Granted Patent B2

US 12,234,454 · App. 15/331,792 · Granted Feb 25, 2025

Inventors: Aaron Andrew Smargon (Cambridge, MA); Feng Zhang (Cambridge, MA)

Assignees: THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY

C12N15/113C12N9/22C12N15/102C12N15/111C12N15/63C12N15/74C12Q1/6876G16B25/00G16B30/00G16B30/10G16B30/20C12N2310/20C12Q2600/156

Abstract

Claims (41)

1. A method for identifying and generating novel nucleic acid modifying effectors, comprising:

a computer-implemented method comprising:

(a) identifying putative nucleic acid modifying loci from a set of nucleic acid sequences, the set of nucleic acid sequences obtained from a genomic or metagenomic database and the nucleic acid sequences are long enough to encode a protein with a defined size limit greater than 700 amino acids, wherein the putative nucleic acid modifying loci: are a defined distance between 1 and 25kb of a CRISPR array, the CRISPR array being identified using a repeat or pattern finding analysis of the set of nucleic acid sequences, and

comprise only one sequence encoding a protein with a defined size limit greater than 700 amino acids;

(b) identifying candidate effector proteins that are the protein with greater than 700 amino acids in (a) and homologous proteins thereof;

(c) grouping the candidate effector proteins into subsets based on homology;

(d) selecting the subsets that have at least 10 candidate effector proteins and more than 50% of the candidate effector proteins within 10 kb of the CRISPR array,

(e) identifying a candidate set of novel nucleic acid modifying effectors proteins by selecting from one or more of the subsets selected in (d) based on one or more of the following:

subsets comprising loci of coding sequences for putative candidate effector proteins with no more than 90% homology matches to known protein domains relative to loci in other subsets,

subsets whose loci have same orientations as putative adjacent accessory proteins relative to effector proteins in other subsets,

subsets comprising candidate effector proteins with lower existing nucleic acid modifying classifications relative to other subsets,

subsets comprising loci with a lower proximity to known nucleic acid modifying loci relative to other subsets, and

total number of candidate effector proteins in each subset; and

generating nucleic acid molecules encoding one or more of the nucleic acid modifying proteins in the candidate set identified by the computer-implemented method; and

expressing the one or more nucleic acid modifying proteins from the generated nucleic acid molecules and performing one or more biochemical y assays that validate a level of nucleic acid modifying function of the one or more novel nucleic acid modifying proteins.

2. The method of claim 1 , wherein the genomic or metagenomic database comprises prokaryotic genomic or metagenomic sequences.

3. The method of claim 1 , wherein the defined distance from the CRISPR array is between 10kb and 25 kb.

4. The method of claim 1 , wherein the defined distance is within 10 kb of the CRISPR array.

5. The method of claim 1 , wherein said repeat or pattern finding analysis comprises PILER-CR.

6. The method of claim 1 , wherein said grouping step is based, at least in part, on results of a domain homology search or an HHpred protein domain homology search.

7. The method of claim 6 , wherein the domain homology search or HHpred protein domain homology search is performed using a defined threshold that is a BLAST nearest-neighbor cut-off value of 0 to 1e-7.

8. The method of claim 1 , further comprising a filtering step that includes only loci with coding sequences for putative proteins of a size between 900 and 1800 amino acids.

9. The method of claim 1 , wherein said validation comprises PAM validation in bacterial colonies, in vitro cleavage assays, the Surveyor method, experiments in mammalian cells, PFS validation, or a combination thereof.

10. The method of claim 1 , further comprising preparing a non-naturally occurring or engineered composition for modifying a target nucleic acid sequence, the composition comprising one or more novel nucleic acid modifying effectors proteins.

11. The method of claim 10 , wherein the loci further comprise coding sequences for one or two small accessory proteins within 2 kb to 10kb of the CRISPR array.

12. The method of claim 11 , wherein the small accessory protein comprises multiple predicted transmembrane domains.

13. The method of claim 12 , wherein the small accessory protein comprises four predicted transmembrane domains.

14. The method of claim 11 , wherein the small accessory protein comprises at least one HEPN domain.

15. The method of claim 11 , wherein the small accessory protein comprises at least one HEPN domain and at least one transmembrane domain.

16. The method of claim 1 , wherein the one or more novel nucleic acid modifying effectors proteins is one or more Class 2 CRISPR effectors.

17. The method of claim 1 , wherein the one or more novel nucleic acid modifying effectors proteins have loci that the identified loci lack coding sequences for Cas1 or Cas2.

18. The method of claim 1 , wherein the single effector protein is greater than 900, or greater than 1100 amino acids in length.

19. The method of claim 18 , wherein the single effector comprises at least one HEPN domain.

20. The method of claim 19 , wherein the at least one HEPN domain is near an N- or C-terminus of the effector protein.

21. The method of claim 20 , wherein the at least one HEPN domain is located in an interior position of the effector protein.

22. The method of claim 19 , wherein the single large effector protein comprises a HEPN domain at the N- and C-terminus and two HEPN domains internal to the protein.

23. The method of claim 1 , wherein the loci comprise no additional coding sequence for proteins out to 25 kb from the CRISPR array.

24. The method of claim 1 , wherein the CRISPR array comprises direct repeat sequences comprising about 36 nucleotides in length.

25. The method of claim 24 , wherein the direct repeat comprises a GTTG/GUUG at the 5′ end that is reverse complementary to a CAAC at the 3′ end.

26. The method of claim 1 , wherein the CRISPR array comprises spacer sequences comprising about 30 nucleotides in length.

27. The method of claim 1 , wherein said loci lack a coding sequence for a small accessory protein.

Assignments (4)

Continuity (5)

References Cited (171)