EA041676B1 - BUILDING MAIS RESISTANT TO NORTHERN LEAF SPOT - Google Patents

Description

Field of invention

The present invention relates to the field of molecular biology and, more specifically, to methods for editing the genome of a plant cell to obtain northern leaf spot resistant maize.

Link to the sequence listing provided electronically

An official copy of this sequence listing is provided electronically via EFS-Web as a sequence listing in an ASCII file named 7156WO_SEQLIST.txt, created on October 13, 2016, with a size of 240 kilobytes, and submitted simultaneously with this description. The sequence listing contained herein in ASCII format is part of the present specification and is incorporated herein by reference in its entirety.

Background of the invention

Northern leaf spot (NLB), caused by the pathogenic fungus Exserohilum turcicum (formerly called Helminthosporium turcicum), is a serious vascular disease of maize leaves in many tropical and temperate environments. Symptoms can range from cigar-shaped lesions on the lower leaves to complete destruction of the foliage, thereby reducing the amount of leaf area available for photosynthesis. A decrease in photosynthetic capacity leads to a lack of carbohydrates needed to fill the grain, which affects the grain yield. The mid-latitude regions of the tropics at about 900-1600 m above sea level have a particularly favorable climate for northern leaf spot due to long periods of dew and moderate temperatures. However, northern leaf spot can also result in yield losses of 30-50% under moderate environmental conditions, such as in the United States during the rainy season, especially if the infection establishes on the upper leaves of the plant before the pistillate stage.

The most effective and most preferred way to control northern leaf spot is to grow resistant hybrids. Resistance to certain strains of the pathogen can be controlled by certain native maize disease resistance genes such as Et1, Ht2, Et3, Html, Htnl, EtN, HtP, ht4 and rt (Welz and Geiger 2000. Plant Breeding. 119 (1): 1-14 Ogliari et al 2005 Genet Mol Biol 28:435-439 Hurni et al 2015 PNAS 112(28):8780-5). However, introgression of resistance genes into other inbreds is a difficult task, which may or may not lead to poor yield due to linkage plume. There is a need to produce northern leaf spot resistant maize plants with greater efficiency and in a way that reduces the linkage plume associated with the introgression of multiple resistance loci into elite maize lines through conventional methods.

Short description

The limitations of conventional breeding with respect to northern leaf spot resistance introgression in the maize line can be overcome by editing genes that confer increased northern leaf spot resistance, such as Et1 and NLB18, for example, or by moving the resistance-associated alleles of Et1 and NLB18 to another site in the genome so that increased resistance to northern leaf spot can be achieved by introgression of a single genomic locus containing multiple nucleotide sequences each conferring increased resistance to northern leaf spot into maize plants.

Provided herein are methods for producing a maize plant cell with a modified Et1 nucleotide sequence. The methods include introducing a double-strand break or site-specific modification at one or more target sites in an endogenous HT1 coding sequence in a maize plant cell and obtaining a maize plant cell having a modified Et1 nucleotide sequence. The method may further comprise introducing an Et1 replacement template into a maize plant cell, wherein said Et1 replacement template contains at least one nucleic acid change from an endogenous HT1 coding sequence, and wherein said Et1 replacement template is included in the endogenous coding sequence. HT 1 A double strand break can be induced with a nuclease such as, but not limited to, TALEN, meganuclease, zinc finger nuclease, or CRISPR associated nuclease. The method may further comprise growing a maize plant from a maize plant cell having a modified Ht1 nucleotide sequence, wherein the maize plant may exhibit increased resistance to northern leaf spot.

In some aspects, the modified Ht1 nucleotide sequence provides for a deletion in the promoter of the endogenous sequence encoding HT1. The methods may involve the use of Cas9 endonuclease and one or more guide RNAs. In one embodiment, at least two guide RNAs are used, wherein the first guide RNA contains a variable targeting domain that is complementary to SEQ ID NO:1 [Ht1-TS2] and the second guide RNA contains a variable targeting domain that is complementary to SEQ ID NO:2

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[Ht1-TS4]. In another embodiment, the first guide RNA contains a variable targeting domain that is complementary to SEQ ID NO:1 [Ht1-TS2], and the second guide RNA contains a variable targeting domain that is complementary to SEQ ID NO:3 [Ht1-ST1-TS1].

In other aspects, an Ht1 replacement template is used that contains the Ht1 nucleotide sequence from PH4GP (Ht1-PH4GP). The nucleotide sequence of Ht1-PH4GP may contain SEQ ID NO:59. In another embodiment, the Ht1-PH4GP nucleotide sequence may comprise a nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO :52, wherein the polypeptide confers resistance to northern leaf spot. The nucleotide sequence of Ht1-PH4GP may also contain SEQ ID NO:65. The methods may involve the use of Cas9 endonuclease and one or more guide RNAs. In one embodiment, at least two guide RNAs are used, wherein the first guide RNA contains a variable targeting domain that is complementary to SEQ ID NO:14 [Ht1-TS6] and the second guide RNA contains a variable targeting domain that is complementary to SEQ ID NO:16 [ Ht1-TS9]. In another embodiment, the first guide RNA contains a variable targeting domain that is complementary to SEQ ID NO:15 [Ht1-TS7] and the second guide RNA contains a variable targeting domain that is complementary to SEQ ID NO:17 [Ht1-TS10]. Provided herein are methods for producing a maize plant cell with a modified NLB18 nucleotide sequence. The methods include introducing a double strand break or site-specific modification at one or more target sites in an endogenous NLB18 coding sequence in a maize plant cell and obtaining a maize plant having a modified NLB18 nucleotide sequence. The methods may further comprise introducing an NLB18 replacement template into a maize plant cell, wherein said NLB18 replacement template contains at least one nucleic acid change from an endogenous NLB18 coding sequence, and wherein said NLB18 replacement template is included in an endogenous NLB18 coding sequence. NLB18. The double strand break can be induced with a nuclease such as, but not limited to, TALEN, meganuclease, zinc finger nuclease, or CRISPR associated nuclease. The method may further comprise growing a maize plant from a maize plant cell having a modified NLB18 nucleotide sequence, wherein the maize plant may exhibit increased resistance to northern leaf spot.

In some aspects, the modified NLB18 nucleotide sequence comprises a modification in the promoter of the endogenous sequence encoding NLB18. In some embodiments, a modification in the promoter of the endogenous Ht1 coding sequence involves the deletion of a portion of repetitive sequences in the Ht1 promoter. In one embodiment, the modification in the promoter of the endogenous Ht1 coding sequence provides for the deletion of SEQ ID NO:71 from the Ht1 promoter.

In other aspects, an NLB18 replacement template is used that contains the NLB18 nucleotide sequence from PH26N or PH99N (NLB18-PH26N or NLB18-PH99N). In one embodiment, the NLB18-PH26N nucleotide sequence comprises any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO :64, wherein the polypeptide confers resistance to northern leaf spot. In some aspects, the nucleotide sequence of NLB18-PH26N contains SEQ ID NO:70. The nucleotide sequence of NLB18-PH99N may comprise any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90% 91, 92, 93, 94, 95, 96, 97, 98, or 99% of SEQ ID NO:62, where the polypeptide confers resistance to northern leaf spot. The methods may involve the use of Cas9 endonuclease and one or more guide RNAs. In one embodiment, at least two guide RNAs are used, wherein the first guide RNA contains a variable targeting domain that is complementary to SEQ ID NO:30 [NLB18TS1] and the second guide RNA contains a variable targeting domain that is complementary to SEQ ID NO:32 [NLB18- TS4]. In another embodiment, the first guide RNA contains a variable targeting domain complementary to SEQ ID NO:31 [NLB18-TS8] and the second guide RNA contains a variable targeting domain complementary to SEQ ID NO:32 [NLB18TS4].

Provided herein are methods for producing a genomic locus-edited maize plant cell containing at least one nucleotide sequence that confers increased resistance to northern leaf spot. The methods include 1) introducing a double strand break or site-specific modification at one or more target sites in the genomic locus of a maize plant cell; 2) introducing one or more nucleotide sequences encoding a polypeptide that confers increased resistance to northern leaf spot, wherein each nucleotide sequence is flanked by 300-500 contiguous nucleotide sequences of nucleotide sequences located 5' or 3' relative to the respective target sites; and 3) obtaining a maize plant cell having a genomic locus containing one or more nucleotide sequences that confer increased resistance to northern leaf spot. The double-strand break or site-specific modification can be induced with a nuclease such as, but not limited to, TALEN, a meganuclease, a zinc finger nuclease, or a CRISPR-associated nuclease. The method may further comprise growing a maize plant from a maize plant cell having an edited genomic locus containing at least one nucleotide sequence that provides increased resistance to northern leaf spot, and wherein the maize plant can exhibit increased resistance to northern leaf spot.

In some aspects, the edited plant cell contains one or more nucleotide sequences including any of the following: Ht1-PH4GP (SEQ ID NO:51), NLB18PH26N (SEQ ID NO:63), and NLB18-PH99N (SEQ ID NO:61). In other aspects, the genomic locus is CTL1. The Ht1-PH4GP nucleotide sequence may comprise SEQ ID NO:59 or any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 9o, 91, 92, 93, 94, 95, 96, 97, 98, or 99% SEQ ID NO:52 wherein the polypeptide confers increased resistance to northern leaf spot in the maize plant. In some aspects, the nucleotide sequence of Ht1-PH4GP contains SEQ ID NO:65. The nucleotide sequence of NLB18-PH2 6N may comprise any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of SEQ ID NO:64 where the polypeptide confers increased resistance to northern leaf spot in the maize plant. In some aspects, the nucleotide sequence of NLB18-PH26N contains SEQ ID NO:70. The NLB18-PH99N nucleotide sequence may comprise any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:62, wherein the polypeptide confers increased resistance to northern leaf spot in the maize plant.

In still other aspects, the nucleotide sequence encoding NLB18-PH26N is targeted at TS8 CTL1; the nucleotide sequence encoding NLB18-PH4GP targets TS10 CTL1; and/or the nucleotide sequence encoding NLB18-PH26N is targeted at TS45 CTL1.

In one aspect, a method for editing a plant cell involves using Cas9 endonuclease as a DSB inducing agent and one or more guide RNAs to target Cas9 to sites at the CTL1 locus. One guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:36 [CTL1-TS8]; one guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:37 [CTL1TS10], and one guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:38 [CTL1-TS45].

Also contemplated are maize plant cells obtained using the methods provided herein, as well as maize plants producing maize plant cells and seeds produced by maize plants.

This document also provides guide polynucleotides containing variable targeting domains complementary to target sites in the endogenous Ht1 coding sequence, the endogenous NLB18 coding sequence, or the CTL1 genomic locus. The targeting polynucleotides can be RNA sequences, DNA sequences, or combined RNA-DNA sequences. For Ht1, targeting polynucleotides may have a variable targeting domain complementary to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. For NLB18, targeting polynucleotides may have a variable targeting domain complementary to SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. For CTL1, targeting polynucleotides may have a variable targeting domain complementary to SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.

Brief Description of Drawings and Sequence Listings

In FIG. 1 shows a schematic representation of the Ht1 locus; the location of each target site in the Ht1 genomic sequence for the deletion of a repeating sequence in the promoter region; and the positions of the primers used for verification. Introns between exons are highlighted in white, and target sites are indicated by upward triangles. E1 is exon1; e2 is exon 2; a pro is a promoter region.

In FIG. 2A and 2B show the expected sequences of splice boundary regions after deletion of repetitive sequences in the promoter region of the Ht1 gene. In FIG. 2A shows the expected sequence of CR2/CR4 splicing boundary regions (sequences in italics are TS2 sequences and underlined sequences are TS4 sequences). In FIG. 2B shows the expected sequence of CR2/ST1-CR1 splicing boundary regions (sequences in italics are TS2 sequences and underlined sequences are ST1-TS1 sequences). The areas in the box indicate areas of the splicing boundary.

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In FIG. 3 shows the position of each target site and a schematic of Ht1 allele exchange. The Ht1 PH4GP allele is shown in dark gray and the allele it replaces is shown in light gray.

In FIG. 4 shows the primer positions used to evaluate Ht1 allele exchange.

In FIG. 5 shows the position of each target site and a schematic representation of NLB18 allele exchange.

In FIG. 6 shows the position of three loci containing target sites (TS8, TS45 and TS10) for the Cas guide RNA/endonuclease system in CTL1 on maize chromosome 1.

In FIG. 7 shows a schematic of the NLB18-PH26N insert in TS8.

