Modified Guide RNAs
AssigneeIntellia Therapeutics, Inc.
InventorsAmy Madison Rhoden Smith, David V. Morrissey, Walter Strapps
Life of this patent
Estimating the term…
Abstract
This disclosure relates to modified single and dual guide RNAs having improved in vitro and in vivo activity in gene editing methods.
Claims217
A single guide RNA (sgRNA) comprising a 5′ end modification and one or more modification in one or more of:
- a. the upper stem region;
- b. the hairpin 1 region;
- and
- c. the hairpin 2 region,
- wherein the 5′ end modification comprises at least two phosphorothioate (PS) linkages within the first seven nucleotides at the 5′ end of the 5′ terminus.
A single guide RNA (sgRNA) comprising 2′-O-Me modified nucleotides at the following positions:
- a. the first three nucleotides at the 5′ end of the 5′ terminus;
- b. LS1, LS6, LS7, LS8, LS11, and/or LS12 in the lower stem region;
- c. B1 and/or B2 in the bulge region;
- d. each nucleotide in the upper stem region;
- e. N16, N17, and/or N18 in the nexus region;
- f. each nucleotide in the hairpin 1 region;
- g. each nucleotide in the hairpin 2 region;
- and
- h. the last four nucleotides at the 3′ terminus.
The sgRNA of any one of the preceding claims, wherein LS9 and LS10 are modified, e.g. with 2′-F or 2′-OMe.
The sgRNA of any one of the preceding claims, wherein N15, N16, N17, and N18 are modified, e.g. with 2′-F or 2′-OMe.
The sgRNA of any one of the preceding claims, wherein H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15 are modified with 2′-F.
The sgRNA of any one of the preceding claims, wherein the second to last, third to last, and fourth to last nucleotides at the 3′ terminus are modified with 2′-F.
A single guide RNA (sgRNA) comprising 2′-F modified nucleotides at the following positions:
- a. LS9 and LS10 in the lower stem region;
- b. N15, N16, N17, and N18 in the nexus region;
- and
- c. H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15 in the hairpin 2 region.
A single guide RNA (sgRNA) comprising:
- a. 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ end of the 5′ terminus;
- b. 2′-O-Me modified nucleotides at US1-US12;
- c. 2′-O-Me modified nucleotides at H1-1-H1-12;
- d. 2′-O-Me modified nucleotides at H2-1-H2-15;
- and
- e. 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ end of the 3′ terminus.
A single guide RNA (sgRNA) comprising:
- a. 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ end of the 5′ terminus;
- b. 2′-F modified nucleotides at LS1-LS6;
- c. 2′-O-Me modified nucleotides at US1-US12;
- d. 2′-O-Me modified nucleotides at H1-1-H1-12;
- e. a 2′-O-Me modified nucleotide between hairpin 1 and hairpin 2;
- f. 2′-O-Me modified nucleotides at H2-1-H2-15;
- and
- g. 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ end of the 3′ terminus.
A single guide RNA (sgRNA) comprising:
- a. 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ end of the 5′ terminus;
- b. 2′-F modified nucleotides at LS2-LS5;
- c. 2′-O-Me modified nucleotides at LS1 and LS6;
- d. 2′-O-Me modified nucleotides at US1-US12;
- e. 2′-O-Me modified nucleotides at H1-1-H1-12;
- f. a 2′-O-Me modified nucleotide between hairpin 1 and hairpin 2;
- g. 2′-O-Me modified nucleotides at H2-1-H2-15;
- and
- h. 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ end of the 3′ terminus.
A single guide RNA (sgRNA) comprising
- a. 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ end of the 5′ terminus;
- b. 2′-O-Me modified nucleotides at US1-US12;
- c. 2′-O-Me modified nucleotides at LS7, LS8, LS11, and LS12;
- d. 2′-O-Me modified nucleotides at H1-1-H1-12;
- e. a 2′-O-Me modified nucleotide between hairpin 1 and hairpin 2;
- f. 2′-O-Me modified nucleotides at H2-1-H2-15;
- and
- g. 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ end of the 3′ terminus.
