Simultaneous gene editing and haploid induction

Patent metadata

Field	Value
Publication number	US-2025115923-A1
Application number	US-202418980635-A
Country	US
Kind code	A1
Filing date	Dec 13, 2024
Priority date	Dec 2, 2016
Publication date	Apr 10, 2025
Grant date	—

Abstract

The presently disclosed subject matter relates to using a haploid inducing line (whether existing or created) and transforming the haploid line so that it encodes cellular machinery capable of editing genes. The transformed haploid inducing line is used as a parent in a cross between two plants. During pollination, the parental gametes fuse to form an embryo; and the gene editing machinery is also delivered to the embryo at this time. During embryonic development, one set of parental chromosomes are lost, and the gene editing machinery operates on the remaining set of chromosomes. Thus, at least one haploid progeny with edited genes is produced from the cross.

First claim

What is claimed is:

1 . A method of editing dicot genomic DNA, comprising:
(a) obtaining a first dicot plant comprising a mutation in a centromeric histone 3 (CENH3) gene and optionally a maize CENH3 tailswap transgene, wherein said first dicot plant expresses a site-directed DNA modification enzyme and optionally at least one guide nucleic acid, wherein the site-directed DNA modification enzyme is a base editor; (b) obtaining a second dicot plant, wherein the second dicot plant comprises the dicot plant genomic DNA to be edited; (c) pollinating the first dicot plant with pollen from the second dicot plant; and (d) selecting at least one haploid progeny produced by the pollination of step (c) wherein the haploid progeny comprises the genome of the second dicot plant but not the first dicot plant, and the genome of the haploid progeny has been modified by the base editor and optional at least one guide nucleic acid delivered by the first dicot plant; wherein the mutation in a CENH3 gene is selected from the group consisting of a loss-of-function mutation, a partial loss-of-function mutation, a restored frameshift mutation, and an in-frame deletion mutation.

2 . The method of claim 1 , wherein the base editor comprises a site-directed nuclease selected from the group consisting of a Cas9 nuclease, Cpf1 nuclease, dCas9-FokI, dCpf1-FokI, a nickase Cas9 (nCas9), chimeric dCas9 non-FokI nuclease and dCpf1 non-FokI nuclease.

3 . The method of claim 1 , wherein the base editor comprises a cytidine deaminase fused to a Cas polypeptide.

4 . The method of claim 1 , wherein the cytidine deaminase is an APOBEC deaminase.

5 . The method of claim 1 , wherein the base editor comprises an adenine deaminase fused to a Cas polypeptide.

6 . The method of claim 1 , wherein the base editor comprises a uracil DNA glycosylase fused to a Cas polypeptide.

7 . The method of claim 1 , wherein an edited haploid progeny selected in (d) is treated with a chromosome doubling agent, thereby creating an edited doubled haploid progeny.

8 . The method of claim 7 , wherein the chromosome doubling agent is colchicine, pronamide, dithipyr, or trifluralin.

9 . The method of claim 1 , wherein the edited haploid progeny undergoes spontaneous chromosome doubling, thereby creating an edited doubled haploid progeny.

10 . The method of claim 1 , wherein the guide nucleic acid is a guide RNA.

11 . The method of claim 10 , wherein the guide RNA is an 18-21 nucleotide sequence and is at least 90% identical to a target sequence.

12 . The method of claim 8 , wherein the target sequence is SEQ ID NO: 103, and wherein the dicot genomic DNA is Arabidopsis genomic DNA.

13 . The method of claim 1 , wherein the first dicot plant expresses a marker gene.

14 . The method of claim 13 , wherein the marker gene is selected from the group consisting of GUS, PMI, PAT, GFP, RFP, CFP, B1, C1, R-nj, and anthocyanin pigments.

13 . The method of claim 1 , wherein the first dicot plant is a transformable dicot plant selected from the group consisting of Arabidopsis, Brassica , soybean, tomato, sunflower, and cotton.

14 . The method of claim 1 , wherein the partial loss-of-function mutation is a frame-shift mutation near the 3′ terminus of the CENH3 gene.

15 . The method of claim 1 , wherein the loss-of-function mutation is a knock-out mutation.

Assignees

• Syngenta Crop Protection Ag

Inventors

• Que Qiudeng
• Kelliher Timothy

Classifications

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/65

  using markers (enzymes used as markers C12N15/52) · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/8218

  Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS] · CPC title