In FIG. 8 shows a schematic of the Ht1-PH4GP insert in TS10.

In FIG. 9 shows a schematic of the NLB18-PH26N insert in the TS45.

In FIG. 10 shows expression of NLB18 using qRT-PCR in T2 TS45 NLB18-BC26N maize plants.

In FIG. 11 shows Ht1 expression using qRT-PCR in T2 TS10 Ht1ED4GP maize plants.

SEQ ID NO:1 is the nucleotide sequence of the target site of Ht1-TS2.

SEQ ID NO:2 is the nucleotide sequence of the Ht2-TS4 target site.

SEQ ID NO:3 is the nucleotide sequence of the target site of Ht1-ST1-TS1.

SEQ ID NO:4 is the nucleotide sequence of the Cas9 gene.

SEQ ID NO:5 is the amino acid sequence of the single amino terminal SV40 intranuclear localization signal.

SEQ ID NO:6 is the nucleotide sequence of the polymerase III U6 promoter.

SEQ ID NO:7 is the nucleotide sequence of DNA capable of expressing the Ht1-CR2 guide RNA.

SEQ ID NO:8 is the nucleotide sequence of DNA capable of expressing the Ht1-CR4 guide RNA.

SEQ ID NO:9 is the nucleotide sequence of DNA capable of expressing the Ht1-ST1-CR1 guide RNA.

SEQ ID NO:10 is the nucleotide sequence of the forward primer for Ht1f3.

SEQ ID NO:11 is the nucleotide sequence of the reverse primer for Ht1r4v2.

SEQ ID NO:12 is the nucleotide sequence of the forward primer used in the second PCR reaction.

SEQ ID NO:13 is the nucleotide sequence of the reverse primer used in the second PCR reaction.

SEQ ID NO:14 is the nucleotide sequence of the target site of Ht1-TS6.

SEQ ID NO:15 is the nucleotide sequence of the target site of HT1-TS7.

SEQ ID NO:16 is the nucleotide sequence of the target site of HT1-TS9.

SEQ ID NO:17 is the nucleotide sequence of the target site of HT1-TS10.

SEQ ID NO:18 is the nucleotide sequence of DNA capable of expressing the HT1-CR6 guide RNA.

SEQ ID NO:19 is the nucleotide sequence of DNA capable of expressing the HT1-CR9 guide RNA.

SEQ ID NO:20 is the nucleotide sequence of DNA capable of expressing the HT1-CR7 guide RNA.

SEQ ID NO:21 is the nucleotide sequence of DNA capable of expressing the HT1-CR10 guide RNA.

SEQ ID NO:22 is the nucleotide sequence of the forward primer for Ht1HR1f1.

SEQ ID NO:23 is the nucleotide sequence of the reverse primer for Ht1HR1r1.

SEQ ID NO:24 is the nucleotide sequence of the forward primer for Ht1HR2f1.

SEQ ID NO:25 is the nucleotide sequence of the reverse primer for Ht1HR2r1.

SEQ ID NO:26 is the nucleotide sequence of the forward primer for hdr2b f.

SEQ ID NO:27 is the nucleotide sequence of the reverse primer for hdr2br.

SEQ ID NO:28 is the nucleotide sequence of the hdr2b PV probe.

SEQ ID NO:29 is the nucleotide sequence of the hdr2b PG probe.

SEQ ID NO:30 is the nucleotide sequence of the target site of NLB18-TS1.

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SEQ ID NO:31 is the nucleotide sequence of the target site of NLB18-TS8.

SEQ ID NO:32 is the nucleotide sequence of the target site of NLB18-TS4.

SEQ ID NO:33 is the nucleotide sequence of DNA capable of expressing the NLB18-CR1 guide RNA.

SEQ ID NO:34 is the nucleotide sequence of DNA capable of expressing the NLB18-CR8 guide RNA.

SEQ ID NO:35 is the nucleotide sequence of DNA capable of expressing the NLB18-CR4 guide RNA.

SEQ ID NO:36 is the nucleotide sequence of the target site of CTL1-TS8.

SEQ ID NO:37 is the nucleotide sequence of the target site of CTL1-TS45.

SEQ ID NO:38 is the nucleotide sequence of the target site of CTL1-TS10.

SEQ ID NO:39 is the nucleotide sequence of the forward primer for 8HR1f1.

SEQ ID NO:40 is the nucleotide sequence of the reverse primer for PH26NPr.

SEQ ID NO:41 is the nucleotide sequence of the forward primer for PH26NTf.

SEQ ID NO:42 is the nucleotide sequence of the reverse primer for 8HR2r1.

SEQ ID NO:43 is the nucleotide sequence of the forward primer for 10HR1f.

SEQ ID NO:44 is the nucleotide sequence of the Ht1Pr reverse primer.

SEQ ID NO:45 is the nucleotide sequence of the forward primer for Ht1Tf.

SEQ ID NO:46 is the nucleotide sequence of the reverse primer for 10HR2r.

SEQ ID NO:47 is the nucleotide sequence of the forward primer for 45hr1f1.

SEQ ID NO:48 is the nucleotide sequence of the reverse primer for PH26NPr.

SEQ ID NO:49 is the nucleotide sequence of the forward primer for PH26NTf.

SEQ ID NO:50 is the nucleotide sequence of the reverse primer for 45hr2r1.

SEQ ID NO:51 is the Ht1 cDNA nucleotide sequence found in the PH4GP inbred line.

SEQ ID NO:52 is the amino acid sequence of the polypeptide encoded by SEQ ID NO:51.

SEQ ID NO:53 is the Ht1 cDNA nucleotide sequence found in the PH1W2 inbred line.

SEQ ID NO:54 is the amino acid sequence of the polypeptide encoded by SEQ ID NO:53.

SEQ ID NO:55 is the Htl cDNA nucleotide sequence found in the B73 inbred line and herein referred to as the B73-high allele.

SEQ ID NO:56 is the amino acid sequence of the polypeptide encoded by SEQ ID NO:55.

SEQ ID NO:57 is the Ht1 cDNA nucleotide sequence found in the B73 inbred line and referred to herein as the B734ow allele.

SEQ ID NO:58 is the amino acid sequence of the polypeptide encoded by SEQ ID NO:57.

SEQ ID NO:59 is the Ht1 genomic DNA nucleotide sequence found in the PH4GP inbred line.

SEQ ID NO:60 is the amino acid sequence of the region found in the Ht1 polypeptides of alleles associated with resistance.

SEQ ID NO:61 is the cDNA sequence of NLB18 from PH99N.

SEQ ID NO:62 is the amino acid sequence of the protein encoded by SEQ ID NO:61.

SEQ ID NO:63 is the cDNA sequence of NLB18 from PH26N.

SEQ ID NO:64 is the amino acid sequence of the protein encoded by SEQ ID NO:63.

SEQ ID NO:65 is the nucleotide sequence of ZM-HT1-PH4GP containing the ZM-HT1-PH4GP promoter, exon 1, intron 1 and terminator.

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SEQ ID NO:66 is the nucleotide sequence of NLB18 from PH184C, including 5' NLB18-CR8 to 3' NLB18-CR4.

SEQ ID NO:67 is the homology arm sequence flanking 5' of NLB18TS1 in PH184C.

SEQ ID NO:68 is the homology arm sequence flanking 3' of NLB18TS4 in PH184C.

SEQ ID NO:69 is the homology arm sequence flanking 5' of NLB18TS8 in PH184C.

SEQ ID NO:70 is the nucleotide sequence of NLB18 from PH2 6N containing the NLB18 PH26N promoter, exon 1, intron 1, exon 2, intron 2, exon 3, and terminator.

SEQ ID NO:71 is the nucleotide sequence of a portion of repetitive sequences in the PH184C Ht1 promoter.

SEQ ID NO:72 is the nucleotide sequence of the expression cassette comprising the Zea mays ubiquitin promoter, ZM ubiquitin 5'-UTR, ZM ubiquitin intron 1, SV40 intranuclear localization signal, Cas9 exon 1 (ST1), potato LS1 intron, exon 2 Cas9 (ST1), VirD2 endonuclease intranuclear localization signal, and pinII terminator.

SEQ ID NO:73 is the nucleotide sequence containing Cas9 used in Example 4; SEQ ID NO:73 contains cas9 exon 1 (SP), ST-LS1 intron 2, Cas9 exon 2 (SP), and VirD2 intranuclear localization signal.

SEQ ID NO:74 is the nucleotide sequence of DNA capable of expressing the ZM-U6:08CR1 guide RNA.

SEQ ID NO:75 is the nucleotide sequence of DNA capable of expressing the ZM-U6:45CR1 guide RNA.

SEQ ID NO:76 is the nucleotide sequence of DNA capable of expressing the ZM-U6:10CR3 guide RNA.

SEQ ID NO:77 is the nucleotide sequence of the 8CR1HR2 repair template of the 08CR1HR1-NLB18 (PH26N) genomic sequence targeting TS8 CTL1.

SEQ ID NO:78 is the nucleotide sequence of the 45CR1HR2 repair template of the 45CR1HR1-NLB18 (PH26N) genomic sequence targeting TS45 CTL1.

SEQ ID NO:79 is the nucleotide sequence of the 10CR3HR2 repair template of the 10CR3HR1-HT1 (PH4GP) genomic sequence targeting TS10 CTL1.

SEQ ID NO:80 is the amino acid sequence of the VirD2 endonuclease carboxyl terminus intranuclear localization signal for Agrobacterium tumefaciens T-DNA border regions.

SEQ ID NO:81 is the 5' HT1-TS6 nucleotide in PH184C (Example 2).

SEQ ID NO:82 is the 3' HT1-TS9 nucleotide in PH184C (Example 2).

SEQ ID NO:83 is the 5' HT1-TS7 nucleotide in PH184C (Example 2).

SEQ ID NO:84 is the 5' HT1-TS10 nucleotide in PH184C (Example 2).

SEQ ID NO:85 is the 5' CTL1-TS8 nucleotide in PH184C (Example 4).

SEQ ID NO:86 is the 3' CTL1-TS8 nucleotide in PH184C (Example 4).

SEQ ID NO:87 is the 5' CTL1-TS45 nucleotide in PH184C (Example 4).

SEQ ID NO:88 is the 3' CTL1-TS45 nucleotide in PH184C (Example 4).

SEQ ID NO:89 is the 5' CTL1-TS10 nucleotide in PH184C (Example 4).

SEQ ID NO:90 is the 3' CTL1-TS10 nucleotide in PH184C (Example 4).

homology arm sequence, flankshomology arm sequence, flankshomology arm sequence, flankshomology arm sequence, flankshomology arm sequence, flankshomology arm sequence, flankshomology arm sequence, flankshomology arm sequence, flankshomology arm sequence, flankshomology arm sequence, flanksDetailed description

It should also be understood that the terminology used herein is only intended to describe specific embodiments and is not intended to be limiting. As used herein and in the appended claims, singular terms and singular forms, for example, include plural entities unless the meaning clearly implies otherwise. Thus, for example, a reference to a plant, a specific plant, or some plant also includes more than one plant; also, depending on the context, the use of the term plant also includes genetically similar or identical offspring of such

- 6 041676 plants; the use of the term nucleic acid optionally in practice includes multiple copies of such a nucleic acid molecule; similarly, the term probe optionally (and typically) encompasses a plurality of similar or identical probe molecules. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention belongs, unless expressly stated otherwise.

Compositions and methods are provided herein for editing the maize genome to produce maize plants that have increased resistance to northern leaf spot.

The term allele refers to one of two or more different nucleotide sequences that are at a particular locus.

Exserohilum turcicum, formerly Helminthosporium turcicum, is a pathogenic fungus that causes northern leaf spot infection. The pathogenic fungus is also referred to herein as Exserohilum or Et.

Disease resistance (such as northern leaf spot resistance) is a characteristic of plants where disease symptoms resulting from plant-pathogen interactions such as maize and Exserohilum turcicum interactions are eliminated, minimized, reduced in the plant. In other words, pathogens do not cause plant diseases and associated disease symptoms or, alternatively, disease symptoms caused by the pathogen are minimized or reduced.

A locus is a position on a chromosome where a gene or marker is located.

