Search

EP-4735590-A1 - MODIFIED TYPE I CRISPR COMPONENTS WITH ENHANCED GENE EDITING ACTIVITY

EP4735590A1EP 4735590 A1EP4735590 A1EP 4735590A1EP-4735590-A1

Abstract

The present invention relates to components and systems for modifying nucleic acids and gene expression. In particular, the present invention relates to engineered Cas proteins, fusion proteins and systems including the engineered Cas proteins, and methods for recruiting effector domains to target nucleic acids, modulating expression of a target gene, and altering a target nucleic acid sequence.

Inventors

  • ZHANG, YAN
  • CHEN, Jue
  • HOU, Zhonggang

Assignees

  • The Regents of The University of Michigan

Dates

Publication Date
20260506
Application Date
20240627

Claims (20)

  1. 1. An engineered Cas protein from a Type I-C CRISPR- Associated Complex for Anti-viral Defense (Cascade) complex having at least 70% identity to a wild-type protein and comprising one or more substitutions of a glutamate residue or aspartate residue within 10A of a nucleic acid bound by the Cascade complex with a positively charged amino acid.
  2. 2 The engineered Cas protein of claim 1, wherein the positively charged amino acid is arginine, histidine, or lysine.
  3. 3 The engineered Cas protein of claim 1 or 2, wherein the positively charged amino acid is arginine.
  4. 4 The engineered Cas protein of any of claims 1-3, wherein the wild-type protein is a Neisseria lactamica type I-C Cas protein.
  5. 5 The engineered Cas protein of any of claims 1-4, wherein the wild-type protein is a Cas8 protein comprising an amino acid sequence of SEQ ID NO: 1 and the one or more substitutions are selected from residues: E76, E230, E235, D268, D387, E391, D396, E426, E479, E484, D495, E524, E526, or combinations thereof, relative to SEQ ID NO: 1.
  6. 6 The engineered Cas protein of claim 5, wherein the one or more substitutions comprise: E76R, E230R, E235R, D268R, D387R, E391R, D396R, E426R, E479R, E484R, D495R, E524R, E526R, or combinations thereof, relative to SEQ ID NO: 1.
  7. 7 The engineered Cas protein of claim 5 or 6, wherein the one or more substitutions are selected from residues: E235/D387, E235/E391, D387/E391, or E235/D387/E391, relative to SEQ ID NO: 1.
  8. 8 The engineered Cas protein of any of claims 5-7, wherein the one or more substitutions comprise: E235R/D387R, E235R/E391R, D387R/E391R, or E235R/D387R/E391R, relative to SEQ ID NO: 1.
  9. 9 The engineered Cas protein of any of claims 1-4, wherein the wild-type protein is a Cas5 protein comprising an amino acid sequence of SEQ ID NO: 2 and the one or more substitutions are selected from residues: E18, E22, E69, E76, E84, D85, or combinations thereof, relative to SEQ ID NO: 2.
  10. 10 The engineered Cas protein of claim 9, wherein the one or more substitutions comprise: E18R, E22R, E69R, E76R, E84R, D85R, or combinations thereof, relative to SEQ ID NO: 2.
  11. 11. The engineered Cas protein of any of claims 1-4, wherein the wild-type protein is a Cas7 protein comprising an amino acid sequence of SEQ ID NO: 3 and the one or more substitutions are selected from residues: D23, D25, E69, D78, E90, D108, E144, E155, D157, E160, D163, or combinations thereof, relative to SEQ ID NO: 3.
  12. 12. The engineered Cas protein of claim 11, wherein the one or more substitutions comprise: D23R, D25R, E69R, D78R, E90R, D108R, E144R, E155R, D157R, E160R, D163R, or combinations thereof, relative to SEQ ID NO: 3.
  13. 13. The engineered Cas protein of claim 11 or 12, wherein the one or more substitutions are selected from residues: E90/D78, D78/E160, E90/E160, D78/E90/E160, relative to SEQ ID NO: 3.
  14. 14. The engineered Cas protein of any of claims 11-13, wherein the one or more substitutions comprise: E90R/D78R, D78R/E160R, E90R/E160R, D78R/E90R/E160R, relative to SEQ ID NO: 3.
  15. 15. The engineered Cas protein of any of claims 1-4, wherein the wild-type protein is a Casl 1 protein comprising an amino acid sequence of SEQ ID NO: 4 and the one or more substitutions are selected from residues: E22, E27, D38, E67, E69, or combinations thereof, relative to SEQ ID NO: 4.
  16. 16. The engineered Cas protein of claim 15, wherein the one or more substitutions comprise: E22R, E27R, D38R, E67R, E69R, or combinations thereof, relative to SEQ ID NO: 4.
  17. 17. A fusion protein comprising a Type I Cas protein of any of claims 1-16 and at least one effector domain.
  18. 18. The fusion protein of claim 17, wherein the at least one effector domain comprises a transcription activator, a transcription repressor, a base editor, an epigenetic modifier, or a combination thereof.
  19. 19. A system comprising: Cas3, or a nucleic acid encoding thereof; one or more engineered Cas protein of any of claims 1-16, a fusion protein of claims 17-18, and/or one or more nucleic acids encoding thereof; and at least one guide RNA (gRNA), wherein each gRNA is configured to hybridize to a portion of a target nucleic acid sequence.
  20. 20. The system of claim 19, wherein the one or more Type I Cas protein comprises a Cas8 protein comprising an amino acid sequence having one or more substitutions selected from: E235R, D387R, E391R, or combinations thereof, relative to SEQ ID NO: 1.

