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US 2026/0286329 A1Published application
Compositions, Methods, and Systems for Genomic Editing
AssigneeIntellia Therapeutics, Inc.
InventorsSabin Mulepati, Christian Dombrowski, Jasmine Josephine Bonanno, Kritika Hanamshet, Anita Ramachandran, Darwin Yang, Sung Hee Choi, Pei Wang, Weijun Chen, Rubina Giare Parmar
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Abstract
The present disclosure relates to compositions, methods, and systems for genomic editing. In certain aspects, the compositions, methods, and systems provided herein can incorporate a SpyCas9 nickase, a DNA-dependent DNA polymerase, and/or a template guide RNA for template-based genome editing applications.
Claims310
A system comprising a DNA-dependent DNA polymerase, a SpyCas9 nickase (nCas9), and a template guide RNA (tgRNA), wherein the tgRNA comprises a first polynucleotide and a second polynucleotide, wherein:
- a) the first polynucleotide comprises a SpyCas9 guide RNA comprising, from 5′ to 3′:
- i) a spacer that is complementary to a target region of a target strand of a DNA duplex target nucleic acid;
- and
- ii) a scaffold;
- and
- b) the second polynucleotide is operably linked to the first polynucleotide and comprises, from 5′ to 3′:
- i) a template sequence that is at least 10 nucleotides in length and complementary to at least a portion of a non-target strand of the DNA duplex target nucleic acid, wherein the template sequence comprises a 3′ end and a 5′ end;
- and wherein the template comprises DNA nucleotides;
- and
- ii) a DNA-dependent DNA polymerase recruiting sequence (DRS) that is 6-20 nucleotides in length, optionally 10-14 nucleotides in length and having a 3′ end and a 5′ end;
- wherein the 5′ end of the DRS is covalently attached to the 3′ end of the template sequence, and wherein the DRS comprises a sequence that is complementary to at least 6 nucleotides of a DRS-complementary region of the non-target strand of the DNA duplex target nucleic acid, wherein the DRS comprises RNA nucleotides;
- and
- iii) wherein the DRS comprises at least one of:
- 1) a sequence not fully complementary to the DRS-complementary region of the non-target strand of the DNA duplex target nucleic acid;
- 2) a 1-5 nucleotide, optionally a 1-3 nucleotide 3′ terminal tail comprising a 3′ terminal modified uridine nucleotide;
- or
- 3) a 5′ terminal (D1) DNA nucleotide and a penultimate 5′ terminal (D2) RNA nucleotide wherein the 3′ end of the first polynucleotide is covalently linked to the 5′ end of the second polynucleotide by a phosphorothioate or phosphodiester linkage.
14 more elements
A template guide RNA (tgRNA), wherein the tgRNA comprises a first polynucleotide and a second polynucleotide, wherein:
- i) the first polynucleotide comprises a SpyCas9 guide RNA, comprising, from 5′ to 3′,
- 1. a spacer that is complementary to a target region of a target strand of a DNA duplex target nucleic acid;
- and
- 2. a scaffold;
- ii) the second polynucleotide is operably linked to the first polynucleotide and comprises, from 5′ to 3′:
- 1) a template sequence that is at least 10 nucleotides in length and complementary to at least a portion of a non-target strand of the DNA duplex target nucleic acid, wherein the template sequence comprises a 3′ end and a 5′ end;
- and wherein the template comprises DNA nucleotides;
- and
- 2) a DNA-dependent DNA polymerase recruiting sequence (DRS) having a 3′ end and a 5′ end;
- wherein the 5′ end of the DRS is covalently attached to the 3′ end of the template sequence, and wherein the DRS comprises a sequence that is complementary to at least 6 nucleotides of a DRS-complementary region of the non-target strand of the DNA duplex target nucleic acid, optionally wherein the DRS is 6-20 nucleotides in length, further optionally 6-17, 8-17, 10-12, 10-14, 10-16, or 12-14 nucleotides in length.
7 more elements
A system comprising:
- a) a first polynucleotide encoding a first polypeptide comprising a DNA-dependent DNA polymerase, or portion thereof, operably linked to a first split-intein;
- and
- b) a second polynucleotide encoding a second polypeptide comprising a SpyCas9 nickase (nCas9), or portion thereof, operably linked to a second split-intein that is complementary to the first split-intein, wherein the first split-intein and the second split-intein undergo trans-splicing reaction, resulting in the formation of a single fusion polypeptide comprising from N-terminus to C-terminus, (i) the DNA-dependent DNA polymerase and the nCas9, or (ii) the nCas9 and the DNA-dependent DNA polymerase.
A system comprising:
- (a) a DNA-dependent DNA polymerase and a SpyCas9 nickase (nCas9);
- and (b) at least one chromatin remodeler operably linked to the nCas9 and the DNA-dependent DNA polymerase.
