Compositions comprising nucleic acids and methods of using the same
The disclosure relates to compositions comprising and methods for chemical modification of single guide RNA (sgRNA), tracrRNA and/or crRNA used individually or in combination with one another or Cas system components. Compositions comprising modified ribonucleic acids have been designed with chemical modification for even higher efficiency as unmodified native strand of sgRNA. Administration of modified ribonucleic acids will allow decreased immune response when administered to a subject, increased stability, increased editing efficiency and facilitated in vivo delivery of sgRNA via various delivery platforms. The disclosure also relates to methods of decreasing off-target effect of CRISPR and a CRISPR complex.
1. A composition comprising a nucleic acid molecule comprising from a 5′ to 3′ orientation: a Cas protein-binding domain and a transcription terminator domain wherein:
a) the Cas-protein binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 11;
b) one or more nucleotides in the Cas-protein binding domain has a 2′-O-methyl modification and the nucleotides at positions 2, 3, 4, 23, 24, 25, 27, 31, 38 and 42 of SEQ ID NO: 11 are not chemically modified;
c) the transcription terminator domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 114; and
d) the transcription terminator domain comprises at least one phosphorothioate modification and the nucleotides at positions 1, 2, 3, and 6 of SEQ ID NO: 114 are not chemically modified.
2. The composition of claim 1 , wherein the Cas protein-binding domain is from about 30 to about 55 nucleotides in length and the transcription terminator domain is from about 17 to about 45 nucleotides in length.
3. The composition of claim 1 , wherein the Cas-protein binding domain comprises at least one 2′ O-fluoro modified nucleotide.
4. The composition of claim 1 , wherein the transcription terminator domain comprises at least one 2′ O-fluoro modified nucleotide, and a conserved nucleotide at position 1, 2, 3 and/or 6 of SEQ ID NO:114.
5. The composition of claim 1 , wherein the Cas protein-binding domain consists of from 38 to 42 contiguous ribonucleotides; and wherein the transcription terminator domain consists of from 38 to 42 contiguous ribonucleotides.
6. The composition of claim 1 , wherein the nucleic acid molecule is at least 70% homologous to SEQ ID NO:2.
7. The composition of claim 1 , wherein the nucleic acid molecule is at least 70% homologous to SEQ ID NO:4.
8. The composition of claim 1 further comprising:
a nucleic acid comprising a regulatory element operable in a eukaryotic cell operably linked to at least one nucleic acid encoding a Cas protein or functional fragment thereof.
9. The composition of claim 8 further comprising a lipid or polymer that encapsulates the nucleic acid molecule.
10. The composition of claim 1 , wherein the nucleic acid molecule consists of from 80 to 101 nucleotides.
11. The composition of claim 1 , wherein the Cas-protein binding domain comprises the nucleic acid sequence of SEQ ID NO: 11.
12. The composition of claim 1 , wherein the nucleotides at positions 2, 3, 4, 23, 24, 25, 27, 31, 38 and 42 of SEQ ID NO: 11 are conserved.
13. The composition of claim 1 , wherein the transcription terminator domain comprises at least one 2′-O-methyl modified nucleotide.
14. The composition of claim 1 , wherein the transcription terminator domain comprises three phosphorothioate modifications.
15. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.
16. A kit comprising: (a) one or more vectors comprising: a first regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Cas protein or a functional fragment thereof and (b) the composition of claim 1 .
17. A composition comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule and the second nucleic acid molecule are sufficiently complementary to form a duplex and in combination comprise a series of contiguous domains from a 5′ to 3′ orientation: a Cas protein-binding domain and a transcription terminator domain, and wherein:
a) the Cas-protein binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 11;
b) one or more nucleotides in the Cas-protein binding domain has a 2′-O-methyl modification and the nucleotides at positions 2, 3, 4, 23, 24, 25, 27, 31, 38 and 42 of SEQ ID NO: 11 are not chemically modified;
c) the transcription terminator domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 114; and
d) the transcription terminator domain comprises at least one phosphorothioate modification, and the nucleotides at positions 1, 2, 3, and 6 of SEQ ID NO: 114 are not chemically modified.
18. The composition of claim 17 , wherein the composition further comprises a nucleic acid comprising a regulatory element operable in a eukaryotic cell operably linked to at least one nucleic acid encoding a Cas protein or functional fragment thereof.
19. The composition of claim 18 , wherein the composition further comprises a lipid or polymer that encapsulates the nucleic acid molecule.
20. A pharmaceutical composition comprising the composition of claim 17 and a pharmaceutically acceptable carrier.