IP Library › Granted Patent US 12,264,314
Granted Patent B1
US 12,264,314 · App. 18/392,905 · Granted Apr 1, 2025

CasZ compositions and methods of use

Inventors: Jennifer A. Doudna (Berkeley, CA); David Burstein (Berkeley, CA); Janice S. Chen (Berkeley, CA); Lucas B. Harrington (Berkeley, CA); David Paez-Espino (Walnut Creek, CA); Jillian F. Banfield (Berkeley, CA)
Assignee: The Regents of the University of California
C12N15/102C12N9/1007C12N15/86C12N2750/14143
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Quick Facts
Patent No.
US 12,264,314
App. No.
18/392,905
Granted
Apr 1, 2025
Kind
B1
Abstract

Provided are compositions and methods that include one or more of: (1) a “CasZ” protein (also referred to as a CasZ polypeptide), a nucleic acid encoding the CasZ protein, and/or a modified host cell comprising the CasZ protein (and/or a nucleic acid encoding the same); (2) a CasZ guide RNA that binds to and provides sequence specificity to the CasZ protein, a nucleic acid encoding the CasZ guide RNA, and/or a modified host cell comprising the CasZ guide RNA (and/or a nucleic acid encoding the same); and (3) a CasZ transactivating noncoding RNA (trancRNA) (referred to herein as a “CasZ trancRNA”), a nucleic acid encoding the CasZ trancRNA, and/or a modified host cell comprising the CasZ trancRNA (and/or a nucleic acid encoding the same).

Claims (39)

1. A method of modifying a target locus of interest comprising delivering to said locus:

a) an engineered polypeptide or a nucleic acid encoding the engineered polypeptide, wherein the engineered polypeptide is a variant of the polypeptide according to SEQ ID NO: 3, wherein the variant has reduced nucleic acid cleavage activity relative to the polypeptide according to SEQ ID NO: 3, wherein the variant comprises an amino acid sequence at least 80% identical to SEQ ID NO: 3, and wherein the variant comprises at least one amino acid substitution at a position selected from a group consisting of D326, E422, R490, and D510 of SEQ ID NO: 3; and

b) a guide nucleic acid or DNA molecule encoding the guide nucleic acid, the guide nucleic acid comprising:

i) a first sequence that is bound by the polypeptide, wherein the first sequence comprises a nucleotide sequence that is at least 90% identical to residues 28-37 of SEQ ID NO: 53, and

ii) a second sequence that hybridizes to a target sequence of a target nucleic acid, wherein the first sequence is located 5′ of the second sequence.

2. The method of claim 1 , wherein the engineered polypeptide recognizes a PAM of 5′-TTTN-3′, wherein T is thymine and N is any nucleotide.

3. The method of claim 1 , wherein the engineered polypeptide recognizes a PAM of 5′-TTTR-3′, wherein T is thymine and R is selected from guanine and adenine.

4. The method of claim 1 , wherein the engineered polypeptide is at least 90% identical to SEQ ID NO: 3.

5. The method of claim 1 , wherein the engineered polypeptide is fused to a heterologous protein, and wherein modifying the target locus comprises introducing, removing, or altering an epigenetic modification of the target locus.

6. The method of claim 1 , wherein the engineered polypeptide is linked to a methyltransferase.

7. The method of claim 1 , wherein the engineered polypeptide is catalytically inactive.

8. The method of claim 1 , wherein the engineered polypeptide comprises at least one amino acid substitution at an aspartic acid residue or a glutamic acid residue relative to a protein that is 100% identical to SEQ ID NO: 3.

9. The method of claim 8 , wherein the at least one amino acid substitution is located in a RuvC domain.

10. The method of claim 1 , wherein the engineered polypeptide comprises at least one amino acid substitution that is selected from an amino acid corresponding to D326A, E422A, D510A, and R490A of SEQ ID NO: 3, or any combination thereof.

11. The method of claim 1 , comprising modifying the target locus in a cell.

12. The method of claim 11 , wherein the cell is a eukaryotic cell.

13. The method of claim 12 , wherein the eukaryotic cell is selected from a stem cell, a germ cell, a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a myofibroblast, a cardiac myoblast, a skeletal myoblast, and a T cell.

14. The method of claim 1 , comprising modifying the target locus in a human subject.

15. The method of claim 14 , comprising delivering the nucleic acid encoding the engineered polypeptide and the guide nucleic acid to the human subject via a lipid nanoparticle.

16. The method of claim 15 , wherein the nucleic acid encoding the engineered polypeptide comprises a messenger RNA.

17. The method of claim 14 , comprising delivering the nucleic acid encoding the engineered polypeptide and the DNA molecule encoding the guide nucleic acid to the human subject via an adeno-associated viral vector.

18. The method of claim 1 , comprising delivering a donor nucleic acid to said locus.

19. A composition comprising:

a) an engineered polypeptide or a nucleic acid encoding the engineered polypeptide, wherein the engineered polypeptide is a variant of the polypeptide according to SEQ ID NO: 3, wherein the variant has reduced nucleic acid cleavage activity relative to the polypeptide according to SEQ ID NO: 3, wherein the variant comprises an amino acid sequence at least 80% identical to SEQ ID NO: 3, and wherein the variant comprises at least one amino acid substitution at a position selected from a group consisting of D326, E422, R490, and D510 of SEQ ID NO: 3; and

b) a guide nucleic acid or DNA molecule encoding the guide nucleic acid, the guide nucleic acid comprising:

i) a first sequence that is bound by the polypeptide, wherein the first sequence comprises a nucleotide sequence that is at least 90% identical to residues 28-37 of SEQ ID NO: 53, and

ii) a second sequence that hybridizes to a target sequence of a target nucleic acid, wherein

the first sequence is located 5′ of the second sequence.