A single guide RNA (sgRNA) comprising
- a. 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ end of the 5′ terminus;
- b. 2′-O-Me modified nucleotides at US1-US12;
- c. 2′-O-Me modified nucleotides at LS7, LS8, LS11, and LS12;
- d. 2′-F modified nucleotides at LS9 and LS10;
- e. 2′-O-Me modified nucleotides at H1-1-H1-12;
- f. a 2′-O-Me modified nucleotide between hairpin 1 and hairpin 2;
- g. 2′-O-Me modified nucleotides at H2-1-H2-15;
- and
- h. 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ end of the 3′ terminus.
A single guide RNA (sgRNA) comprising
- a. 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ end of the 5′ terminus;
- b. 2′-O-Me modified nucleotides at US1-US12;
- c. 2′-O-Me modified nucleotides at LS8, LS10, and LS12;
- d. 2′-O—F modified nucleotides at LS7, LS9, and LST11;
- e. 2′-O-Me modified nucleotides at H1-1-H1-12;
- f. a 2′-O-Me modified nucleotide between hairpin 1 and hairpin 2;
- g. 2′-O-Me modified nucleotides at H2-1-H2-15;
- and
- h. 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ end of the 3′ terminus.
A single guide RNA (sgRNA) comprising:
- a. 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ end of the 5′ terminus;
- b. 2′-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11, and LS12
- c. 2′-O-Me modified nucleotides at US1-US12;
- d. 2′-O-Me modified nucleotides at H1-1-H1-12;
- e. a 2′-O-Me modified nucleotide between hairpin 1 and hairpin 2;
- f. 2′-O-Me modified nucleotides at H2-1-H2-15;
- and
- g. 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ end of the 3′ terminus.
A single guide RNA (sgRNA) comprising
- a. 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ end of the 5′ terminus;
- b. 2′-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11, and LS12;
- c. 2′-F modified nucleotides at LS9 and LS10;
- d. 2′-O-Me modified nucleotides at US1-US12;
- e. 2′-O-Me modified nucleotides at H1-1-H1-12;
- f. a 2′-O-Me modified nucleotide between hairpin 1 and hairpin 2;
- g. 2′-O-Me modified nucleotides at H2-1-H2-15;
- and
- h. 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ end of the 3′ terminus.
A single guide RNA (sgRNA) comprising
- a. 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ end of the 5′ terminus;
- b. 2′-O-Me modified nucleotides at US1-US12;
- c. 2′-O-Me modified nucleotides at H1-1-H1-12;
- d. a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin 2;
- e. 2′-O-Me modified nucleotides at H2-1-H2-8;
- f. 2′-F modified nucleotides at H2-9-H2-15;
- g. 2′-F modified nucleotides at the second from last, third from last, and fourth from last nucleotide at the 3′ terminus;
- and
- h. a 2′-O-Me modified nucleotide at the last nucleotide at the 3′ end of the 3′ terminus.
A single guide RNA (sgRNA) comprising
- a. 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ end of the 5′ terminus;
- b. 2′-O-Me modified nucleotides at US1-US12;
- c. 2′-O-Me modified nucleotides at H1-2, H1-4, H1-6, H1-8, H1-10, and H1-12;
- d. 2′-F modified nucleotides at H1-1, H1-3, H1-5, H1-7, H1-9, and H1-11;
- e. a 2′-F modified nucleotide between hairpin 1 and hairpin 2;
- f. 2′-F modified nucleotides at H2-2, H2-4, H2-6, H2-8, H2-10, H2-12;
- and H2-14;
- g. 2′-O-Me modified nucleotides at H2-1, H2-3, H2-5, H2-7, H2-9, H2-11;
- H2-13, and H2-15;
- h. 2′-F modified nucleotides at the second from last, and fourth from last nucleotide at the 3′ terminus;
- and
- i. 2′-O-Me modified nucleotide at the third from last, and last nucleotide at the 3′ end of the 3′ terminus.
