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US-20260125709-A1 - METHOD FOR TARGETED ALTERATION OF DUPLEX DNA

US20260125709A1US 20260125709 A1US20260125709 A1US 20260125709A1US-20260125709-A1

Abstract

The current invention relates to methods of targeted genetic alteration in plant cells, as well as to plant cells and plants thus obtained. In the method, a Cpf1 protein and a crRNA is employed to provide for targeted alteration, in particular increased biallelic alteration, of a DNA duplex with increased efficacy.

Inventors

  • Paul Bundock

Assignees

  • KEYGENE N.V.

Dates

Publication Date
20260507
Application Date
20250515

Claims (14)

  1. 1 .- 16 . (canceled)
  2. 17 . A method for producing plant calli of the genus Solanum, wherein the produced plant calli have a targeted alteration in a target sequence that is comprised within two or more gene copies, the method comprising: (a) providing plant protoplasts comprising said target sequence, wherein said plant protoplasts are of the genus Solanum; (b) exposing said target sequence in the provided protoplasts to: a single RNA-guided endonuclease Cpf1 protein; and a crRNA comprising a guide sequence for targeting said Cpf1 protein to the target sequence comprised within the two or more gene copies, by introducing into the plant protoplasts said Cpf1 protein, or nucleic acid encoding the same, and said crRNA, or nucleic acid encoding the same, using polyethylene glycol and divalent cation mediated transformation, resulting in one targeted alteration of the two or more gene copies by said crRNA; (c) regenerating calli from said protoplasts obtained in (b); (d) selecting the calli comprising the targeted alteration of the two or more gene copies, wherein the Cpf1 protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 7.
  3. 18 . The method of claim 17 , further comprising a step of synchronizing the cell cycle phase of the protoplast, preferably before and/or during performing step (b), preferably wherein synchronizing is performed by contacting the protoplast with a synchronizing agent.
  4. 19 . The method of claim 18 , wherein the step of synchronizing the cell phase synchronizes the protoplast in the S-phase, the M-phase, the G1 and/or G2 phase of the cell cycle.
  5. 20 . The method of claim 17 , wherein the alteration comprises the insertion, deletion or modification of at least one base pair.
  6. 21 . The method of claim 17 , wherein the alteration comprises the deletion of at least one base pair and the insertion of at least one base pair.
  7. 22 . The method of claim 17 , wherein the targeted alteration is biallelic.
  8. 23 . The method of claim 17 , further comprising a step of regenerating a plant of the genus Solanum from the calli, wherein said plant or progeny thereof comprises the targeted alteration.
  9. 24 . The method of claim 17 , wherein said polyethylene glycol and divalent cation are present in a solution wherein the divalent cation is calcium.
  10. 25 . The method of claim 24 , wherein the polyethylene glycol and calcium are present in a solution wherein the calcium is provided as calcium nitrate.
  11. 26 . The method of claim 25 , wherein the polyethylene glycol and calcium nitrate are present in a solution wherein the concentration of calcium nitrate is 0.1-0.6M.
  12. 26 . The method of claim 17 , wherein the polyethylene glycol is PEG 4000.
  13. 27 . The method of claim 17 , further comprising a step of adding an alginate solution to the protoplasts after transformation.
  14. 28 . The method of claim 17 , wherein the targeted alteration results in null alleles and wherein the selected calli is homozygous for said null alleles.

Description

FIELD OF THE INVENTION The invention relates to the field of genome or gene editing tools. BACKGROUND OF THE INVENTION The process of deliberately creating alterations in the genetic material of living cells generally has the goal of modifying one or more genetically encoded biological properties of that cell, or of the organism of which the cell forms part or into which it can regenerate. These changes can take the form of deletion of parts of the genetic material, addition of exogenous genetic material, or changes in the existing nucleotide sequence of the genetic material. Methods of altering the genetic material of eukaryotic organisms have been known for over 20 years, and have found widespread application in plant, human and animal cells and micro-organisms for improvements in the fields of agriculture, human health, food quality and environmental protection. The most common methods consist of adding exogenous DNA fragments to the genome of a cell, which will then confer a new property to that cell or its organism over and above the properties encoded by already existing genes (including applications in which the expression of existing genes will thereby be suppressed). Although many such examples are effective in obtaining the desired properties, these methods are nevertheless not very precise, because there is no control over the genomic positions in which the exogenous DNA fragments may be inserted (and hence over the ultimate levels of expression), and because the desired effect will have to manifest itself over the natural properties encoded by the original and well-balanced genome. On the contrary, methods of genome editing that will result in the addition, deletion or conversion of nucleotides in predefined, i.e. targeted, genomic loci will allow the precise modification of the genome, for example in existing genes. A large part of targeted genome editing described in the literature has been performed using zinc finger nucleases (ZFNs). ZFNs have been used to modify endogenous genes in a wide range of organisms and cell types. Several types of genomic alterations can be introduced with ZFNs including point mutations, deletions, insertions, inversions, duplications, and translocations, thus providing researchers with unprecedented tools to perform genetic manipulations. In the more recent years, transcription activator-like effector nucleases (TALENs) have rapidly emerged as an alternative to ZFNs for genome editing and introducing targeted double-strand breaks (DSBs), i.e. in which both strands of the DNA duplex are severed. TALENs are similar to ZFNs and comprise a non-specific FokI nuclease domain fused to a customizable DNA-binding domain. This DNA-binding domain is composed of highly conserved repeats derived from transcription activator-like effectors (TALEs), which are proteins secreted by Xanthomonas bacteria to alter transcription of genes in host plant cells. In more detail, TALEs consist of a number of repeating protein domains, each of which is able to specifically recognize and bind to one of the 4 DNA nucleotides (A,T,G,C). The domains specific for each nucleotide have been identified and arrays of these domains which have high binding affinity for any DNA sequence can be produced (Christian, 2010, Genetics 186:757-761; Cermak et al., 2011, Nucleic Acids Res 39:e82; Bogdanove and Voytas, 2011, Science 333:1843-1846; Boch, 2011, Nature Biotechnology 29:135-136). These arrays are then fused to the nuclease domain of FokI to create a TALEN and, similar to ZFN, two TALEN proteins are used to induce a DNA DSB in the target DNA duplex. Several papers have described the use of TALENs to create mutations at the target sequence (Curtin (2012) The Plant Genome, 5, 42-50). Joung et al. (Nat Rev Mol Cell Biol. (2013) 14(1): 49-55. doi: 10.1038/nrm3486A) reviewed and compared various techniques employing TALENs in targeted genome editing. Recently, another novel method for targeted genome editing has been reported. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are loci containing multiple short direct repeats and are found in 40% of the sequenced bacteria and 90% of sequenced archaea. The CRISPR repeats form a system of acquired bacterial immunity against genetic pathogens such as bacteriophages and plasmids. When a bacterium is challenged with a pathogen, a small piece of the pathogen genome is processed by CRISPR associated proteins (Cas) and incorporated into the bacterial genome between CRISPR repeats. The CRISPR loci are then transcribed and processed to form so called crRNA's which include approximately 30 nucleotides of sequence identical to the pathogen genome. These RNA molecules form the basis for the recognition of the pathogen upon a subsequent infection and lead to silencing of the pathogen genetic elements through either a RNAi-like process or direct digestion of the pathogen genome. The CRISPR-Cas systems of bacterial and archaeal adaptive immunity show extr