A system comprising a DNA polymerase kappa (polK), a SpyCas9 nickase (nCas9), and a template guide RNA (tgRNA), wherein the tgRNA comprises a first polynucleotide and a second polynucleotide, wherein:
- a) the first polynucleotide comprises a SpyCas9 guide RNA comprising, from 5′ to 3′:
- i) a spacer that is complementary to a target region of a target strand of a DNA duplex target nucleic acid;
- and
- ii) a scaffold;
- and
- b) the second polynucleotide is operably linked to the first polynucleotide and comprises, from 5′ to 3′:
- i) a template sequence that is at least 10 nucleotides in length and complementary to at least a portion of a non-target strand of the DNA duplex target nucleic acid, wherein the template sequence comprises a 3′ end and a 5′ end;
- and wherein the template comprises DNA nucleotides;
- ii) a DNA-dependent DNA polymerase recruiting sequence (DRS) that is 6-18 nucleotides in length, optionally 8-18 or 10-12 nucleotides in length and having a 3′ end and a 5′ end;
- wherein the 5′ end of the DRS is covalently attached to the 3′ end of the template sequence, and wherein the DRS comprises a sequence that is complementary to at least 6 nucleotides of a DRS-complementary region of the non-target strand of the DNA duplex target nucleic acid, wherein the DRS comprises RNA nucleotides.
7 more elements
The system of any one of claims 335-337, wherein the polk comprises a mutation at G411 or E412 relative to SEQ ID NO:
- 1021, optionally wherein the polk comprises a mutation at G411 and E412.
A system comprising a T5 DNA polymerase (T5 pol), a SpyCas9 nickase (nCas9), and a template guide RNA (tgRNA), wherein the tgRNA comprises a first polynucleotide and a second polynucleotide, wherein:
- 1) the first polynucleotide comprises a SpyCas9 guide RNA comprising, from 5′ to 3′:
- a. a spacer that is complementary to a target region of a target strand of a DNA duplex target nucleic acid;
- and
- b. a scaffold;
- and
- 2) the second polynucleotide is operably linked to the first polynucleotide and comprises, from 5′ to 3′:
- a. a template sequence that is at least 10 nucleotides in length and complementary to at least a portion of a non-target strand of the DNA duplex target nucleic acid, wherein the template sequence comprises a 3′ end and a 5′ end;
- and wherein the template comprises DNA nucleotides;
- and
- b. a DNA-dependent DNA polymerase recruiting sequence (DRS) that is 6-20 nucleotides in length, optionally 10-16 or 12-14 nucleotides in length, and having a 3′ end and a 5′ end;
- wherein the 5′ end of the DRS is covalently attached to the 3′ end of the template sequence, and wherein the DRS comprises a sequence that is complementary to at least 6 nucleotides of a DRS-complementary region of the non-target strand of the DNA duplex target nucleic acid comprising nucleotides 5-10 5′ of the PAM in the non-target strand of the DNA duplex target nucleic acid, wherein the DRS comprises RNA nucleotides.
8 more elements
A fusion polypeptide comprising a polK comprising GE411-412RV mutations relative to SEQ ID NO:
- 1021, a SpyCas9 nickase (nCas9), and a heterologous nuclear localization signal (NLS) or a nucleic acid encoding the fusion polypeptide.
A fusion polypeptide comprising a T5 pol comprising D164A and E166A mutations relative to SEQ ID NO:
- 1116, a SpyCas9 nickase (nCas9), a heterologous nuclear localization signal (NLS), and optionally a heterodimerization domain, or a nucleic acid encoding the fusion polypeptide thereof.
A method of producing a ligated template guide RNA (tgRNA), the method comprising:
- providing a first polynucleotide comprising a 3′ end comprising at least 10 nucleotides;
- providing a second polynucleotide comprising a 5′ end comprising at least 10 nucleotides;
- providing a splint oligonucleotide capable of annealing to the at least 10 nucleotides at the 3′ end of the first polynucleotide and the at least 10 nucleotides at the 5′ end of the second polynucleotide;
- and
- providing a ligase to ligate the 3′ end of the first polynucleotide to the 5′ end of the second polynucleotide thereby forming a ligated tgRNA;
- wherein the ligated tgRNA comprises a SpyCas9 guide RNA, a template sequence, and a DNA-dependent DNA polymerase recruiting sequence (DRS).
3 more elements
A method of producing a ligated template guide RNA (tgRNA), the method comprising:
- providing a first polynucleotide comprising a 3′ end comprising at least 10 nucleotides;
- providing a second polynucleotide comprising a 5′ end comprising at least 10 nucleotides and a 3′ end comprising at least 10 nucleotides;
- providing a third polynucleotide comprising a 5′ end comprising at least 10 nucleotides.
- providing a first splint oligonucleotide capable of annealing to the at least 10 nucleotides at the 3′ end of the first polynucleotide and the at least 10 nucleotides at the 5′ end of the second polynucleotide;
- providing a second splint oligonucleotide capable of annealing to the at least 10 nucleotides at the 3′ end of the second polynucleotide and the at least 10 nucleotides at the 5′ end of the third polynucleotide;
- and
- providing one or more ligases to ligate (i) the 3′ end of the first polynucleotide to the 5′ end of the second polynucleotide and (ii) the 3′ end of the second polynucleotide to the 5′ end of the third polynucleotide thereby forming a ligated tgRNA;
- wherein the ligated tgRNA comprises a SpyCas9 guide RNA, a template sequence, and a DNA-dependent DNA polymerase recruiting sequence (DRS).
5 more elements
Dependencies are inferred from the claim text.