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EP-4739779-A2 - GENE EDITING MEDIATED BY REVERSE TRANSCRIPTION OF A MODULAR RNA TEMPLATE ANCHORED BY A TAG GRNA AND USES THEREOF

EP4739779A2EP 4739779 A2EP4739779 A2EP 4739779A2EP-4739779-A2

Abstract

The present disclosure relates to gene editing, related systems, and uses thereof. The gene editing is mediated by reverse transcription of a modular RNA template anchored by a complementary sequence within a modified guide RNA.

Inventors

  • JIN, SHENGKAN

Assignees

  • Rutgers, the State University of New Jersey

Dates

Publication Date
20260513
Application Date
20240701

Claims (20)

  1. CLAIMS WHAT IS CLAIMED IS: 1. A gene editing complex for editing a target site in a target DNA molecule, comprising (A) an RNA-guided nickase; (B) a reverse transcriptase; (C) an RNA template molecule comprising (1) a template segment comprising a template sequence complementary to a desired DNA sequence to be introduced into the target site, and (2) a priming segment complementary to the 3’ end of the nicking strand of the target DNA molecule; and (D) a matching gRNA (magRNA) molecule comprising (1) a guide or spacer sequence complementary to a target sequence on a target strand of the target DNA molecule, (2) an RNA scaffold capable of binding to the RNA-guided nickase, and (3) an anchoring tag sequence complementary to a segment in the RNA template.
  2. 2. The complex of claim 1, wherein the reverse transcriptase is covalently linked to the RNA-guided nickase.
  3. 3. The complex of claim 1, wherein the magRNA further comprises a protein-binding motif capable of binding to an RNA-interacting protein, and the reverse transcriptase is linked to the RNA-interacting protein.
  4. 4. The complex of any one of claims 1-3, wherein the anchoring tag in magRNA is not a polyN, wherein N is a repeating nucleotide A, C, U, or G.
  5. 5. The complex of any one of claims 1-4, wherein the RNA template does not comprise additional sequence other than the said priming segment and template segment. 114 160128159.2
  6. 6. The complex of claim 1, wherein the RNA-guided nickase is a nickase variant of a Cas protein or a nickase variant of transposon-encoded IscB, IsrB, or TnpB family endonuclease protein.
  7. 7. The complex of claim 6, wherein the Cas protein is selected from Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cpf1 (Cas12a), C2c1 (Cas12b), C2c3 (Cas12c), CasY (Cas12d), CasX (Cas12e), Cas14 (Cas12f), CasPhi (Cas12j), Cas13a, Cas13b, Cas13c, Cas13d, Cas13x, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, and the orthologues thereof.
  8. 8. The complex of claim 6, wherein the Cas protein is Cas9 and the orthologues thereof.
  9. 9 The complex of claim 6, wherein the nickase variant of Cas protein is Streptococcus pyogenes nCas9(H840A) or Staphylococcus aureus nCas9 (N580A).
  10. 10. The complex of any one of the preceding claims, wherein the reverse transcriptase is a naturally-occurring reverse transcriptase from a retrovirus or a retrotransposon, or a variant thereof.
  11. 11. The complex of claim 10, wherein the reverse transcriptase is selected from the group consisting of Moloney Murine Leukemia Virus (M-MLV), Human Immunodeficiency Virus (HIV) reverse transcriptase, Avian Sarcoma-Leukosis Virus (ASLV) reverse transcriptase, Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV reverse transcriptase, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, and Myeloblastosis Associated Virus (MAV) reverse transcriptase, Line 1 ORF2, R2Bm, and 115 160128159.2
  12. 12. The complex of claim 10 or claim 11, wherein the reverse transcriptase is an MMLV- RT or a variant thereof.
  13. 13. The complex of any one of claims 1-12, wherein the anchoring tag sequence is at the 3’ or 5’ end of the magRNA.
  14. 14. The complex of claim 13, wherein the anchoring tag sequence is covalently linked to the magRNA via a polynucleotide linker.
  15. 15. The complex of any one of the preceding claims, wherein the anchoring tag sequence in the magRNA is about 6-24 nt. in length.
  16. 16. A system for editing a target site in a target DNA molecule, comprising (I) a first gene editing complex of any one of claims 1-15, and (II) a second gene editing complex.
  17. 17. The system of claim 16, wherein the second gene editing complex comprises (A) a second RNA-guided nickase, and (B) a gRNA molecule comprising a second guide or spacer sequence and a second RNA scaffold capable of binding to the second RNA-guided nickase
  18. 18. The system of claim 16, wherein the second gene editing complex comprises (A) a second RNA-guided nickase; and (B) a second magRNA molecule comprising (1) a second guide or spacer sequence, (2) a second RNA scaffold capable of binding to the second RNA-guided nickase, and (3) a second anchoring tag sequence.
  19. 19. The system of claim 17 and 18, wherein the second gene editing complex comprises (C) a second reverse transcriptase. 116 160128159.2
  20. 20. The system of any one of claims 16 to 19, wherein the guide sequence of the magRNA in the first gene editing complex and the second guide sequence of the gRNA or second magRNA in the second gene editing complex are complementary to two target sequences of the target DNA molecule, respectively.