Description

MODIFIED TYPE I CRISPR COMPONENTS WITH ENHANCED GENE EDITING ACTIVITY FIELD [001] The present invention relates to components and systems for modifying nucleic acids and gene expression. In particular, the present invention relates to engineered Cas proteins, fusion proteins and systems including the engineered Cas proteins, and methods for recruiting effector domains to target nucleic acids, modulating expression of a target gene, and altering a target nucleic acid sequence. CROSS-REFERENCE TO RELATED APPLICATIONS [002] This application claims the benefit of U.S. Provisional Application No. 63/510,425, filed June 27, 2023, the content of which is herein incorporated by reference in its entirety. SEQUENCE LISTING STATEMENT [003] The content of the electronic sequence listing titled UM_42152_601_SequenceListing.xml (Size: 42,561 bytes; and Date of Creation: June 24, 2024) is herein incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [004] This invention was made with Government support under GM137833 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND [005] CRISPR-Cas systems employ diverse RNA-guided nucleases to help microbes fend off bacteriophages and other mobile genetic elements. These systems have also been developed into tools that revolutionized genome engineering. Type I system is the most widespread and diversified type of CRISPR, which accounts for >50% of all identified CRISPR systems in nature, ten times more abundant than the currently widely used CRISPR/Cas9. Type I CRISPR is further classified into eight subtypes (I- A through I-F, I-Fv, and LG) based on their cas gene composition. The Type I CRISPR DNA interference machinery consists of an RNA-guided Cascade complex for target site recognition and a helicase- nuclease enzyme Cas3 for processive target DNA degradation. Unlike widely used CRISPR/Cas9 systems that create a double-strand DNA break locally at the target site, Type I CRISPR creates targeted large chromosomal deletions in human cells. This is especially useful for the removal of disease-causing genomic loci such as toxic repeat expansions, large parasitic sequences, or integrated viral genomes. Furthermore, because Type I CRISPR can create a spectrum of large chromosomal deletions ranging from a few hundred base pairs to up to 30-100 kb, it holds great potential as a screening tool to dissect key regulatory elements (e.g., enhancers, insulators, or repressors) along the mammalian genome. However, Type I based gene editing tools are associated with large target to target variations in editing efficiency, especially for base editing. SUMMARY [006] Provided herein are engineered Cas protein from a Type I CRISPR-Associated Complex for Antiviral Defense (Cascade) complex. In some embodiments, the engineered Cas protein is from a Type I-C Cascade complex. In some embodiments, the engineered Cas protein has at least 70% identity to a wildtype protein and comprises one or more substitutions of a glutamate residue or aspartate residue within 10A of a nucleic acid bound by the Cascade complex with a positively charged amino acid. [007] In some embodiments, the positively charged amino acid is arginine, histidine, or lysine. In some embodiments, the positively charged amino acid is arginine. [008] In some embodiments, the wild-type protein is a Neisseria lactamica (Nla) type I-C Cas protein. [009] In some embodiments, the wild-type protein is a Cas8 protein comprising an amino acid sequence of SEQ ID NO: 1 and the one or more substitutions are selected from residues: E76, E230, E235, D268, D387, E391, D396, E426, E479, E484, D495, E524, E526, or combinations thereof, relative to SEQ ID NO: 1. In some embodiments, the one or more substitutions comprise: E76R, E230R, E235R, D268R, D387R, E391R, D396R, E426R, E479R, E484R, D495R, E524R, E526R, or combinations thereof. In some embodiments, the one or more substitutions are selected from residues: E235/D387, E235/E391, D387/E391, or E235/D387/E391. In some embodiments, the one or more substitutions comprise: E235R/D387R, E235R/E391R, D387R/E391R, or E235R/D387R/E391R. [010] In some embodiments, the wild-type protein is a Cas5 protein comprising an amino acid sequence of SEQ ID NO: 2 and the one or more substitutions are selected from residues: El 8, E22, E69, E76, E84, D85, or combinations thereof, relative to SEQ ID NO: 2. In some embodiments, the one or more substitutions comprise: E18R, E22R, E69R, E76R, E84R, D85R, or combinations thereof. [OH] In some embodiments, the wild-type protein is a Cas7 protein comprising an amino acid sequence of SEQ ID NO: 3 and the one or more substitutions are selected from residues: D23, D25, E69, D78, E90, D108, E144, E155, D157, E160, D163, or combinations thereof, relative to SEQ ID NO: 3. In some embodiments, the one or more substitutions comprise: D23R, D25R, E69R, D78R, E90R, D108R, E144R, E155R, D157R, E160