide sequence capable of hybridizing with a target nucleotide sequence, and a uridine repeat sequence connected to the 3′-end of the guide sequence, or a DNA encoding the same; and a Cpf1 protein or a DNA encoding the same, a method for genome editing using the same, a method for construction of a genetically modified organism, and a genetically modified organism. The present invention can increase an indel efficiency and decrease off-target activity in genome editing of eukaryotic cells using the CRIPSPR/Cpf1 system and thus can easily construct a genetically modified cell or genetically modified animal or plant having a desired gene inserted thereinto (knock-in) or deleted therefrom (knock-out).
1. A composition for genome editing comprising:
a CRISPR RNA (crRNA) comprising a guide sequence complementary to a target nucleotide sequence and a uridine repeat sequence linked to a 3′-end of the guide sequence, or a DNA encoding the crRNA; and
a Cpf1 protein or a DNA encoding the Cpf1 protein,
wherein the length of guide sequence is 18 to 24 nt.
2. The composition for genome editing of claim 1 , wherein the uridine repeat sequence is a nucleotide sequence represented by (U a V) n U b , wherein a and b are an integer from 2 to 20, n is an integer from 1 to 5, and V is adenine (A), cytosine (C), or guanine (G).
3. The composition for genome editing of claim 2 , wherein V is A.
4. The composition for genome editing of claim 2 , wherein n is 1.
5. The composition for genome editing of claim 2 , wherein (U a V) n U b is U 4 AU 4 .
6. The composition for genome editing of claim 1 , wherein the length of guide sequence is 18 to 23 nt.
7. The composition for genome editing of claim 1 , wherein the Cpf1 protein is derived from one or more microorganisms selected from the group consisting of Candidatus genus, Lachnospira genus, Butyrivibrio genus, Peregrinibacteria, Acidominococcus genus, Porphyromonas genus, Prevotella genus, Francisella genus, Candidatus methanoplasma , and Eubacterium genus microorganisms.
8. The composition for genome editing of claim 1 , wherein the composition comprises a PCR amplicon comprising the DNA encoding the crRNA and a recombinant vector comprising the DNA encoding the Cpf1 protein.
9. The composition for genome editing of claim 1 , wherein the composition comprises the DNA encoding the crRNA and the DNA encoding the Cpf1 protein.
10. The composition for genome editing of claim 9 , wherein the DNA encoding the crRNA and the DNA encoding the Cpf1 protein are inserted either in one recombinant vector or separate vectors.
11. The composition for genome editing of claim 1 , wherein the composition is applied for genome editing in a eukaryotic cell or a eukaryotic organism.
12. The composition for genome editing of claim 11 , wherein the eukaryotic organism is a eukaryotic animal or a eukaryotic plant.
13. A method for genome editing, the method including: introduction of the composition of claim 1 into an isolated cell or organism.
14. The method of claim 13 , wherein the introduction of the composition is achieved by local injection, microinjection, electroporation, or a lipofection method.
15. The method of claim 13 , wherein the cell or organism is an isolated eukaryotic cell or a eukaryotic non-human organism.
16. The method of claim 15 , wherein the eukaryotic cell is a cell isolated from a eukaryotic animal or a eukaryotic plant.