Description

Gene Editing Mediated by Reverse Transcription of a Modular RNA Template Anchored by a Tag gRNA and Uses Thereof CROSS REFERENCE TO RELATED APPLICATION This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Application No. 63/511,710 filed on July 3, 2023. The content of the application is incorporated herein by reference in its entirety. GOVERNMENT INTERESTS This invention was made with government support under MD200088 awarded by the Department of Defense. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (096738.00780SeqList.xml; Size: 162,826 bytes; and Date of Creation: June 28, 2024) is herein incorporated by reference in its entirety. FIELD OF THE INVENTION This disclosure relates to gene editing, related systems, and uses thereof. BACKGROUND Targeted gene editing has been used for genetic manipulation of eukaryotic cells, embryos, and animals. Sequence-specific nucleases, including Talen, Zinc finger nucleases, and RNA- guided nucleases such as CRISPR/Cas9, have provided tools for precision genome editing. Upon binding to the target sequence, the native nuclease generates a DNA double- strand break (DSB), eliciting cellular DNA repair pathways including non-homologous end joining (NHEJ) and homology-directed repair (HDR). As a result, a desired sequence change, or gene editing, could be achieved. In the editing process, the on-target and off-target DNA DSB intermediates can lead to chromosomal translocation and other mutagenic events with potential oncogenic liability. To avoid the requirement of DNA DSBs, base editing platforms were developed. The CRISPR base editors use a nuclease-null or nickase version of CRISPR proteins. Proficient in DNA sequence recognition, the mutant CRISPR proteins do not cause DSBs. Instead, the mutant CRISPR protein-gRNA complex recruits a cytidine deaminase or adenine deaminase, which in 1 160128159.2 turn converts a C to U or A to G at the target, respectively, leading to sequence-specific point mutations. Avoiding DSBs, base editing reduces the oncogenic liability and is broadly used for precision genome editing for both basic research and therapeutic development. As nucleotide deamination is the underlying mechanism for base change, base editors edit transition point mutations but not transversion mutations. In addition, base editors require the target editing nucleotide within the R-loop adjacent to the PAM motif. The requirement excludes the accessibility of target sites that do not have an adjacent PAM motif. Moreover, within the editing activity window, the deamination is generally promiscuous, which causes bystander base editing within the window. While bystander base editing may be innocuous in some therapeutic developments, such as correcting a loss-of-function mutation, precision editing free of bystander editing is generally preferred under many other circumstances. Prime editing is another precision gene editing platform that does not require double strand DNA break (Anzalone, A.V., et al. Search-and-replace genome editing without double- strand breaks or donor DNA. Nature 576, 149–157 (2019)). The Prime Editor complex comprises a nickase version of CRISPR protein fused with a reverse transcriptase (RT) and a modified gRNA called pegRNA containing an RNA template and a primer binding sequence, commonly at the 3’ end of gRNA. After binding to the target DNA sequence, the Prime Editor creates a DNA nick. The nicked DNA strand serves as a primer, which binds to the primer binding sequence within the pegRNA and synthesizes a new DNA sequence using the RNA template within the pegRNA. The sequence information of the template RNA is then copied from RNA to DNA by the reverse transcriptase. In turn, the DNA sequence is further incorporated into the target site. By copying a desired RNA template at the 3’ end of pegRNA, Prime Editing is capable of achieving bystander-free precision genome editing of base pair change. The base change can be both transition and transversion. Moreover, by designing insertion and deletion in the pegRNA template, Prime Editing can also create insertion and deletion at the target location. At the center of Prime Editing system is the modified gRNA named pegRNA, which contains both gRNA scaffold, a primer binding sequence, and an editing template in the same RNA molecule. This configuration, however, limits the size of the editing template, as a long RNA template sequence within the same RNA molecule of the gRNA would create secondary structures that may interfere with the secondary structure of the gRNA CRISPR protein binding scaffold, for example. The longest template tried in Anzalone, A.V., et al. Nature 576, 149– 157 (2019) within pegRNA molecules is 34 nt in length. The template size restriction makes 2 160128159.2 some target sites in the genome inaccessible by Prime Editors due to the