Provided are a composition for ribonucleoprotein delivery, comprising a guide RNA free of 5′-terminal phosphates, and a method for ribonucleoprotein delivery, using the same.
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1. A composition for delivering an RNA-guided endonuclease ribonucleoprotein having decreased cytotoxicity into an organism, comprising a guide RNA free of a diphosphate residue and a triphosphate residue at a 5′ end thereof, wherein the guide RNA is at least one selected from the group consisting o
1. A composition for delivering an RNA-guided endonuclease ribonucleoprotein having decreased cytotoxicity into an organism, comprising a guide RNA free of a diphosphate residue and a triphosphate residue at a 5′ end thereof, wherein the guide RNA is at least one selected from the group consisting of CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and single guide RNA (sgRNA), andthe guide RNA free of a diphosphate residue and a triphosphate residue at a 5′ end thereof is chemically synthesized or prepared by removing a di- or triphosphate residue from three phosphate residues of the 5′ end after in-vitro transcription using a prokaryotic RNA polymerase. 2. The composition of claim 1, further comprising a Cas9 protein or a Cpf1 protein. 3. The composition of claim 2, wherein the Cas9 protein is derived from Streptococcus pyogenes. 4. The composition of claim 2, wherein the Cpf1 protein is derived from Parcubacteria bacterium, Peregrinibacteria bacterium, Acidaminococcus sp., Porphyromonas macacae, Lachnospiraceae bacterium, Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi, Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida, Candidatus Methanoplasma termitum, or Eubacterium eligens. 5. The composition of claim 1, wherein the organism is a eukaryotic cell, a eukaryotic animal, or a eukaryotic plant. 6. A method for delivering an RNA-guided endonuclease ribonucleoprotein having decreased cytotoxicity into an organism, comprising administering a mixture of a guide RNA free of both a diphosphate residue and a triphosphate residue at a 5′ end thereof, and an RNA-guided endonuclease into the organism, wherein the guide RNA is at least one selected from the group consisting of CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and single guide RNA (sgRNA), andthe guide RNA free of both a diphosphate residue and a triphosphate residue at a 5′ end thereof is chemically synthesized or prepared by removing a di- or triphosphate residue from three phosphate residues of the 5′ end after in-vitro transcription using a prokaryotic RNA polymerase. 7. The method of claim 6, wherein the RNA-guided endonuclease is a Cas9 protein or a Cpf1 protein. 8. The method of claim 7, wherein the Cas9 protein is derived from Streptococcus pyogenes. 9. The method of claim 7, wherein the Cpf1 protein is derived from Parcubacteria bacterium, Peregrinibacteria bacterium, Acidaminococcus sp., Porphyromonas macacae, Lachnospiraceae bacterium, Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi, Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida, Candidatus Methanoplasma termitum, or Eubacterium eligens. 10. The method of claim 6, wherein the prokaryotic RNA polymerase is a bacteriophage RNA polymerase. 11. The method of claim 10, wherein the bacteriophage RNA polymerase is at least one selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. 12. The method of claim 6, wherein the organism is a eukaryotic cell, a eukaryotic animal, or a eukaryotic plant. 13. A method for preparing a guide RNA having decreased cytotoxicity, comprising: (1) providing a guide RNA that is prepared through in vitro transcription in presence of a prokaryotic RNA polymerase; and(2) removing a di- or triphosphate residue from a 5′ end of the guide RNA,wherein the guide RNA is at least one selected from the group consisting of CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and single-stranded guide RNA (sgRNA). 14. The method of claim 13, wherein the step of removing the di- or triphosphate residue from the 5′ end of the guide RNA is performed by treatment with a phosphatase. 15. The method of claim 14, wherein the phosphatase is at least one selected from the group consisting of calf intestinal alkaline phosphatase (CIP), shrimp alkaline phosphatase (SAP), and Antarctic phosphatase. 16. The method of claim 13, wherein the prokaryotic RNA polymerase is a bacteriophage RNA polymerase. 17. The method of claim 16, wherein the bacteriophage RNA polymerase is at least one selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. 18. A method for preparing an RNA-guided endonuclease ribonucleoprotein having decreased cytotoxicity, comprising mixing an RNA free of both a diphosphate residue and a triphosphate residue at a 5′ end thereof with a Cas9 protein or a Cpf1 protein. 19. The method of claim 18, wherein the guide RNA free of both a diphosphate residue and a triphosphate residue at the 5′ end thereof is either chemically synthesized or is prepared using the method of claim 13.
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