A single guide RNA (sgRNA) comprising
- a. 2′-O-Me modified nucleotides LS8, LS10, LS12, H1-2, H1-4, H1-6, H1-8, H1-10, H1-12, H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13, and H2-15;
- and
- b. 2′-F modified nucleotides at LS7, LS9, LS11;
- H1-1, H1-3, H1-5, H1-7, H1-9, H1-11, H1-13, H2-2, H2-4, H2-6, H2-8, H2-10, H2-12, and H2-14.
An sgRNA comprising any of SEQ ID Nos:
- 228-332, including the modifications of Table 4.
An sgRNA comprising any of SEQ ID Nos:
- 235-240, 265-285, and 309-329, including the modifications of Table 4.
An sgRNA comprising SEQ ID No:
- 240.
An sgRNA comprising SEQ ID No. 240, including the modifications of Table 4.
An sgRNA comprising SEQ ID No:
- 242, including the modifications of Table 4.
An sgRNA comprising the modifications of SEQ ID No:
- 242, as depicted in Table 4.
An sgRNA comprising SEQ ID No:
- 358.
An sgRNA comprising nucleic acids having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to the nucleic acids of any one of SEQ ID Nos:
- 235-240, 265-285, and 309-329, wherein the modification at each nucleotide of the sgRNA that corresponds to a nucleotide of the reference sequence identifier in Table 4, is identical to or equivalent to the modification shown in the reference sequence identifier in Table 4.
A guide RNA comprising 2′-O-Me modifications at each nucleotide in the upper stem region.
A guide RNA comprising 2′-O-Me modifications at one, two, three, or four, five, six, seven, eight, nine, ten, or eleven of the following nucleotides:
- US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12.
A guide RNA comprising 2′-O-Me modifications at 50% or more of the nucleotides in the hairpin 1 and hairpin 2 regions.
A guide RNA comprising 2′-O-Me modifications at each nucleotide in the upper stem region and 2′-O-Me modifications at 50% or more of the nucleotides in the hairpin 1 and hairpin 2 regions.
A guide RNA comprising or consisting of 2′-O-Me modifications at each nucleotide starting at H2-1 or H2-2 through the last nucleotide of the 3′ terminus.
A guide RNA comprising or consisting of a 2′-O-Me modification at LS1 and/or LS6 and no modification at LS2-LS5.
A guide RNA comprising 2′-O-Me modified nucleotides at 2 or more of LS8, LS9, LS10, LS11, and/or LS12.
A guide RNA comprising 2′-O-Me modifications at up to 50% of the nucleotides in the nexus region.
A guide RNA comprising 2′-O-Me modifications at four or more nucleotides in the nexus region.
A guide RNA comprising 2′-O-Me modified nucleotides at ten or more nucleotides in the nexus region.
A guide RNA comprising 2′-O-Me modifications at 50% or more of the nucleotides in the bulge region.
A guide RNA comprising 2′-O-Me modified nucleotides at three or more nucleotides in the bulge region.
A guide RNA comprising the nucleotides of SEQ ID NO:
- 357 with the modification pattern of the guide RNA of any preceding claim.
The guide RNA comprising the nucleotides of SEQ ID NO:
- 356 with the modification pattern of the guide RNA of any preceding claim.
A single guide RNA (sgRNA) comprising or consisting of 2′-O-Me modified nucleotides at:
- a. the first three nucleotides at the 5′ end of the 5′ terminus;
- b. each nucleotide in the upper stem region;
- c. each nucleotide in the hairpin 1 region;
- d. the nucleotide between hairpin 1 and hairpin 2;
- e. each nucleotide in the hairpin 2 region;
- and
- f. the last four nucleotides at the 3′ end of the 3′ terminus.
An sgRNA having the modification pattern shown in SEQ ID NO:
- 350, 351, 352, or 353, as depicted in Table 4.