Resistance is a relative term indicating that an infected plant has better plant health or produces a better maize crop than another similarly treated, more susceptible plant. In other words, the conditions cause a reduction in the decline in maize survival, growth and/or yield in a resistant maize plant compared to a susceptible maize plant. One skilled in the art will appreciate that the resistance of maize plants to northern leaf spot or the pathogen that causes it may involve a spectrum of more resistant or less resistant phenotypes, and may vary depending on the severity of the infection. However, by simple observation, one skilled in the art can determine the relative resistance or susceptibility of various plants, plant lines or plant families to northern leaf spot, and moreover, will also recognize phenotypic gradations of resistant. For example, a visual score of 1 to 9 can be used to determine the level of resistance to northern leaf spot. A higher score indicates higher resilience. Data should only be collected when sufficient selection pressure exists in the measurement experiment. The terms tolerance and resistance are used interchangeably in this document.

Resistance can be newly acquired or increased. Newly acquired or enhanced resistance refers to an increased level of resistance to a particular pathogen, a broad spectrum of pathogens, or infection caused by the pathogen(s). An increased level of resistance to a particular pathogenic fungus, such as Et, for example, is an increased or improved resistance to fungi. Embodiments may provide increased or improved plant resistance to a pathogenic fungus.

I. Gene editing.

In some embodiments, gene editing can be facilitated by inducing a double strand break (DSB) at a specific position in the genome near the desired change. DSB can be induced using any available DSB inducing agent, including, but not limited to, TALEN, meganucleases, zinc finger nucleases, Cas9-gRNA systems (based on bacterial CRISPR-Cas systems), and the like. In some embodiments, the introduction of DSB can be combined with the introduction of a template for modifying a polynucleotide.

The polynucleotide modification template may be introduced into a cell by any method known in the art, such as, without limitation, transient administration, transfection, electroporation, microinjection, particle-mediated delivery, topical application, whisker-mediated delivery, cell-penetrating peptide delivery, or direct delivery. , mediated by mesoporous silica (MSN) nanoparticles.

The polynucleotide modification template can be introduced into the cell as a single-stranded polynucleotide molecule, a double-stranded polynucleotide molecule, or as part of a circular DNA (vector DNA). The polynucleotide modification template may also be linked to a guide RNA and/or a Cas endonuclease. Linked DNAs may provide co-localization of target and template DNA suitable for genome editing and targeted genome regulation, and may also be suitable for targeting post-mitotic cells where the function of the endogenous homologous recombination HR mechanism is expected to be greatly reduced (Mali et al. 2013 Nature Methods Vol 10: 957-963.) The polynucleotide modification template may be temporarily present in the cell, or it may be introduced via a viral replicon.

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A modified nucleotide or edited nucleotide refers to a nucleotide sequence of interest that contains at least one change compared to an unmodified nucleotide sequence. Such changes include, for example, (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii). An edited cell or edited plant cell refers to a cell containing at least one change in genomic sequence compared to a control cell or plant cell that does not include such a change in genomic sequence.

As used herein, the term polynucleotide modification template or modification template refers to a polynucleotide that contains at least one nucleotide modification relative to the target nucleotide sequence to be edited. A nucleotide modification may be at least one substitution, addition or deletion of a nucleotide. Optionally, the polynucleotide modification template may further comprise homologous nucleotide sequences flanking at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.

A method for editing a genomic sequence combining DSB and modification templates typically provides the host cell with a DSB inducing agent or a nucleic acid encoding a DSB inducing agent that recognizes a target sequence in a chromosomal sequence, and wherein the DSB inducing agent is capable of inducing DSB in genomic sequence; and providing at least one polynucleotide modification template containing at least one nucleotide change compared to the nucleotide sequence to be edited. The endonuclease can be provided to the cell by any method known in the art, for example, without limitation, via transient administration, transfection, microinjection, and/or topical application, or indirectly via recombination constructs. The endonuclease can be provided as a protein or as a targeting polynucleotide complex directly into the cell or indirectly via recombination constructs. The endonuclease may be introduced into the cell transiently or may be incorporated into the genome of the host cell using any method known in the art. In the case of a CRISPR-Cas system, the uptake of an endonuclease and/or targeting polynucleotide into a cell can be facilitated by a cell penetrating peptide (CPP) as described in WO2016073433.

As used herein, the term genomic region refers to a segment of a chromosome in a cell's genome. In one embodiment, the genomic region includes a segment of a chromosome in the plant cell genome that is present on either side of the target site, or alternatively also contains part of the target site. The genomic region may contain at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60 , 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5 -600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1300, 5-1400, 5-1500, 51600, 5-1700, 5-1800, 5 -1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800. 52900, 5-3000, 5-3100 or more bases, so that the genomic region has sufficient homology to undergo homologous recombination with the corresponding homology region.

TAL effector nucleases (TALENs) are a class of sequence-specific nucleases that can be used to generate double-strand breaks at specific target sequences in the genome of a plant or other organism. (See Miller et al. (2011) Nature Biotechnology 29:143-148).

Endonucleases are enzymes that cleave the phosphodiester bond in a polynucleotide chain. Endonucleases include restriction endonucleases, which cleave DNA at specific sites without base damage, and meganucleases, also known as homing endonucleases (HEases), which, like restriction endonucleases, bind and cut specific recognition sites, however recognition sites for meganucleases are usually longer, about 18 bp. .O. or more (patent application PCT/US12/30061, filed March 22, 2012). The meganucleases have been classified into four families based on conserved sequence motifs, with the families being the LAGLIDADG, GIY-YIG, H-N-H, and His-Cys-box families. These motifs are involved in the coordination of metal ions and the hydrolysis of phosphodiester bonds. HEases are notable for their long recognition sites and for allowing some sequence polymorphisms in their DNA substrates. The nomenclature rules for meganucleases are similar to those for other restriction endonucleases. Meganucleases are also characterized by the prefix F-, I-, or PI- for enzymes encoded by autonomous ORFs, introns, and inteins, respectively. One step in the recombination process involves cleaving the polynucleotide at or near the recognition site. This splitting activity can be used to produce a double strand break. For reviews of site-specific recombinases and their restriction sites, see Sauer (1994) Curr Op Biotechnol 5:521-7 and Sadowski, (1993) FASEB 7:760-7. In some examples, recombinases belong to the integrase or resolvas families.

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Zinc finger nucleases (ZFNs) are engineered double-strand break-inducing agents containing a zinc finger DNA-binding domain and a double-strand gap-inducing agent domain. Recognition site specificity is provided by the zinc finger domain, which typically contains two, three or four zinc fingers, eg having the C2H2 structure, however other zinc finger structures are known and constructed. Zinc finger domains are engineerable polypeptides that specifically bind a selected recognition polynucleotide sequence. The ZFNs comprise an engineered zinc finger DNA binding domain associated with a non-specific endonuclease domain, for example a nuclease domain from a type II endonuclease such as FokI. Additional functionalities can be attached to the zinc finger binding domain, including transcriptional activator domains, transcriptional repressor domains, and methylases. In some examples, dimerization of the nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a 3-finger domain recognizes a sequence of 9 contiguous nucleotides; if nuclease dimerization is required, two sets of zinc finger triplets are used to bind the 18 nucleotide recognition sequence.

Genome editing using DSB-inducing agents such as Cas9gRNA complexes is described, for example, in US Patent Application US 2015-0082478 A1, WO2015/026886 A1, WO2016007347 and WO201625131, all of which are incorporated by reference herein.

The term Cas gene, as used herein, refers to a gene that is typically fused, linked, or located near or near flanking CRISPR loci in bacterial systems. The terms Cas gene, CRISPR-associated (Cas) gene are used interchangeably herein. The term Cas endonuclease as used herein refers to a protein or complex of proteins encoded by the Cas gene. The Cas endonuclease disclosed herein, when complexed with a suitable polynucleotide component, is capable of recognizing, binding, and optionally cutting or cleaving all or part of a specified target DNA sequence. The Cas endonuclease described herein contains one or more nuclease domains. The Cas endonucleases of the present invention include those having an HNH or HNH-like nuclease domain and/or a RuvC or RuvC-like nuclease domain. The Cas endonuclease of the present invention may include Cas9 protein, Cpf1 protein, C2c1 protein, C2c2 protein, C2c3 protein, Cas3, Cas5, Cas7, Cas8, Cas10, or complexes thereof.

As used herein, the targeting polynucleotide/Cas endonuclease complex, targeting polynucleotide/Cas endonuclease system, targeting polynucleotide/Cas complex, targeting polynucleotide/Cas system, targeting Cas system are used interchangeably herein and refer to at least one targeting polynucleotide and to at least one Cas endonuclease that is capable of forming a complex, wherein said guide polynucleotide/Cas endonuclease complex can direct the Cas endonuclease to a target DNA site, allowing recognition, binding, and optionally cutting or cleaving (introducing a single-strand or double-strand break) of the target DNA site by Cas endonuclease . The targeting polynucleotide/Cas endonuclease complex herein may comprise the Cas protein(s) and the suitable polynucleotide(s) component(s) of any of the four known CRISPR systems (Horvath and Barrangou, 2010, Science 327: 167-170), such as a type I, II, or III CRISPR system. Cas endonuclease unwinds a DNA duplex at the site of a target sequence and optionally cleaves at least one strand of DNA, which is mediated by recognition of the target sequence by a polynucleotide (such as, but not limited to, crRNA or guide RNA) that is complexed with a Cas protein. Such recognition and cleavage of the target sequence by the Cas endonuclease generally occurs when the correct protospacer adjacent motif (PAM) is located at or near the 3' end of the target DNA sequence. Alternatively, the Cas protein of this document may not have DNA cleavage or nicking activity, but may continue to specifically bind to the target DNA sequence when complexed with a suitable RNA component. (See also US 2015-0082478 A1 and US 2015-0059010 A1, both of which are incorporated herein by reference in their entirety.)

The guide polynucleotide/Cas endonuclease complex can cleave one or both strands of the target DNA sequence. A guide polynucleotide/Cas endonuclease complex that can cleave both strands of the target DNA sequence typically contains a Cas protein in which all of its endonuclease domains are in a functional state (for example, they are wild-type endonuclease domains or variants thereof that retain some degree of activity or total activity of each endonuclease domain). Thus, a wild-type Cas protein (e.g., the Cas9 protein disclosed herein) or a variant thereof that retains some or all of the activity of each endonuclease domain of a Cas protein is a suitable example of a Cas endonuclease that can cleave both strands of the target sequence. 9 041676 DNA. The Cas9 protein containing the functional nuclease domains of RuvC and HNH is an example of a Cas protein that can cleave both strands of a target DNA sequence. A guide polynucleotide/Cas endonuclease complex that can cleave one strand of a target DNA sequence can be characterized herein as having no case activity (eg, partial cleavage ability). Cas nickase typically contains a single functional endonuclease domain that allows Cas to cleave only one strand (ie, make a single strand break) of the target DNA sequence. For example, a Cas9 nickase may contain (i) a mutant, dysfunctional RuvC domain and (ii) a functional HNH domain (eg, a wild-type HNH domain). As another example, the Cas9 nikase may contain (i) a functional RuvC domain (eg, a wild-type RuvC domain) and (ii) a mutant, dysfunctional HNH domain. Non-limiting examples of Cas9 nicases suitable for use herein are disclosed in US Patent Application Publication No. 2014/0189896, which is incorporated herein by reference.

A pair of Cas9 nicases can be used to increase the specificity of DNA targeting. In general, this can be done by providing two Cas9 nikases that, by being associated with RNA components with different guide sequences, target and form a single strand break in adjacent DNA sequences on opposite strands at the desired targeting site. This closely spaced cleavage of each DNA strand produces a double strand break (i.e. DSB with single strand overhangs) which is then recognized as a substrate for NHEJ nonhomologous end joining (prone to incomplete repair leading to mutations) or HR homologous recombination. In these embodiments, each single strand break may be spaced, for example, at least about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 (or any integer from 5 to 100) bases apart. One or two Cas9 nickase proteins according to this document can be used in a pair of Cas9 nickases. For example, a Cas9 nickase with a mutant RuvC domain but a functioning HNH domain (ie Cas9 HNH+/RuvC-) (eg Cas9 HNH+/RuvC- from Streptococcus pyogenes) can be used. Each Cas9 nickase (e.g., Cas9 HNH+/RuvC-) will target specific DNA sites located close to each other (up to 100 bp apart) using appropriate RNA components according to this document with RNA guide sequences targeting each nickase to each specific DNA site.

