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US-20260125666-A1 - METHODS AND COMPOSITIONS FOR IMPROVING CYTOSINE BASE EDITOR GENOME EDITING SPECIFICITY AND EFFICIENCY

US20260125666A1US 20260125666 A1US20260125666 A1US 20260125666A1US-20260125666-A1

Abstract

The present invention is directed toward improved materials and methods related to cytosine and adenosine base editing of genomic DNA.

Inventors

  • Erin Brettmann
  • Fuqiang Chen
  • Graeme Garvey

Assignees

  • SIGMA-ALDRICH CO. LLC

Dates

Publication Date
20260507
Application Date
20231205

Claims (20)

  1. 1 . A composition suitable for modifying a cytosine residue in a DNA sequence, comprising: a single stranded DNA binding domain (SSB), a deaminase, a catalytically modified Cas protein, a guide RNA and, optionally, one or more free and/or fused uracil glycosylase inhibitors (UGI) to form a base editing RNP complex optionally with UGI.
  2. 2 . The composition of claim 1 , wherein the SSB is linked directly to said deaminase.
  3. 3 . The composition of claim 1 , wherein the SSB is linked indirectly to said deaminase.
  4. 4 . The composition of claim 1 , wherein said SSB is not covalently linked to any of the deaminase, the catalytically modified Cas protein, the guide RNA or the optional UGI.
  5. 5 . The composition of claim 1 , wherein said deaminase is covalently linked to the catalytically modified Cas protein.
  6. 6 . The composition of claim 1 , wherein said one or more UGI are not covalently linked to any of the deaminase, the catalytically modified Cas protein or the guide RNA.
  7. 7 . The composition of claim 1 , wherein the one or more SSB are from viruses.
  8. 8 . The composition of claim 1 , wherein the one or more SSB are from prokaryotes.
  9. 9 . The composition of claim 1 , wherein the one or more SSB are from eukaryotes.
  10. 10 . The composition of claim 1 , wherein said deaminase is a cytosine deaminase.
  11. 11 . The composition of claim 1 , wherein said an adenosine deaminase.
  12. 12 . The composition of claim 1 , wherein the catalytically modified Cas protein contains a catalytically inactive RuvC nuclease domain.
  13. 13 . The composition of claim 1 , wherein the catalytically modified Cas protein contains a catalytically inactive RuvC nuclease domain and a catalytically inactive HNH nuclease domain.
  14. 14 . The composition of claim 1 , wherein the catalytically modified Cas protein is a Cas9.
  15. 15 . The composition of claim 1 , wherein the catalytically modified Cas protein is a Cas12.
  16. 16 . At least one nucleic acid encoding one or more of the SSB, the deaminase, the catalytically modified Cas protein, the guide RNA and, optionally, the one or more free and/or fused UGI of the composition of claim 1 .
  17. 17 . At least one expression vector comprising a nucleic acid encoding at least one or more of the SSB, the deaminase, the catalytically modified Cas protein, the guide RNA and, optionally, the one or more free and/or fused UGI of the composition of claim 1 .
  18. 18 . The composition of claim 1 further comprising a Nuclear Location Sequence (NLS).
  19. 19 . A composition suitable for modifying a cytosine residue in a DNA sequence, comprising: a deaminase, a catalytically modified Cas protein, a guide RNA and one or more free UGI to form a base editing RNP complex with free UGI.
  20. 20 . The composition of claim 19 , wherein said deaminase is covalently linked to the catalytically modified Cas protein.