A single guide RNA (sgRNA) comprising or consisting of 2′-O-Me modified nucleotides at:
- a. the first three, four, five, or seven nucleotides at the 5′ end of the 5′ terminus;
- b. each nucleotide in the upper stem region;
- c. each nucleotide in the hairpin 1 region;
- d. the nucleotide between hairpin 1 and hairpin 2;
- e. each nucleotide in the hairpin 2 region;
- and
- f. the last four nucleotides at the 3′ end of the 3′ terminus.
A single guide RNA (sgRNA) comprising or consisting of 2′-O-Me modified nucleotides at:
- a. the first three, four, five, or seven nucleotides at the 5′ end of the 5′ terminus;
- b. each of nucleotides US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11 and US12;
- c. each of nucleotides H1-1, H1-2, H1-3, H1-4, H1-5, H1-6, H1-7, H1-8, H1-9, H1-10, H1-11, and H1-12;
- d. the nucleotide between hairpin 1 and hairpin 2;
- e. each of nucleotides H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-8, H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15;
- and
- f. the last four nucleotides at the 3′ end of the 3′ terminus.
An sgRNA having the modification pattern of SEQ ID NO:
- 350, 351, 352, or 353 as shown in Table 4.
A crispr RNA (crRNA) comprising one or more modifications within one or more of the following regions:
- a. the first five nucleotides at the 5′ end of the 5′ terminus;
- b. the lower stem region;
- c. the bulge region;
- d. the upper stem region;
- and
- e. the last five nucleotides at the 3′ end of the crRNA.
A crispr RNA (crRNA) comprising 2′-O-Me modified nucleotides at:
- a. LS1 and LS6 in the lower stem region;
- and
- b. each nucleotide in the upper stem region.
A crispr RNA (crRNA) comprising 2′-F modified nucleotides at:
- a. LS1, LS2, and LS6 in the lower stem region;
- and
- b. each nucleotide in the bulge region.
A crRNA comprising any one of SEQ ID Nos:
- 1-187, including the modifications of Table 4.
A crRNA comprising any one of SEQ ID Nos:
- 19-31, 53-73, 104-130, and 161-187, including the modifications of Table 4.
A crRNA comprising nucleic acids having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to any one of SEQ ID Nos:
- 19-31, 53-73, 104-130, and 161-187, wherein the modification at each nucleotide of the crRNA that corresponds to a nucleotide of the reference sequence identifier in Table 4, is identical to or equivalent to the modification shown in the reference sequence identifier in Table 4.
A tracr RNA (trRNA) comprising one or more modifications within one or more of the following regions:
- a. the first five nucleotides at the 5′ end of the 5′ terminus;
- b. the upper stem region;
- c. the bulge region;
- d. the lower stem region;
- e. the nexus region;
- f. the hairpin 1 region;
- g. the hairpin 2 region;
- and
- h. the last five nucleotides at the 3′ end of the 3′ terminus.
A tracr RNA (trRNA) comprising 2′-O-Me modified nucleotides at:
- a. each nucleotide in the upper stem;
- b. B1 and/or B2 within the bulge region;
- c. N3, N4, N5, N15, N16, N17, and/or N18 in the nexus region;
- d. each nucleotide in the hairpin 1 region;
- and
- e. each nucleotide in the hairpin 2 region.
A trRNA comprising any one of SEQ ID Nos:
- 188-227, including the modifications of Table 4.
A trRNA comprising nucleic acids having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to any one of SEQ ID Nos:
- 188-227, wherein the modification at each nucleotide of the trRNA that corresponds to a nucleotide of the reference sequence identifier in Table 4, is identical to or equivalent to the modification shown in the reference sequence identifier in Table 4.
A dual guide comprising a crRNA and a trRNA, wherein the crRNA comprises any one of SEQ ID Nos:
- 1-187 as depicted in Table 4, and wherein the trRNA comprises the nucleic acids of any one of SEQ ID Nos:
- 188-227 as depicted in Table 4.
The crRNA of any one of claims 737-163 for use in preparing a medicament for treating a disease or disorder.
Dependencies are inferred from the claim text.