The Cas protein may be part of a fusion protein containing one or more heterologous protein domains (eg, 1, 2, 3 or more domains in addition to the Cas protein). Such a fusion protein may contain any additional protein sequence and optionally a linker sequence between any two domains, such as between Cas and the first heterologous domain. Examples of protein domains that can be fused to the Cas protein according to this document include, without limitation, epitope tags (e.g., histidine [His], V5, FLAG, influenza virus hemagglutinin [HA], myc, VSV-G, thioredoxin [Trx]) , reporters (eg, glutathione 5-transferase [GST], horseradish peroxidase [HRP], chloramphenicol acetyltransferase [CAT], betagalactosidase, beta-glucuronidase [GUS], luciferase, green fluorescent protein [GFP], HcRed, DsRed, cyan fluorescent protein [CFP], yellow fluorescent protein [YFP], blue fluorescent protein [BFP]) and domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity (e.g. VP16 or VP64), downregulation activity transcription, transcription termination factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. The Cas protein can also be fused to a protein that binds DNA molecules or other molecules, such as the maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD), GAL4A DNA binding domain, and VP16 herpes simplex virus (HSV). ). See PCT patent applications PCT/US16/32073 filed May 12, 2016 and PCT/US16/32028 filed May 12, 2016 (both applications incorporated herein by reference) for additional examples of Cas proteins. .

In certain embodiments, the guide polynucleotide/Cas endonuclease complex can bind to the DNA target site sequence but does not cleave any strand in the target site sequence. Such a complex may contain the Cas protein, in which all of its nuclease domains are mutant and dysfunctional. For example, as used herein, a Cas9 protein that can bind to a DNA target site sequence but does not cleave any strand in the target site sequence may contain both a mutant, dysfunctional RuvC domain and a mutant, dysfunctional HNH domain. A Cas protein according to this document that binds but does not cleave a target DNA sequence can be used to modulate gene expression, for example, in this case, the Cas protein can be fused to a transcription factor (or part thereof) (for example, a repressor or an activator, such as any disclosed in this document). In other aspects, the inactivated Cas protein may be fused to another protein having endonuclease activity, such as the Fok I endonuclease.

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The Cas endonuclease gene herein may encode a type II Cas9 endonuclease, such as, without limitation, the Cas9 genes listed in SEQ ID NOs: 462, 474, 489, 494, 499, 505, and 518WO2007/025097 and incorporated herein by reference. In another embodiment, the Cas9 endonuclease gene is a microbial or optimized Cas9 endonuclease gene. The Cas endonuclease gene may be operably linked to an SV40 intranuclear targeting signal upstream of the Cas codon site and a bipartite VirD2 intranuclear localization signal (Tinland et al. (1992) Proc. Natl. Acad. Sci. USA 89:7442-6) downstream of the Cas codon site .

Other Cas endonuclease systems have been described in PCT patent applications PCT/US16/32073 and PCT/US16/32028, both applications are incorporated herein by reference.

Cas9 (formerly referred to as Cas5, Csn1 or Csx12) as used herein refers to the Cas endonuclease from the type II CRISPR system, which forms a complex with crNucleotide and tracrNucleotide or with a guide polynucleotide to specifically recognize and cleave all or part of a target DNA sequence. The Cas9 protein contains a RuvC nuclease domain and an HNH (H-N-H) nuclease domain, each of which can cleave one strand of DNA in the target sequence (the coordinated action of both domains results in double-stranded DNA cleavage, while the activity of one domain results in a single-strand break). In general, the RuvC domain contains subdomains I, II, and III, with domain I located near the N-terminus of Cas9, and subdomains II and III located in the middle of the protein, flanking the HNH domain (Hsu et al, Cell 157:1262-1278 ). The type II CRISPR system includes a DNA cleavage system using the Cas9 endonuclease in complex with at least one polynucleotide component. For example, Cas9 can be complexed with CRISPR RNA (crRNA) and CRISPR transactivating RNA (tracrRNA). In another example, Cas9 may be complexed with a single guide RNA.

A Cas protein of this document, such as Cas9, may contain a heterologous nuclear localization sequence (NLS). The heterologous amino acid sequence of NLS according to this document may be sufficiently effective, for example, to lead to the accumulation of the Cas protein in a detectable amount in the nucleus of the yeast cell according to this document. NLS contains one (single) or several (e.g. double) short sequences (e.g. having 2 to 20 residues) of basic, positively charged residues (e.g. lysine and/or arginine) and can be located anywhere in the Cas amino acid sequence , but so that it is located on the surface of the protein. The NLS may be operably linked to, for example, the N-terminus or C-terminus of the Cas protein as described herein. Two or more NLS sequences can be associated with a Cas protein, such as, for example, at both the N- and C-terminus of the Cas protein. Non-limiting examples of suitable NLS sequences include those disclosed in US Pat. No. 7,309,576, which is incorporated herein by reference.

The Cas endonuclease may comprise a modified form of the Cas9 polypeptide. A modified form of a Cas9 polypeptide may include an amino acid change (eg, deletion, insertion, or substitution) that reduces the native nuclease activity of the Cas9 protein. For example, in some instances, a modified form of a Cas9 protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 polypeptide (US Patent Application US20140068797 A1). In some cases, the modified form of the Cas9 polypeptide does not have sufficient nuclease activity and is referred to as catalytically inactivated Cas9 or deactivated cas9 (dCas9). Catalytically inactivated Cas9 variants include Cas9 variants that contain mutations in the HNH and RuvC nuclease domains. Such catalytically inactivated Cas9 variants are able to interact with sgRNA and bind to the target site in vivo, but cannot cleave any of the target DNA strands.

Catalytically inactive Cas9 can be fused to a heterologous sequence (US Patent Application US20140068797 A1). Suitable fusion partners include, without limitation, a polypeptide that provides an activity that indirectly increases transcription by acting directly on the target DNA or on a polypeptide (eg, a histone or other DNA-binding protein) associated with the target DNA. Additional suitable fusion partners include, without limitation, a polypeptide that provides methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity , deribosylation activity, myristoylation activity, or demyristoylation activity. Other suitable fusion partners include, without limitation, a polypeptide that directly provides for increased transcription of the target nucleic acid (e.g., a transcription activator or fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug sensitive transcription regulator, etc. ). Catalytically inactive Cas9 can also be fused to FokI nuclease to form double strand breaks (Guilinger et al. Nature Biotechnology, Volume 32, Number 6, June 2014).

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The terms functional fragment, fragment that is functionally equivalent, and functionally equivalent fragment of a Cas endonuclease are used interchangeably herein and refer to a portion or subsequence of the Cas endonuclease sequence of the present invention that retains the ability to recognize, bind, and optionally cut or cleave (administer a single or double strand break) target site.

The terms functional variant, variant that is functionally equivalent, and functionally equivalent variant of the Cas endonuclease are used interchangeably herein and refer to a variant of the Cas endonuclease of the present invention that retains the ability to recognize, bind, and optionally cut or cleave (introduce a single or double strand break). ) target site. Fragments and variants can be generated using methods such as site-directed mutagenesis and synthetic sequence engineering.

Any targetable endonuclease can be used in the methods disclosed herein. Such endonucleases include, without limitation, the Cas9 and Cpf1 endonucleases. So far, many endonucleases have been described that can recognize specific PAM sequences (see, for example, Jinek et al. (2012) Science 337 p 816-821, PCT patent applications PCT/US16/32073 and PCT/US16/32028 and Zetsche B et al 2015 Cell 163, 1013) and cleave the target DNA at certain positions. It will be understood that based on the methods and embodiments described herein that use a targeted Cas system, these methods can be adapted to use any targeted endonuclease system in a given situation.

As used herein, the term guide polynucleotide refers to a polynucleotide sequence that can form a complex with a Cas endonuclease and allows the Cas endonuclease to recognize, bind to, and optionally cleave a target DNA site. The targeting polynucleotide can be a single molecule or a dimeric molecule. The targeting polynucleotide sequence may be an RNA sequence, a DNA sequence, or a combination thereof (combined DNA-RNA sequence). Optionally, the targeting polynucleotide may contain at least one nucleotide, a phosphodiester bond, or a bond modification such as, but not limited to, a closed nucleic acid (LNA), 5-methyl-dC, 2,6-diaminopurine, 2'-fluoro-A, 2'- fluoro-U, 2'-O-methyl-RNA, a thiophosphate bond, a bond to a cholesterol molecule, a bond to a polyethylene glycol molecule, a bond to a spacer 18 molecule (hexaethylene glycol chain), or a 5'-3'-covalent bond resulting in circularization. A guide polynucleotide that contains only ribonucleic acids is also referred to as a guide RNA or gRNA (see also US 2015-0082478 A1 and US 20150059010 A1, both of which are incorporated herein by reference in their entirety).

The targeting polynucleotide may be a dimeric molecule (also referred to as a duplex targeting polynucleotide) containing a crNucleotide sequence and a tracrNucleotide sequence. CrNucleotide comprises a first nucleotide sequence domain (referred to as the variable target domain or VT domain) that can hybridize to a nucleotide sequence in the target DNA and a second nucleotide sequence (also referred to as a paired sequence for tracr) that is part of an endonuclease recognition domain (CER). A paired sequence for tracr can hybridize to tracrNucleotide along the complementarity region and together form a Cas endonuclease recognition domain or a CER domain. The CER domain is capable of interacting with the Cas endonuclease polypeptide. The CrNucleotide and tracrNucleotide of the duplex guide polynucleotide may be combined RNA, DNA and/or RNA-DNA sequences. In some embodiments, the duplex guide polynucleotide crNucleotide molecule is referred to as crDNA (when it consists of a continuous stretch of DNA nucleotides), or crRNA (when it consists of a continuous stretch of RNA nucleotides), or crDNA-RNA (when it consists of a combination of DNA - and RNA nucleotides). The crnucleotide may contain a cRNA fragment naturally occurring in Bacteria and Archaea. The size of the naturally occurring cRNA fragment in Bacteria and Archaea that may be present in the crNucleotide disclosed herein may vary without limitation in the range of 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, tracrNucleotide is called tracrRNA (if it consists of a continuous stretch of RNA nucleotides), or tracrDNA (if it consists of a continuous stretch of DNA nucleotides), or tracrDNA-RNA (if it consists of a combination of DNA and RNA nucleotides) . In one embodiment, the RNA that directs the RNA/Cas9 endonuclease complex is a duplex RNA containing a crRNA-tracrRNA duplex. tracrRNA (trans-activating CRISPR RNA) contains in the 5'-3' direction (i) a sequence that undergoes crRNA type II CRRPR repetitive annealing and (ii) a portion containing a stem-loop structure (Deltcheva et al., Nature 471 : 602607). The duplex guide polynucleotide may form a complex with a Cas endonuclease, where said polynucleotide/Cas endonuclease complex (also referred to as the guide polynucleotide/Cas endonuclease system) may direct the Cas endonuclease to a genomic target site, allowing recognition, binding, and optionally cutting or cleaving (introducing a single stranded

- 12 041676 or double-strand break) of the target site by Cas endonuclease. (See also US Patent Application

2015-0082478 A1, published March 19, 2015, and US 2015-0059010 A1, both of which are incorporated herein by reference in their entirety).

A single target polynucleotide can form a complex with a Cas endonuclease, where said polynucleotide/Cas endonuclease complex (also referred to as the target polynucleotide/Cas endonuclease system) can direct the Cas endonuclease to a genomic target site, allowing for recognition, binding, and optionally cutting or cleaving (introducing single or double stranded gap) of the target site by Cas endonuclease. (See also US Patent Application US 2015-0082478 A1 and US 2015-0059010 A1, both of which are incorporated herein by reference in their entirety.)

The terms variable targeting domain or VT domain are used interchangeably herein and include a nucleotide sequence that can hybridize (is complementary) to one strand (nucleotide sequence) of a double-stranded DNA target site. The percent complementarity between the first domain of the nucleotide sequence (VT domain) and the target sequence can be at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. The variable targeting domain may be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the implementation of the variable targeting domain contains a continuous stretch of 12-30 nucleotides. The variable targeting domain may be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.

The terms Cas endonuclease recognition domain or CER (guide polynucleotide) domain are used interchangeably herein and include a nucleotide sequence that interacts with a Cas endonuclease polypeptide. The CER domain contains a paired sequence for tracrNucleotide followed by a tracrNucleotide sequence. A CER domain may consist of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, for example, US 2015-0059010 A1, incorporated herein by reference in its entirety), or any combination thereof.

The terms functional fragment, fragment that is functionally equivalent and functionally equivalent guide RNA fragment, crRNA or tracrRNA are used interchangeably herein and refer to a portion or subsequence of the guide RNA, crRNA or tracrRNA, respectively, of the present invention, wherein the ability to function as a guide RNA, crRNA or tracrRNA, respectively, is preserved.

