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EP-4735609-A2 - COMPOSITIONS, CONSTRUCTS, PLANT VIRAL VECTORS, AND METHODS FOR PLANT GENE EDITING

EP4735609A2EP 4735609 A2EP4735609 A2EP 4735609A2EP-4735609-A2

Abstract

Disclosed herein are compositions, constructs, viral vectors, and methods for plant gene editing. The compositions can include a TnpB bacterial enzyme and a polynucleotide having a first portion adapted to bind to at least a portion of the TnpB bacterial enzyme and a second portion for binding to a plant genomic target.

Inventors

  • JACOBSEN, Steven Erik
  • LI, ZHENG
  • WEISS, Trevor John
  • KAMALU, Maris
  • AMERASEKERA, Jasmine
  • DOUDNA, Jennifer
  • ADLER, Benjamin
  • SHI, Honglue

Assignees

  • The Regents of the University of California

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A composition, comprising: a TnpB bacterial enzyme; and a polynucleotide comprising a first portion adapted to bind to at least a portion of the TnpB bacterial enzyme and a second portion for binding to a plant genomic target.
  2. 2. The composition of claim 1, wherein the polynucleotide is RNA.
  3. 3. The composition of claim 1 or 2, wherein the TnpB bacterial enzyme comprises less than about 500 amino acids, less than about 450 amino acids, less than about 420 amino acids, or less than about 400 amino acids.
  4. 4. The composition of any one of claims 1-3, wherein the TnpB bacterial enzyme is from, or derived from, tnpB from Brevibacillus agri.
  5. 5. The composition of any one of claims 1-3, wherein the TnpB bacterial enzyme comprises ISBagOl, ISYMul, or TnpB30.
  6. 6. The composition of any one of claims 1-5, wherein the TnpB bacterial enzyme comprises a peptide tag.
  7. 7. The composition of claim 6, wherein the peptide tag comprises a FLAG tag.
  8. 8. A construct comprising a plant promoter operably connected to a polynucleotide encoding for a TnpB bacterial enzyme.
  9. 9. The construct of claim 8, wherein the polynucleotide further encodes for an coRNA having a first portion adapted to bind to at least a portion of the TnpB bacterial enzyme and a second portion for binding to a plant genomic target.
  10. 10. The construct of claim 9, wherein the coRNA is about 100-400 nucleotides in length.
  11. 11. The construct of any one of claims 8-10, wherein the polynucleotide encoding for a TnpB bacterial enzyme is codon optimized for expression in plant cells.
  12. 12. The construct of any one of claims 8-11, wherein the plant promotor comprises a UBQ10 gene promoter.
  13. 13. The construct of any one of claims 9-12, wherein a coding sequence of the coRNA and a coding sequence of the TnpB bacterial enzyme at least partly overlap.
  14. 14. The construct of any one of claims 8-13, wherein the TnpB bacterial enzyme comprises less than about 500 amino acids, less than about 450 amino acids, less than about 420 amino acids, or less than about 400 amino acids.
  15. 15. The construct of any one of claims 8-14, wherein the TnpB bacterial enzyme is from, or derived from, tnpB from Brevibacillus agri.
  16. 16. The construct of any one of claims 8-14, wherein the TnpB bacterial enzyme comprises ISBagOl, ISYMul, or TnpB30.
  17. 17. The construct of any one of claims 8-16, wherein the TnpB bacterial enzyme comprises a peptide tag.
  18. 18. The construct of claim 17, wherein the peptide tag comprises a FLAG tag.
  19. 19. A plant viral vector comprising the construct of any one of claims 8-18, 26, 27, 29, 31, or 32.
  20. 20. The plant viral vector of claim 19, wherein the plant viral vector is derived from the Tobacco rattle virus (TRV).