20. The composition of claim 19 , wherein the engineered polypeptide recognizes a PAM of 5′-TTTN-3′, wherein T is thymine and N is any nucleotide.

21. The composition of claim 19 , wherein the engineered polypeptide recognizes a PAM of 5′-TTTR-3′, wherein T is thymine and R is selected from guanine and adenine.

22. The composition of claim 19 , wherein the engineered polypeptide is at least 90% identical to SEQ ID NO: 3.

23. The composition of claim 19 , wherein the engineered polypeptide is fused to a heterologous protein.

24. The composition of claim 19 , wherein the engineered polypeptide is linked to a methyltransferase.

25. The composition of claim 19 , wherein the engineered polypeptide is catalytically inactive.

26. The composition of claim 19 , wherein the engineered polypeptide further comprises at least one amino acid substitution at an aspartic acid residue or a glutamic acid residue relative to a protein that is 100% identical to SEQ ID NO: 3.

27. The composition of claim 19 , wherein the engineered polypeptide comprises at least one amino acid substitution that is selected from an amino acid corresponding to D326A, E422A, D510A, and R490A of SEQ ID NO: 3, or any combination thereof.

28. The composition of claim 19 , further comprising a lipid nanoparticle or an adeno-associated viral vector.

29. The composition of claim 19 , wherein the nucleic acid encoding the engineered polypeptide comprises a messenger RNA.

30. The composition of claim 19 , further comprising a donor nucleic acid.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 9, 2024
From: DOUDNA, JENNIFER A.; BURSTEIN, DAVID; CHEN, JANICE S.; HARRINGTON, LUCAS B.; PAEZ-ESPINO, DAVID; BANFIELD, JILLIAN F.
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 067053/0207 →
Continuity (5)
Continuation 17935521 · Sep 26, 2022
Continuation 16896711 · Jun 9, 2020
Continuation 16694720 · Nov 25, 2019
Continuation PCTUS2018058545 · Oct 31, 2018
Provisional Application 62580395 · Nov 1, 2017
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Transposase, JGI Accession No. 3300025308.a:Ga0209211_100536734, Nov. 9, 2021, 2 pages. [cited by applicant]
Transposase, JGI Accession No. 3300025317.a:Ga0209541_100096836, Nov. 9, 2021, 2 pages. [cited by applicant]
Transposase, JGI Accession No. 3300025323.a:Ga0209542_1000010711, Nov. 9, 2021, 2 pages. [cited by applicant]
Transposase, JGI Accession No. 3300025323.a:Ga0209542_10000107204, Nov. 9, 2021, 2 pages. [cited by applicant]
Transposase, JGI Accession No. (Taxon ID:Gene ID) 3300013125.a:Ga0172369_100104642, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase, JGI Accession No. (Taxon ID:Gene ID) 3300013130.a:Ga0172363, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase, JGI Accession No. (Taxon ID:Gene ID) 3300025317.a:Ga0209541_100217848, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase, JGI Accession No. (Taxon ID:Gene ID) 3300025323.a:Ga0209542_100271699, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase, JGI Accession No. (Taxon ID:Gene ID) 3300013127.a:Ga0172365_100044211, Nov. 5, 202, 2 pages. [cited by applicant]
Transposase, JGI Accession No. (Taxon ID:Gene ID) 3300013123.a:Ga0172368_100090142, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase, JGI Accession No. (Taxon ID:Gene ID) 3300025317.a:Ga0209541_1000016152, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase, JGI Accession No. (Taxon ID:Gene ID) 3300025317.a:Ga0209541_1000046133, Nov. 5, 2021, 1 pages. [cited by applicant]
Transposase and inactivated derivatives, JGI Accession No. (Taxon ID:Gene ID) 3300005573.a:Ga0078972_100101520, Nov. 5, 2021, 1 pages. [cited by applicant]
Transposase and inactivated derivatives, JGI Accession No. 3300002502.a:C687J35174_100538264, Sep. 1, 2021, 2 pages. [cited by applicant]
Transposase and inactivated derivatives, JGI Accession No. (Taxon ID:Gene ID) 3300002966.a:JG|24721J44947_100297402, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase and inactivated derivatives, JGI Accession No. (Taxon ID:Gene ID) 3300002502.a:C687J35174_100502431, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase and inactivated derivatives, JGI Accession No. (Taxon ID:Gene ID) 3300001245.a:JG|12048J13642_102012859, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase and inactivated derivatives, JGI Accession No. (Taxon ID:Gene ID) 3300001245.a:JG|12048J13642_102012865, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase and inactivated derivatives, JGI Accession No. (Taxon ID:Gene ID) 3300001256.a:JG|12210J13797_103875826, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase and inactivated derivatives, JGI Accession No. (Taxon ID:Gene ID) 3300001256.a:JG|12210J13797_103875833, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase and inactivated derivatives, JGI Accession No. (Taxon ID: Gene ID) 3300000353.a:ElkS_mat_MD6ADRAFT_10068983, Nov. 5, 2021, 2 pages. [cited by applicant]
Transposase and inactivated derivatives, JGI Accession No. 3300002105.a:C687J26635_100228363, Nov. 9, 2021, 2 pages. [cited by applicant]
Sawamura, et al., “Generation of biallelic F0 mutants in medaka using the CRISPR/Cas9 system”, Genes Cells, Aug. 2017, 22(8):756-763. [cited by applicant]
Wright, et al., “Rational design of a split-Cas9 enzyme complex”, PNAS, Mar. 10, 2015, 112(10):2984-2989. [cited by applicant]
Harrington et al., (2020) “A scoutRNA Is Required for Some Type V CRISPRCas Systems.” Molecular Cell, vol. 79, pp. 416-424. [cited by applicant]