The terms functional variant, variant that is functionally equivalent, and functionally equivalent guide RNA, crRNA, or tracrRNA variant are used interchangeably herein and refer to a guide RNA, crRNA, or tracrRNA variant, respectively, of the present invention, wherein the ability to function as a guide RNA, crRNA, or tracrRNA is accordingly preserved.

The terms single guide RNA and sgRNA are used interchangeably herein and refer to the synthetic fusion of two RNA molecules, crRNA (CRISPR RNA), containing a variable targeting domain (linked to a pairing sequence for tracr that hybridizes to tracrRNA), fused to tracrRNA (trans- CRISPR activating RNA). A single guide RNA may contain a crRNA or a fragment of a crRNA and a tracrRNA or a fragment of a tracrRNA of the CRISPR/Cas type II system that can form a complex with a type II Cas endonuclease, where said guide RNA/Cas endonuclease complex can guide the Cas endonuclease to a target DNA site, allowing recognition , binding and optionally cutting or cleaving (introducing a single-strand or double-strand break) of the target DNA site with Cas endonuclease.

The terms guide RNA/Cas endonuclease complex, guide RNA/Cas endonuclease system, guide RNA/Cas complex, gRNA/Cas system, RNA-directed endonuclease are used interchangeably herein and refer to at least one RNA component, and at least to one Cas endonuclease that are capable of forming a complex, wherein said guide RNA/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, allowing the Cas endonuclease to recognize, bind, and optionally cut or cleave (introduce a single or double strand break) the target DNA site. The Cas guide RNA/endonuclease complex herein may comprise Cas protein(s) and suitable RNA component(s) of any of the four known CRISPR systems (Horvath and Barrangou, 2010, Science 327: 167-170), such as type I, II, or III CRISPR system. The guide RNA/Cas endonuclease complex may comprise a Cas9 type II endonuclease and at least one RNA component (eg crRNA and tracrRNA or gRNA). (See also US 2015-0082478 A1 and US 2015-0059010 A1, both of which are incorporated herein by reference in their entirety.)

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The targeting polynucleotide can be temporarily introduced into the cell as a single-stranded polynucleotide or a double-stranded polynucleotide using any method known in the art, such as, but not limited to, particle bombardment, Agrobacterium transformation, or topical applications. A targeting polynucleotide can also be introduced indirectly into a cell by introducing a recombinant DNA molecule (using methods such as, but not limited to, particle bombardment or Agrobacterium transformation) containing a heterologous nucleic acid fragment encoding a targeting polynucleotide operably linked to a specific promoter that is capable of transcribing the targeting polynucleotide. RNA in the specified cell. The specific promoter may be, but is not limited to, an RNA polymerase III promoter that provides transcription of RNA with well-defined, unmodified 5' and 3' ends (DiCarlo et al., Nucleic Acids Res. 41: 4336-4343; Ma et al., Mol. Ther. Nucleic Acids 3:e161) as described in WO2016025131, incorporated herein by reference in its entirety.

The terms target site, target sequence, target site sequence, target DNA, target locus, genomic target site, genomic target sequence, genomic target locus, and protospacer are used interchangeably herein and refer to a polynucleotide sequence, including but not limited to a nucleotide sequence in a chromosome , episome or any other DNA molecule in the genome (including chromosomal, chloroplastic, mitochondrial DNA, plasmid DNA) of a cell in which the guide polynucleotide/Cas endonuclease complex can recognize, bind and optionally cut or cleave. The target site may be an endogenous site in the cell's genome, or alternatively, the target site may be heterologous to the cell and thus not naturally occurring in the cell's genome, or the target site may be at a heterologous location in the genome compared to with the one in which it occurs in nature. As used herein, the terms endogenous target sequence and native target sequence are used interchangeably herein to refer to a target sequence that is endogenous or native to the genome of a cell. Cells include, without limitation, human cells, non-human cells, animal cells, bacteria, fungi, insects, yeast, non-traditional yeasts, and plant cells, as well as plants and seeds obtained using the methods described herein. The terms artificial target site or artificial target sequence are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a cell. Such an artificial target sequence may be identical in sequence to an endogenous or native target sequence in the cell's genome, but located at a different position (ie, non-endogenous or non-native position) in the cell's genome.

The terms altered target site, altered target sequence, modified target site, modified target sequence are used interchangeably herein and refer to a target sequence disclosed herein that contains at least one change compared to an unchanged target sequence. Such changes include, for example, (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

The length of the target DNA sequence (target site) can vary and includes, for example, target sites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. It is also possible that the target site may be palindromic, meaning that the sequence of one strand reads exactly the same backwards on the complementary strand. The nick/cleavage site may be within the target sequence, or the nick/cleavage site may be outside the target sequence. In another embodiment, cleavage may occur at nucleotide positions directly opposite each other to produce a blunt-ended cut, or, in other cases, the cuts may be asymmetrically spaced to produce single-stranded overhangs, also called cohesive ends, which may be 5' cohesive ends. or 3' sticky ends. You can also use active variants of genomic target sites. Such active variants may contain at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the target site, wherein active variants retain biological activity and are therefore able to be recognized and cleaved by Cas endonuclease. Assays for measuring single-strand or double-strand break of an endonuclease target site are known in the art and generally measure the overall activity and specificity of an agent on DNA substrates containing recognition sites.

A protospacer adjacent motif (PAM) as used herein refers to a short nucleotide sequence adjacent to a target sequence (protospacer) that is recognized (targeted) by the Cas guide polynucleotide/endonuclease system described herein. Cas endonuclease may not successfully recognize the target DNA sequence if the target DNA sequence is not followed by a PAM sequence.

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The sequence and length of the PAM according to this document may differ depending on the Cas protein or Cas protein complex used. The PAM sequence can be any length, but is usually 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.

The terms targeting, gene targeting, and DNA targeting are used interchangeably herein. DNA targeting according to this document may be the specific introduction of a knockout, edit, or knockin to a specific DNA sequence located on a chromosome or plasmid of a cell. In general, DNA targeting according to this document can be accomplished by cleaving one or both strands in a specific DNA sequence in a cell with an endonuclease associated with a suitable polynucleotide component. Such DNA cleavage, in the case of a double strand break (DSB), can stimulate NHEJ or HDR processes, which can lead to modifications at the target site.

The method of targeting according to this document can be carried out in such a way that, for example, two or more DNA target sites are targeted in the method. Such a method can optionally be characterized as a multiplex method. In certain embodiments, two, three, four, five, six, seven, eight, nine, ten or more target sites may be targeted at the same time. The multiplex method is typically performed using the targeting method of this document, in which a plurality of different RNA components are provided, each designed to direct the targeting polynucleotide/Cas endonuclease complex to a unique DNA target site.

The terms knockout, gene knockout and gene knockout are used interchangeably herein. A knockout according to this document corresponds to a DNA sequence in a cell that has become partially or completely inoperable due to the targeting of the Cas protein; moreover, such a DNA sequence before knockout, for example, could encode an amino acid sequence or could have a regulatory function (for example, a promoter). Knockout can be done by insertion/deletion (insertion or deletion of nucleotide bases in the target DNA sequence by NHEJ) or by specific deletion of a sequence that reduces or completely destroys the function of the sequence at or near the target site.

The targeting polynucleotide/Cas endonuclease system can be used in combination with a co-delivered polynucleotide modification template to allow editing (modification) of the genomic nucleotide sequence of interest. (See also US Patent Application US 2015-0082478 A1 and W02015/026886 A1, both of which are incorporated herein by reference in their entirety.)

The terms knockin, gene knockin, gene insert, and gene knockin are used interchangeably herein. Knockin corresponds to the substitution or insertion of a DNA sequence into a specific DNA sequence in a cell by targeting with the Cas protein (by HR, where a suitable donor DNA polynucleotide is also used). Examples of knockins include, without limitation, specific insertion of a heterologous amino acid coding sequence into a coding region of a gene, or specific insertion of a transcriptional regulatory element into a gene locus.

Various methods and compositions can be used to obtain a cell or organism having a polynucleotide of interest inserted into the target site for the Cas endonuclease. In such methods, homologous recombination can be used to ensure integration of the polynucleotide of interest into the target site. In one proposed method, a polynucleotide of interest is provided to an organism cell in a donor DNA construct. As used herein, the term donor DNA is a DNA construct that contains a polynucleotide of interest to be inserted into the target site of the Cas endonuclease. The donor DNA construct may further comprise first and second homology regions that flank the polynucleotide of interest. The first and second regions of donor DNA homology have homology with respect to the first and second regions of the genome, respectively, present in the target site of the cell or organism or flanking it. By homology is meant DNA sequences that are similar. For example, a region of homology to a region of the genome that is in the donor DNA is a region of DNA that has a similar sequence to that region of the genome in a cell or genome of an organism. The homology region may be of any length that is sufficient to allow homologous recombination at the cleaved target site. For example, a region of homology may contain at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5 -60, 5-65, 5-70, 5-75, 580, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5 -600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 51300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5 -1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 52600, 5-2700, 5-2800, 5-2900, 5-3000, 5-3100 or more bases in length, so that the region of homology has sufficient homology to undergo homologous recombination with the corresponding region of the genome. Sufficient homology indicates that two polynucleotide sequences have sufficient structural similarity to act as substrates for a homologous recombination reaction. Structural similarity includes the overall length of each polynucleotide fragment as well as the sequence similarity of the polynucleotides. Sequence similarity can be described by percent sequence identity over the entire length of the sequences, and/or by conserved regions containing localized similarities, such as contiguous nucleotides having 100% sequence identity and percent sequence identity over the length of the sequences.

Percent (%) sequence identity relative to the control sequence (considered) is defined as the percentage of amino acid residues or nucleotides in the candidate sequence (requested) that are identical to the corresponding amino acid residues or nucleotides in the control sequence after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity and without considering any amino acid conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in a variety of ways that are known to one of skill in the art, for example using publicly available computer software such as BLAST, BLAST-2. Suitable parameters for sequence alignment can be determined by those skilled in the art, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for purposes of optimal comparison. The percent identity of two sequences is a function of the number of identical positions present in both sequences (e.g., percent identity of the requested sequence=number of identical positions between the requested sequence and the sequence in question/total number of positions in the requested sequence (e.g., overlapping positions)x100).

The amount of homology or sequence identity separated by the target and donor polynucleotide can vary and includes total lengths and/or regions characterized by integer units in the ranges of approximately 1-20 bp, 20-50 bp, 50-100 bp .o., 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp ., 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 t.b., 1.5-3 t.b., 2-4 t.b., 2.5-5 t .o., 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including the total length of the target site. Such ranges include every integer within the range, such as the range of 1-20 bp. includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 p.o. The amount of homology can also be described by percent sequence identity over the entire aligned length of two polynucleotides, which includes percent sequence identity of at least about 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77 , 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. Sufficient homology includes any combination of polynucleotide length, overall percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, e.g. sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity with region of the target locus. Sufficient homology can also be described by the predicted ability of two polynucleotides to specifically hybridize under high stringency conditions, see, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al., Eds (1994) Current Protocols (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes (Elsevier, New York).

Structural similarity between a given region of the genome and the corresponding region of homology found in the donor DNA may represent any degree of sequence identity that allows homologous recombination to occur. For example, the amount of homology or sequence identity shared by the homology region of the donor DNA and the genomic region of the organism's genome may be at least 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86 , 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity such that sequences undergo homologous recombination

A region of homology in the donor DNA may share homology with any sequence flanking the target site. Whereas, in some embodiments, regions of homology have significant sequence homology to the genomic sequence immediately flanking the target site, it should be appreciated that regions of homology can be designed to provide sufficient homology to regions that, in addition, may be located 5'- or 3'- relative to the target site. In still other embodiments, regions of homology may also share homology with a fragment of the target site along with downstream regions of the genome. In one embodiment, the first homology region further comprises a first target site fragment, and the second homology region comprises a second target site fragment, where the first and second fragments are different.