Description

COMPOSITIONS, CONSTRUCTS, PLANT VIRAL VECTORS, AND METHODS FOR PLANT GENE EDITING CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional App. No. 63/511,070, filed on June 29, 2023. U.S. Provisional App. No. 63/520.258, filed on August 17, 2023, and U.S. Provisional App. No. 63/566,666, filed on March 18, 2024. The entire contents of each of the aforementioned applications are incorporated by reference herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with government support under award number 2334027 awarded by the National Science Foundation. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [0003] The contents of the electronic sequence listing (790482.00489.xml; Size: 785,615 bytes; and Date of Creation: June 26, 2024) is herein incorporated by reference in its entirety'. BACKGROUND [0004] Given current crop productivity projections, agricultural practices will be inadequate to meet the global food demands by the year 2050 (Ray et al., 2013). Our ability to address this problem will largely depend on the efficiency that novel genetic diversity can be created and introduced into current plant breeding pipelines. Historically, novel phenotypes were generated through random mutation or transgenic breeding, followed by successive backcrossing to an elite crop variety, taking upwards of 10-12 years to complete (Chen et al., 2019). With the advent of genome editing, we now have the ability to more efficiently genetically modify' crop genomes, resulting in beneficial phenotypes(Haun et al., 2014; Lemmon et al., 2018; Liu et al., 2021; Rodriguez-Leal et al., 2017; Zsogon et al., 2018). Despite this advance, a primary bottleneck remains: fast and efficient delivery of the gene editing reagents into crop plants (Altpeter et al.. 2016). SUMMARY [0005] In one aspect, a composition is provided. The composition can include a TnpB bacterial enzy me. The composition can also include a polynucleotide including a first portion adapted to bind to at least a portion of the TnpB bacterial enzyme and a second portion for binding to a plant genomic target. [0006] In another aspect, a construct is provided. The construct can include a plant promotor operably connected to a polynucleotide encoding for a TnpB bacterial enzyme. [0007] In yet another aspect, a plant viral vector is provided. The plant viral vector can include a construct that can include a plant promotor operably connected to a polynucleotide encoding for a TnpB bacterial enzyme. [0008] In another aspect, a plant cell is provided. The plant cell can include a construct that can include a plant promotor operably connected to a polynucleotide encoding for a TnpB bacterial enzyme, or a plant viral vector that includes the construct. [0009] In another aspect, a method for gene editing a plant is provided. The method can include introducing a construct or a plant viral vector into one or more plant cells. The construct can include a plant promotor operably connected to a polynucleotide encoding for a TnpB bacterial enzyme. The plan viral vector can include the construct. BRIEF DESCRIPTION OF THE FIGURES [0010] FIGS. 1 A and IB. (FIG. lA)The '‘DOTS’’ pipeline for detecting bona-fide RNA- guided TnpB systems in metagenomes. (FIG. IB) A phylogenetic tree of TnpBs from metagenomes (red) as well as a public IS database, ISfinder (black), with the corresponding amino acid length shown in the bar graph below. Distinct TAM sequences and small-sized TnpB enzymes from metagenomes are also highlighted in red circles and black arrows, respectively. [0011] FIGS. 2A-2C. (FIG. 2A) Representative loci architectures for TnpB-associated systems characterized in this study. (FIG. 2B) The Web logo of the TAMs for ISBagOl and ISXfaOl TnpB systems. (FIG. 2C) Comparable E. coli interference activities between Casl2a, ISBagOl and ISXfaOl, when the target site is flanked by a PAM/TAM at 26°C. [0012] FIGS. 3A-3C. (FIG. 3A) Schematics of the constructs used to test gene editing activity by ISBagOl. Top panel, ISBagOl coRNA with a 20bp spacer targeting the AtPDS3 gene were driven by the AtU6-26 promoter and followed by the HDV ribozyme. 2X indicates that the construct also contains a second similar AtU6-26 driven coRNA cassette with a second gRNA sequence that is not shown. Zea mays codon optimized ISBagOl coding sequence was expressed in a separate transcription cassette driven by the UBQ10 gene promoter and an rbcS- E9 terminator. Bottom panel, native ISBagOl sequence encoding the ISBagOl protein and the overlapping coRNA were driven by the UBQ10 gene promoter, followed by a 20bp spacer targeting the AtPDS3 gene the HDV ribozyme, and the rbcS-E9 terminator. NLS, nuclear localization signal. (FIG. 3B) Left panel, editing efficiency of ISBagOl at the AtPDS3 gene at regions targeted by four different guide RNAs in Arabidopsis protoplasts, with ISBagOl pr