Description

CROSS REFERENCE TO RELATED APPLICATIONS The present application is a U.S. National Stage Application of International Application No. PCT/US2023/082569, filed Dec. 5, 2023, which claims the benefit of priority of U.S. provisional patent application No. 63/386,187, filing date Dec. 6, 2022, the entire content of each of which is incorporated herein by reference. SEQUENCE LISTING The present application contains a Sequence Listing that has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. The XML copy, created on Dec. 6, 2023, is named P22-236-WO-PCT_SL, and is 75.3 kilobytes in size. BACKGROUND Targeted genome modification is a powerful tool for genetic manipulation of DNA, including the manipulation of eukaryotic cells, embryos and animals. For example, exogenous sequences can be integrated at targeted genomic locations and/or specific endogenous DNA (e.g., chromosomal) sequences can be deleted, inactivated or modified. CRISPR-Cas9 genome editing systems have become a widespread method for introducing genetic modifications into a diverse range of cell types and organisms. While simple double-stranded DNA cleavage by Cas9 allows for gene inactivation through the formation of insertions and deletions, precision editing can be achieved through homology-directed repair (HDR) or base editing. The rate of installation of the desired edit is often quite challenging by HDR, the donor DNA can be toxic in some cell types, and the requirement for double-stranded DNA breaks (DSBs) introduces the risk of unintended deleterious repair outcomes. In contrast, base editing utilizes a deaminase domain to install a C to T or an A to G substitution at the target site without the need for a donor DNA. For cytosine base editors (CBEs), the cytosine base is deaminated to uracil, which is “read” as thymine by DNA polymerase, resulting in the installation of cytosine-to-thymine substitution. Furthermore, base editing utilizes a CRISPR effector, such as Cas9 and Cas12a, with fully or partially inactive DNA cleavage function, which prevents the formation of DSBs. Since its first publication by Komor, et al. (Nature, 2016), cytosine base editing has been widely used in diverse organisms from bacteria to humans and is being explored for correction of pathogenic mutations in a therapeutic context. The cytosine-base editing system of Komor utilizes a catalytically dead Cas9 (dCas9) that contains Asp10Ala and His840Ala mutations that inactivate its nuclease activity while still retaining its ability to bind DNA in a guide RNA-programmed manner without cleaving the DNA backbone. As mentioned above, the deamination of cytosine is catalyzed by cytosine deaminases and results in the conversion of a cytosine to an uracil. Uracil has the base-pairing properties of thymine and, upon replication or repair, creates an A-T base pair. However, there still exist certain drawbacks in this editing technology that need to be addressed. One of the drawbacks is the so-called “bystander editing,” wherein a neighboring cytosine residue other than the intended one within the R-loop is converted to thymine, causing an off-target effect. Several efforts to address this issue have been previously attempted with some success. These include the use of rigid peptide linkers between a deaminase and a Cas9 nickase to narrow the window of editing and thus minimize bystander editing (Tan, et al., Nat. Commun., 2019) and protein engineering on the deaminase domain to restrict the C to T editing to certain nucleotide motifs (Gehrke, et al., Nat Biotechnol, 2018; Kim, et al., Nat Biotechnol, 2017) or to reduce the frequency of editing on multiple cytosine residues within the editing window (Jin, et al., Molecular Cell, 2020). However, each of these approaches carries its own limitations. Another issue associated with cytosine base editing is the undesired excision of the uracil intermediate during the process of C to T conversion. Uracil does not naturally occur in DNA. The most common causes of the presence of deoxyuridine in DNA are misincorporation in place of deoxythymidine and spontaneous deamination of cytosine, both of which are mutagenic. Therefore, the presence of deoxyuracil will lead to DNA damage response by uracil DNA glycosylase, resulting in excision of the uracil base. Translesion synthesis at the abasic site can lead to the installation of undesired cytosine-to-adenine or cytosine-to-guanine substitutions. Abasic sites are also prone to DNA strand breakage and, in the context of CBEs that contain Cas9 nickase activity, this can lead to double-stranded DNA breaks (DSBs), which frequently result in DNA insertions and deletions. To address these undesired repair events that might occur in cytosine base editing, the second-generation cytosine base editor (BE2) (Komor, et al., Nature, 2016) carries a phage-derived uracil glycosylase inhibitor (UGI) covalently linked to the C terminus of Cas9, which was sho