The term homologous recombination as used herein includes the exchange of DNA fragments between two DNA molecules at sites of homology. The frequency of homologous recombination is affected by a number of factors. Different organisms differ in the amount of homologous recombination and the relative ratio of homologous to non-homologous recombination. As a rule, the length of the homology region affects the frequency of homologous recombination events, the longer the homology region, the greater the frequency. The length of the homology region required to observe homologous recombination also varies with species. In many cases homology of at least 5 kb was used, but homologous recombination was observed at only 25-50 bp homology. See, for example, Singer et al. (1982) Cell 31:25-33; Shen and Huang (1986) Genetics 112:441-57; Watt et al. (1985) Proc. Natl. Acad. sci. USA 82:4768-72, Sugawara and Haber (1992) Mol Cell Biol 12:563-75, Rubnitz and Subramani (1984) Mol Cell Biol 4:2253-8; Ayares et al. (1986) Proc. Natl. Acad. sci. USA 83:5199-203; Liskay et al. (1987) Genetics 115:161-7.

Homology-driven repair (HDR) is a mechanism in cells for the repair of double-strand and single-strand DNA breaks. Homology driven repair includes homologous recombination (HR) and single strand annealing (SSA) (Lieber. 2010 Annu. Rev. Biochem. 79:181-211). The most common form of HDR is called homologous recombination (HR), which has a requirement for the longest sequence homology between donor and acceptor DNA. Other forms of HDR include single-strand annealing (SSA) and break-induced replication, and they require shorter sequence homology compared to HR. Homology-driven repair of nicks (single-strand breaks) can occur by a mechanism different from HDR in double-strand breaks (Davis and Maizels. (2014) PNAS (0027-8424), 111(10), p. E924-E932).

Rearranging the plant cell genome, for example through homologous recombination (HR), is a powerful tool for genetic engineering. Homologous recombination has been demonstrated in plants (Halfter et al. (1992) Mol Gen Genet 231: 186-93) and insects (Dray and Gloor, 1997, Genetics 147: 689-99). Homologous recombination has also been carried out in other organisms. For example, at least 150-200 p. homology was necessary for homologous recombination in the parasitic protozoan Leishmania (Papadopoulou and Dumas, (1997) Nucleic Acids Res25:4278-86). In the micelle fungus Aspergillus nidulans, gene replacement was performed with as little as 50 bp. flanking homology (Chaveroche et al., (2000) Nucleic Acids Res 28:e97). Targeted gene replacement has also been demonstrated in the ciliate Tetrahymena thermophila (Gaertig et al., (1994) Nucleic Acids Res 22:5391-8). In mammals, homologous recombination has been most successful in mice using pluripotent embryonic stem (ES) cell lines that can be cultured, transformed, selected, and introduced into the mouse embryo (Watson et al., 1992, Recombinant DNA, 2nd Ed., ( Scientific American Books distributed by W. H. Freeman & Co.).

Error-prone DNA repair mechanisms can cause mutations at double-strand break sites. Non-homologous end joining (NHEJ) repair pathways are the most common repair mechanisms for joining broken ends together (Bleuyard, et al., (2006) DNA Repair 5:1-12). Structural integrity of chromosomes is usually preserved during repair, but deletions, insertions, or other rearrangements are possible. The two ends of a single double strand break are the most predominant substrates for NHEJ (Kirik, et al., (2000) EMBO J 19:5562-6), however, if two different double strand breaks occur, free ends from different breaks can bind and form as a result chromosomal deletions (Siebert and Puchta, (2002) Plant Cell 14:1121-31), or chromosomal translocations between different chromosomes (Pacher, et al., (2007) Genetics 175:21-9).

Donor DNA can be administered using any method known in the art. Donor DNA can be provided by any transformation method known in the art, including, for example, Agrobacterium-mediated transformation or particle bombardment during bioballistic transformation. The donor DNA may be temporarily present in the cell, or it may be introduced via a viral replicon. In the presence of the Cas endonuclease and the target site, the donor DNA is inserted into the genome of the transformed plant (see manual).

Further use of Cas guide RNA/endonuclease systems has been described (see US Patent Application US 2015-0082478 A1, WO2015/026886 A1, US 2015-0059010 A1, US Application 62/023246 and US Application 62/036652, all of which are included in herein by reference), which include, without limitation, modification or substitution of nucleotide sequences of interest (such as regulatory elements), insertion of polynucleotides of interest, gene knockout, gene knockin, modification of splice sites and/or introduction of alternative splice sites, modification of nucleotide sequences, encoding a protein of interest, amino acid and/or protein fusions, and gene silencing by expression of an inverted repeat in the gene of interest.

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II. Methods for producing maize plants with modified Ht 1 and/or NLB18 nucleotide sequences.

A. Ht1.

Mapping of QTL associated with northern leaf spot resistance on chromosome 2 is described in US Patent Application US2010095395. The Ht1 gene was cloned and identified as the putative CC-NB-LRR (supercoiled, nucleotide-binding, leucine-rich repeat) gene (US 62/242691). The Ht1 cDNA sequences from PH4GP and PH1W2 (another source of resistance-associated Ht1 allele; US Patent Application US2010095395) are shown under SEQ ID NO:51 and 53, respectively, while the amino acid sequences encoding the polypeptides are shown under SEQ ID NO:52 and 54 and are 99.6% identical. B73 (which has a susceptibility-associated allele) has two splicing variants and the new variant is expressed at a much higher level (also referred to herein as B73-high) than the known variant (also referred to herein as B73-low). SEQ ID NO:55 is the cDNA sequence of the B73-high allele, while the amino acid sequence of the encoded polypeptide is shown under SEQ ID NO:56. SEQ ID NO:57 is the cDNA sequence of the B73-low allele, while the amino acid sequence of the encoded polypeptide is shown under SEQ ID NO:58. The genomic sequence of the PH4GP (resistant) allele is provided herein as SEQ ID NO:59. The CC and NB domains are very similar between the susceptibility-associated allele (B73) and the resistance-associated alleles (from PH4GP and PH1W2) as shown in US 62/242691. However, B73 has a deletion in the LRR. The amino acid sequence of this region in resistance-associated Ht1 alleles is shown under SEQ ID NO:60.

Methods for producing a maize plant cell with a modified Ht1 nucleotide sequence include introducing a double strand break at one or more target sites in an endogenous HT1 coding sequence in a maize plant cell and obtaining a maize plant cell having a modified Ht1 nucleotide sequence. In one aspect, the methods include introducing a double strand break at one or more target sites in an endogenous Ht1 coding sequence in a maize plant cell and obtaining a maize plant cell having a modified Ht1 nucleotide sequence. The method may further comprise introducing an NLB18 replacement template into a maize plant cell, wherein said Ht1 replacement template contains at least one nucleic acid change from an endogenous Ht1 coding sequence, and wherein said Ht1 replacement template is included in an endogenous Ht1 coding sequence. Ht1. The method may further comprise introducing an Ht1 replacement template into a maize plant cell, wherein said Ht1 replacement template contains at least one nucleic acid change from an endogenous HT1 coding sequence, and wherein said Ht1 replacement template is included in an endogenous coding sequence. NT1. The double strand break can be induced by a nuclease, including but not limited to TALEN, meganuclease, zinc finger nuclease, or CRISPR-associated nuclease. The method may further comprise growing a maize plant from a maize plant cell having a modified Ht1 nucleotide sequence, wherein the maize plant may exhibit increased resistance to northern leaf spot.

The Ht1 nucleotide sequence provided herein may refer to the Ht1 promoter, exons, introns, and/or terminator sequences, either as a whole or as fragments.

An endogenous HT1 coding sequence refers to a nucleotide sequence that is present in an unmodified maize plant cell and encodes an HT1 polypeptide.

The Ht1 replacement template is a polynucleotide modification template containing a favorable version of the Ht1 nucleotide sequence (ie one that confers increased resistance to northern leaf spot).

Maize plants show increased resistance to northern leaf spot compared to equivalent maize plants lacking the modified Ht1 nucleotide sequence. Equivalent means that maize plants are genetically similar except for the Ht1 sequence.

In some aspects, the modified Ht1 nucleotide sequence provides for a deletion in the promoter of the endogenous sequence encoding HT1. In this case, the use of Cas9 endonuclease and one or more guide RNAs may be envisaged. If two guide RNAs are used, the first guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:1 [Ht1-TS2] and the second guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:2 [Ht1-TS4] ; or the first guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:1 [Ht1-TS2], and the second guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:3 [Ht1-ST1-TS1].

In other aspects, an Ht1 replacement template is used that contains an Ht1 nucleotide sequence from PH4GP, or a fragment thereof, or an Ht1 nucleotide sequence that, when introduced into a maize plant cell, encodes a polypeptide with an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO:52. In this case, the use of Cas9 endonuclease and one or more guide RNAs may be envisaged. If two guide RNAs are used, the first guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:14 [Ht1-TS6] and the second guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:16 [Ht1TS9]; or the first guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:15 [Ht1-TS7], and the second guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:17 [Ht1-TS10].

B. NLB18.

Mapping of QTL associated with northern leaf spot resistance on chromosome 8 is described in international patent application WO2011163590. Two protein kinase (PK)-like genes with highly conserved kinase catalytic domains have been identified in close proximity and referred to in international patent application WO2011163590 as NLB17 and NLB18. NLB18 has been confirmed as a gene conferring increased resistance to northern leaf spot (unpublished). The NLB18 cDNA sequences from PH26N and PH99N, two stable sources described in WO2011163590, are shown under SEQ ID NO:61 and 63, respectively, while the amino acid sequences of the encoded polypeptides are shown under SEQ ID NO:62 and 64. SEQ ID NO:62 and SEQ ID NO:64 are 92.4% identical.

Provided herein are methods for producing a maize plant cell with a modified NLB18 nucleotide sequence. The methods include introducing a double strand break at one or more target sites in an endogenous NLB18 coding sequence in a maize plant cell and obtaining a maize plant cell having a modified NLB18 nucleotide sequence. The method may further comprise introducing an NLB18 replacement template into a maize plant cell, wherein said NLB18 replacement template contains at least one nucleic acid change from an endogenous NLB18 coding sequence, and wherein said NLB18 replacement template is included in an endogenous NLB18 coding sequence. NLB18. The double strand break can be performed with a nuclease such as, but not limited to, TALEN, meganuclease, zinc finger nuclease, or CRISPR associated nuclease. The method may further comprise growing a maize plant from a maize plant cell having a modified NLB18 nucleotide sequence, wherein the maize plant may exhibit increased resistance to northern leaf spot.

The NLB18 nucleotide sequence provided herein may refer to the NLB18 promoter, exons, introns, terminator sequences, and/or any other genomic nucleotide sequence located at the NLB18 genomic locus, either as a whole or as fragments.

An endogenous sequence encoding NLB18 refers to a nucleotide sequence that is present in an unmodified maize plant cell and encodes an NLB18 polypeptide.

The NLB18 replacement template is a polynucleotide modification template containing a favorable version of the NLB18 nucleotide sequence (ie one that confers increased resistance to northern leaf spot).

Maize plants show increased resistance to northern leaf spot compared to equivalent maize plants lacking the modified NLB18 nucleotide sequence. Equivalent means that maize plants are genetically similar except for the NLB18 sequence.

In some aspects, the modified NLB18 nucleotide sequence comprises a modification in the promoter of the endogenous sequence encoding NLB18.

In other aspects, an NLB18 replacement template is used that contains an NLB18 nucleotide sequence of PH26N or PH99N, or an NLB18 nucleotide sequence that, when introduced into a maize plant cell, encodes a polypeptide with an amino acid sequence that is at least 90, 91, 92, 93, 94 , 95, 96, 97, 98 or 99% identical to SEQ ID NO:62 or SEQ ID NO:64. In some aspects, the NLB18 replacement matrix contains SEQ ID NO:70. In some embodiments, the use of an NLB replacement template may include the use of a Cas9 endonuclease and one or more guide RNAs. If two guide RNAs are used, the first guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:30 [NLB18-TS1] and the second guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:32 [NLB18-TS4] ; or the first guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:31 [NLB18TS8] and the second guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:32 [NLB18-TS4].

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III. Methods for obtaining maize plant cells with a genomic locus containing nucleotide sequences that confer increased resistance to northern leaf spot.

Polynucleotides of interest and/or features can be combined together in a complex feature locus as described in US 2013/0263324-A1 and PCT/US13/22891, both of which are incorporated herein by reference.

This document provides methods for producing a maize plant cell with a genomic locus containing at least one nucleotide sequence that confers increased resistance to northern leaf spot. The disclosed methods include introducing a double strand break at one or more target sites in the genomic locus of a maize plant cell; the introduction of one or more nucleotide sequences that confer increased resistance to northern leaf spot, where each nucleotide sequence is flanked by 300-500 p. nucleotide sequences located 5' or 3' relative to the respective target sites; and providing a maize plant cell having a genomic locus containing one or more nucleotide sequences that confer increased resistance to northern leaf spot. The double strand break can be induced with a nuclease such as, but not limited to, TALEN, meganuclease, zinc finger nuclease, or CRISPR associated nuclease. The method may further comprise growing a maize plant from a maize plant cell having a genomic locus containing at least one nucleotide sequence that provides increased resistance to northern leaf spot, and wherein the maize plant can exhibit increased resistance to northern leaf spot.

Maize plants exhibit increased resistance to northern leaf spot compared to equivalent maize plants lacking nucleotide sequences conferring increased resistance to northern leaf spot at the genomic locus of interest. Equivalent means that maize plants are genetically similar except for the genomic locus of interest.

In some aspects, one or more nucleotide sequences that confer increased resistance to northern leaf spot include any of the following: Ht1PH4GP, NLB18-PH26N, and NLB18-PH99N. The Ht1-PH4GP nucleotide sequence may comprise SEQ ID NO:59 or any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% SEQ ID NO:52 wherein the polypeptide confers increased resistance to northern leaf spot in the maize plant. In some aspects, the nucleotide sequence of Ht1-PH4GP is SEQ ID NO:65. The nucleotide sequence of NLB18-PH26N may comprise any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:64, wherein the polypeptide confers increased resistance to northern leaf spot in the maize plant. In some aspects, the nucleotide sequence of NLB18-PH26N is SEQ ID NO:70. The NLB18-PH99N nucleotide sequence may comprise any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:62, wherein said polypeptide confers increased resistance to northern leaf spot in a maize plant.

In other aspects, the genomic locus that confers increased resistance to northern leaf spot contains CTL1. In still other aspects, the nucleotide sequence encoding NLB18-PH26N is targeted at TS8 CTL1; the nucleotide sequence encoding NLB18-PH4GP targets TS10 CTL1; and/or the nucleotide sequence encoding NLB18-PH26N is targeted at TS45 CTL1.

The guide polynucleotide/Cas9 endonuclease system described herein provides an efficient system for the formation of double strand breaks and allows features to be stacked into a complex feature locus. Thus, in one aspect, Cas9 endonuclease is used as a DSB inducing agent and one or more guide RNAs are used to target Cas9 to sites at the CTL1 locus. One guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:36 [CTL1-TS8]; one guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:37 [CTL1-TS10], and one guide RNA may contain a variable targeting domain that is complementary to SEQ ID NO:38 [CTL1-TS45].

Producing maize plants using the methods described herein can confer long-term and broad spectrum tolerance to northern leaf spot and can promote the breeding of northern leaf spot resistant maize plants. For example, since the nucleotide sequences that confer increased resistance to northern leaf spot are in close association with each other (at the same locus), this results in a reduction in the number of specific loci that require trait introgression by backcrossing and minimization of undesirable linked traits from non-elite stable donors.

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As used herein, the term "heterologous with respect to sequence" means that the sequence originates from a foreign species or, if it originates from the same species, is substantially modified in composition and/or location in the genome compared to its native form as a result of deliberate human intervention. . For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide is derived, or if it is from the same/similar species, one or both of them are substantially modified from their original form and/or location in the genome, or the promoter is not a native promoter for the operably linked polynucleotide.

IV. Maize plant cells, plants and seeds.

Maize belongs to the plant Zea mays L. ssp. mays, also known as corn. The use of ZM in front of an object described in this document refers to the fact that the object is derived from Zea mays.

Also contemplated are maize plants, maize plant cells, maize plant parts and seeds, and maize grains having the modified Ht1 or NLB18 sequences disclosed herein.

As used herein, the term plant includes plant cells, plant protoplasts, tissue cultures of plant cells from which plants can be regenerated, plant calluses, aggregations of plant cells, and plant cells that are intact in plants or plant parts such as germs, pollen, ovules. , seeds, leaves, flowers, grains, spikelets, cobs, leaf wrappers, stems, roots, root tips, anthers, etc. Grain is intended to mean mature seed obtained by commercial growers for purposes other than cultivation or reproduction of a species.

V. Guide polynucleotides.

This document also provides guide polynucleotides containing variable targeting domains complementary to target sites in the endogenous Ht1 coding sequence, the endogenous NLB18 coding sequence, or the CTL1 genomic locus. Such guide polynucleotides can be RNA sequences, DNA sequences, or combined RNA-DNA sequences. For Ht1, targeting polynucleotides may have a variable targeting domain complementary to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. For NLB18, targeting polynucleotides may have a variable targeting domain complementary to SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. For CTL1, targeting polynucleotides may have a variable targeting domain complementary to SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.

Examples

The following examples are provided to illustrate and not to limit the appended claims. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that those skilled in the art will appreciate that various reagents or parameters may be changed without departing from the spirit of the present invention or the scope of the appended claims.

Example 1

Editing the HT1 gene by removing repetitive sequences in the promoter region.

Target site selection.

The site-directed gRNA/Cas9 nuclease system described in WO2015026885, WO20158026887, WO2015026883 and WO2015026886 was used to edit the Htl gene in maize (WO2017066597, which is incorporated herein by reference). The following target site pairs were used to delete a repeat sequence in the Ht1 promoter region of PH184C (represented by SEQ ID NO:71): HT1-TS2 and HT1-TS4, and HT1-TS2 and HT1ST1-TS1. The location of each target site in the Htl genomic sequence and a schematic representation of the result of the deletion are shown in FIG. 1, and the target sequences are listed in table. 1.

Table 1. Promoter sequences of Ht1 genomic target sites

Designation target site Maize genomic target site sequence Subsequence PAM Htl-TS2 SEQID NO:1 TGG html-ts4 SEQ ID NO:2 AGG Htl-STl-TSl SEQ ID NO:3 TTAGAAA

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Cas9 vector design.

The Cas9 gene from Streptococcus pyogenes Ml GAS (SF370) (SEQ ID NO:4) was codon optimized for maize using standard techniques known in the art, and the potato ST-LS1 intron was introduced to abolish its expression in E. coP and Agrobacterium. To facilitate nuclear localization of the Cas9 protein in maize cells, a single amino-terminal simian virus 40 (SV40) intranuclear localization signal (SEQ ID NO:5) was included at the amino end of the Cas9 open reading frame. The optimized maize Cas9 gene was operably linked to the maize ubiquitin promoter using standard molecular biology techniques. In addition to the SV40 amino-terminal nuclear localization signal, Agrobacterium tumefaciens VirD2 endonuclease C-terminal two-part nuclear localization signal was fused at the end of exon 2. The resulting sequence is SEQ ID NO:72 including the Zea mays ubiquitin promoter, 5'-UTR of the ubiquitin gene ZM, intron 1 of the ZM ubiquitin gene, SV40 intranuclear localization signal, Cas9 exon 1 (ST1), potato LS1 intron, Cas9 exon 2 (ST1), VirD2 endonuclease intranuclear localization signal, and pinII terminator.

Guide RNA vector design.

In order to direct the Cas9 nuclease to the designated genomic target sites (in Table 1), the polymerase III U6 promoter (SEQ ID NO:6; see WO2015026885, WO20158026887, WO2015026883 and WO2015026886) and its related polymerase III U6 terminator sequences (TTTTTTTT) were used to control the initiation and termination of gRNA expression. Variable targeting domains of the HT1 gene editing guide RNA have been identified as HT1-CR2 and HT1CR4, which correspond to the genomic target sites HT1-TS2, HT1-TS4 and HT1-ST1-CR1 correspond to HT1-ST1-TS, respectively. The DNA encoding each of the nucleotide targeting variable domains was cloned into the gRNA expression cassette via BsbI sites using double stranded oligos. Each guide RNA expression cassette consists of a maize U6 polymerase III promoter operably linked to one of the DNA versions of the guide RNA (Table 2) and then a related U6 polymerase III terminator sequence. The DNA version of the guide RNA consists of an appropriate nucleotide variable targeting domain followed by a polynucleotide sequence capable of interacting with a double-strand break-inducing endonuclease. The HT1-ST1-CR1 guide RNA expression cassette was inserted into the ST1 Cas9 expression cassette using standard procedures.

Table 2. Guide RNA expression cassettes

Name DNA version of guide RNA Htl-CR2 SEQ ID NO:7 Htl-CR4 SEQ ID NO:8 Htl-STl-CRl SEQ ID NO:9

Delivery of the DNA guide RNA/Cas9 endonuclease system to maize.

Plasmids containing the expression cassettes of Cas9 and the guide RNA described above were co-bombarded with plasmids containing the selectable transformation marker NPTII and development-enhancing ODP2 genes (ODP2 transcription factor with AP2 domains (responsible for ovule development protein 2)) and Wuschel (20151030-6752 USPSP) into the genomes of elite maize lines. Transformation of immature maize germ can be performed using any method known in the art or the method described below.

In one transformation method, cobs were stripped of their leaf wrap, surface sterilized in 30-50% Clorox bleach along with 0.5% Micro detergent for 10 minutes, and rinsed twice with sterile water. Immature embryos were isolated and placed rib down (shield up), 25 embryos per plate, on 13224E medium for 2-4 hours and then aligned within a 2.5 cm target zone in the bombardment preparation.

Plasmid DNA was glued to 0.6 μm (average diameter) gold beads using the proprietary TransIT®-2020 lipid polymer blend (Cat. No. MIR 5404, Mirus Bio LLC, Madison, WI 5371). A DNA solution was prepared using 1 μg of plasmid DNA and optionally other constructs were prepared for co-bombardment using 10 ng (0.5 μl) of each plasmid. 50 µl of prepared gold particles (30 mg/ml) and 1 µl of TransIT®-2020 were added to the premixed DNA and mixed gently. The final mixture was allowed to incubate with constant vortexing at low speed for 10 minutes. After a settling period, the tubes were rapidly centrifuged and the liquid removed. Gold particles were precipitated in a microcentrifuge at 10,000 rpm for 1 min and the supernatant was removed. 120 μl 100% EtOH was added and the particles were resuspended by rapid sonication. Then 10 μl was applied to the center of each macrocarrier and allowed to dry for approximately 2 minutes before bombardment, a total of ten aliquots were withdrawn from each prepared particle/DNA tube.

Sample plates were bombarded with a Biolistic PDA-1000/He (Bio-Rad). Embryos

- 22 041676 are 6 cm from the macro carrier with a 1/8 inch gap between the 200 psi burst pressure disc and the macro carrier. All samples received a single shot.

After bombardment, embryos were incubated on a bombardment plate for ~20 h, then transferred to 13266L (rest/induction medium) for 7-9 days at temperatures ranging from 26-30°C. Embryos were then transferred to 289H maturation medium at ~21 days. Then, mature somatic embryos were transferred to 272G germination medium and exposed to light. After about 1-2 weeks, seedlings containing viable shoots and roots were selected for analysis and sent to the greenhouse where they were transferred to trays (equivalent to a 2.5-inch pot) containing potting soil. After 1-2 weeks, the plants were transferred to 600 classic pots (1.6 gal) and grown to maturity.

Wednesday.

Bombardment medium (13224E) contains 4.0 g/l basic N6 salts (SIGMA C-1416), 1.0 ml/l Ericsson's vitamin mixture (1000X SIGMA-1511), 0.5 mg/l thiamine-HCl, 190 0 g/l sucrose, 1.0 mg/l 2,4-D and 2.88 g/l L-proline (brought to volume with DI H ₂ O after adjusting the pH to 5.8 with KOH); 6.3 g/l Sigma agar (added after making up to volume with DI H2O) and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).

Sampling medium (13266L) contains 1650 mg/l ammonium nitrate, 277.8 mg/l ammonium sulfate, 5278 mg/l potassium nitrate, calcium chloride, 407.4 mg/l anhydrous calcium chloride, 234.92 mg/l anhydrous magnesium sulfate, 410 mg/l anhydrous potassium phosphate, 8 mg/l monobasic boric acid, 8.6 mg/l, zinc sulfate 7H2O, 1.28 mg/l potassium iodide, 44.54 mg/l iron sulfate-7H ₂ O, 59.46 mg / l Na2edta-2H2O, 0.025 mg / l cobalt chloride FN ₂ O, 0.4 mg / l molybdic acid (sodium salt / 2H ₂ O, 0.025 mg / l copper sulfate 5H ₂ O, 6 mg / l manganese sulfate monohydrate, 2 mg/l thiamine, 0.6 ml/l b5h microsalts 1000x, 0.4 ml/l Eriksson vitamins 1000x, 6 ml/l s&h stock solution of vitamins 100x, 1.98 g/l Lproline, 3 .4 mg/l silver nitrate, 0.3 g/l casein hydrolyzate (acid), 20 g/l sucrose, 0.6 g/l glucose, 0.8 mg/l 2,4-d, 1.2 mg /l dicamba, b g/l tc agar, 100 mg/l agribio carbenicillin, 25 mg/l cefotaxime and 150 mg/l geneticin (g418).

Plant regeneration medium (289H) contained 4.3 g/L MS salts (GIBCO 11117-074), 5.0 mL/L MS vitamin stock solution (0.100 g nicotinic acid, 0.02 g/L thiamine-HCL , 0.10 g/l pyridoxine-HCL and 0.40 g/l glycine, brought to volume with purified DI H ₂ O) (Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l myoinositol, 0.5 mg/l zeatin, 60 g/l sucrose and 1.0 ml/l 0.1 mM abscisic acid (brought to volume with purified DI H2O after adjusting the pH to 5.6); 8.0 g/l Sigma agar (added after making up to volume with DI H ₂ O), as well as 1.0 mg/l indoleacetic acid and 150 mg/l geneticin (G418) (added after sterilizing the medium and cooling to 60 °C).

Hormone-free medium (272G) contained 4.3 g/L MS salts (GIBCO 11117-074), 5.0 mL/L MS vitamin stock solution (0.100 g/L nicotinic acid, 0.02 g/L thiamine-HCl , 0.10 g/l pyridoxine-HCl and 0.40 g/l glycine, brought to volume with purified DI H2O), 0.1 g/l myoinositol and 40.0 g/l sucrose (brought to volume with purified DI H2O after adjusting the pH to 5.6); and 0.5 mg/l IBA and 150 mg/l geneticin (G418) and 6 g/l bactoagar (added after making up to volume with purified DI H ₂ O), it was sterilized and cooled to 60°C.

Screening of T0 Plants and Transformant Characterization To identify positive transformants with deletion of repeat sequences, genomic DNA was isolated from leaf tissue of T0 plants and PCR was performed using the Phusion Master Mix (Thermo Scientific F-581) and the primers listed in Table 1. 3. Primer arrangements are shown in FIG. 1. Amplicons were obtained by digesting TS2/TS4 or TS2/ST1-TS1; the sequence (~35 kb) between the two sites was deleted, and the remaining sequences were combined. Received options with a deletion with the expected size of the product.

Next generation sequencing (NGS) was used to evaluate the sequences of the splice boundary regions of the deletion positive transformants. Splice boundary regions were amplified by PCR with the PHUSION® High Fidelity PCR Master Mix (Thermo Scientific, F-531). The same primers can be used for both CR2/CR4 and CR2/ST1-CR1 deletions. The primers used in the primary PCR reaction are shown in Table 1. 3 and the primers used in the secondary PCR reaction are shown in SEQ ID NO:12 and SEQ ID NO:13. NNNNNNNN in the reverse primer is a barcode sequence corresponding to the location of the sample on the plate. In FIG. 2A shows the sequence of splicing boundary regions resulting from a TS2/TSS4 deletion; and in FIG. 2B shows sequences of splicing boundary regions obtained from a TS2/ST1-TS1 deletion. A brief description of the resulting T0 transformants with a deletion is presented in Table 1. 4.

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Table 3. Primers used to screen for deletion of repeat sequences

Name deletions Primer name Primer orientation Primer SEQ ID NO: Htl-CR2-CR4de Htlf3 Straight SEQ ID NO:10 Htlr4v2 Back SEQ ID NO:11

Table 4. Brief description of deletion T0 transformants

guide Number of embryos bombarded Number of TO plants screened Number of plants with a deletion CR2/CR4 2000 178 59 CR2/ST1 1000 108 29

T1 analysis.

T0 plants with a repetitive Ht1 sequence were transferred to a controlled environment. Pollen from T0 plants was transferred to recurrent parent plants to obtain seeds. T1 plants were subjected to more extensive molecular characterization, not only to confirm that the mutations observed in the T0 plant were stably inherited, but also to ensure that T1 or later generation plants were free of any foreign DNA elements used in the process. transformations. First, qPCR was performed on all helper genes, including Cas9, guide RNA, selective marker of transformation (NPTII), and transformation-enhancing genes ODP2 and WUS2, to ensure that the genes were separated from the resulting mutant alleles. T1 plants will be sampled using Southern Sequencing (SbS) analysis to further demonstrate that the plants do not contain any foreign DNA.

Example 2

HT1 gene editing by allele substitution.

Target site crashes.

The site-directed gRNA/Cas9 nuclease system described in WO2015026885, WO20158026887, WO2015026883 and WO2015026886 was used to edit the Ht1 gene by replacing the native allele with the resistance-associated Ht1 allele from PH4GP (US2010095395; SEQ ID NO:65). The following target site pairs were used to remove the entire Ht1 allele from the PH184C line (US 8445763), including the putative promoter, coding sequence and 1 kb. 3'-UTR: HT1-TS6 and HT1-TS9, as well as HT1-TS7 and HT1-TS10. The location of each target site and a schematic representation of the allele exchange are shown in FIG. 3. Target sequences are listed in table. 5. DNA repair template co-delivered with Cas9 plasmids and guide RNA.

Table 5. Target site sequences for HT1 allelic substitution

Destination site designation Maize genomic target site sequence Subsequence PAM HT1-TS6 SEQ ID NO:14 TGG HT1-TS7 SEQ ID NO:15 CGG HT1-TS9 SEQ ID NO:16 AGG HT1-TS10 SEQ ID NO:17 AGG

Cas9 vector design.

See example 1.

Guide RNA vector design.

In order to direct the Cas9 nuclease to the designated genomic target sites (in Table 4), the polymerase III U6 promoter (SEQ ID NO:6; see WO2015026885, WO20158026887, WO2015026883 and WO2015026886) and its related polymerase III U6 terminator sequences (TTTTTTTT) were used to control the initiation and termination of gRNA expression. HT1 gene editing guide RNA variable targeting domains have been identified as HT1-CR6, HT1CR7, HT1-CR9, and HT1-CR10, which correspond to the genomic target sites HT1-TS6, HT1-TS7, HT1TS9 and HT1-TS10, HT1-TS10, respectively. Oligo containing DNA encoding each of the nucleotide targeting variable domains were synthesized and cloned into an expression cassette.

- 24 041676 gRNA as described in Example 1. Each guide RNA expression cassette consists of the maize polymerase III U6 promoter operably linked to one of the DNA versions of the guide RNA (Table 6) and then a cognate U6 polymerase III terminator sequence. The DNA version of the guide RNA consists of an appropriate nucleotide variable targeting domain followed by a polynucleotide sequence capable of interacting with a double-strand break-inducing endonuclease.

Table 6. Guide RNA expression cassettes for Ht1 allele exchange

Name DNA version of guide RNA SEQID NO: HT1-CR6 SEQ ID NO:18 HT1-CR9 SEQ ID NO:19 HT1-CR7 SEQ ID NO:20 HT1-CR10 SEQ ID NO:21

Vector design of matrix for reparation.

The CR6/CR9 substitution/replacement template contains the ZM-HT1(PH4GP) resistance-associated allele and homology sequences flanking 5'HT1-TS6 and 3'HT1-TS9; the CR7/CR10 replacement template contains the same resistance-associated ZM-HT1-(PH4GP) allele and homology sequences flanking 5'HT1-TS7 and 3'HT1-TS11 in the PH184C lineage. Homology arm sequences (SEQ ID NOs: 81-84) were synthesized and then cloned with ZM-HT1-(PH4GP) replacement genomic sequences using the standard Gibson seamless cloning method.

Delivery of the DNA guide RNA/Cas9 endonuclease system to maize.

Plasmids containing Cas9 expression cassettes, guide RNA, and the replacement template described above were co-bombarded with plasmids containing the selectable transformation marker NPTII and developmental-enhancing ODP2 genes (0DP2 transcription factor with AP2 domains (responsible for ovule development). protein 2)) and Wuschel (20151030-6752 USPSP) into the genomes of elite maize lines. Transformation of immature maize germ can be performed using any method known in the art or the method described in Example 1.

Screening of T0 plants and characterization of the transformant.

The protocol for extracting DNA from the leaves of the T0 plant is the same as described in Example 1. PCR was performed using a premixed Sigma Extract-N-Amp PCR to identify positive transformants upon exchange. PCR was performed to analyze HR1 splice boundary regions using primer pair Ht1HR1f1/Ht1HR1r1, while primary PCR with primer pair Ht1HR2f1 and Ht1HR2r1 was combined with secondary qPCR with allelic differentiation to screen for HR2 splice boundary regions due to high homology of putative edited variants and unmodified genomic sequences. Primers for primary PCR and primers and probes for the 2nd qPCR are listed in table. 7. The same assay previously described for CR6/CR9 exchange is also used to screen transformants for CR7/CR10 allele exchange.

Table 7. Primers used to screen for Ht1 allele exchange variants

Primer name Primer orientation Primer sequence for the first PCR Plot splicing boundaries HR1 HtlHRlfl Straight SEQ ID NO:22 HtlHRlrl Back SEQ ID NO:23 Plot splicing boundaries HR2 HtlHR2fl Straight SEQ ID NO:24 HtlHR2rl Back SEQ ID NO:25 Plot splicing boundaries HR2 2nd qPCR hdr2b_f Straight SEQ ID NO:26 hdr2b_r Back SEQ ID NO:27 hdr2b_PV Probe 6FAM-SEQ ID NO:28 hdr2b_PG Probe VIC-SEQ ID NO:29

- 25 041676

The allele exchange variants identified will be further molecularly characterized and qPCR will be used to screen T1 (BCO) plants for null segregants that are not expected to contain the plasmid DNA used during transformation initiation. Southern sequencing will also be performed to confirm null segregant plants. In table. 8 shows a summary of the T0 results obtained from allele exchange experiments. Three T0 plants were identified as potential allele exchange variants among the 300 screened T0 plants.

Table 8. Summary of results of T0 allele exchange screening

Number of TOs screened Number of maintenance HR1 Number of maintenance HR2 Number of maintenance HR1+HR2 100 2 2 1 150 13 3 1 50 4 3 1

Example 3

Editing of NLB18 genes through allele substitution.

Target site selection.

The cell wall associated kinase (WAK) gene, NLB18, has been identified and validated as a northern leaf spot resistance gene (WO2011163590). The NLB18 gene is clustered with NLB17 on the long arm of chromosome 8. The NLB18 and NLB17 genes are 6.9 kb apart. and have a high degree of homology; thus, identifying a unique target site for NLB18 allele exchange is challenging. Many sites have been identified and guide RNAs tested. Guides that excised only the NLB18 gene region and not the NLB17 gene region were selected to replace the NLB18 allele.

The site-directed gRNA/Cas9 nuclease system described in WO2015026885, WO20158026887, WO2015026883 and WO2015026886 was used to edit the NLB18 gene. The following target site pairs were used to remove the entire NLB18 allele from the PH184C line, including the potential promoter, coding sequence, and 3'UTR: NLB18-TS1 and NLB18-TS4, and NLB18-TS8 and NLB18-TS4. The location of each target site at the NLB18 locus and a schematic representation of the allele exchange are shown in FIG. 5. Target sequences are listed in table. 9. The deleted allele is replaced by the resistance-associated allele NLB18 from PH26N (US 6765132; SEQ ID NO:70); DNA repair template was co-delivered with Cas9 plasmids and guide RNA.

Table 9. Target site sequences for NLB18 allelic substitution

Destination site designation Maize genomic target site sequence Subsequence PAM NLB18-TS1 SEQ ID NO:30 TGG NLB18-TS8 SEQ ID NO:31 CGG NLB18-TS4 SEQ ID NO:32 GGG

Cas9 vector design.

See example 1.

Guide RNA vector design.

In order to direct the Cas9 nuclease to the designated genomic target sites (in Table 9), the polymerase III U6 promoter (SEQ ID NO:6; see WO2015026885, WO20158026887, WO2015026883 and WO2015026886) and its related polymerase III U6 terminator sequences (TTTTTTTT) were used to control the initiation and termination of gRNA expression. Variable targeting domains of the guide RNA for the NLB18 gene have been identified as NLB18-CR1, NLB18-CR8, and NLB18-CR4, which correspond to the genomic target sites of NLB18-TS1, NLB18-TS8, and NLB18-TS4, respectively. Oligo containing DNA encoding each of the nucleotide-targeting variable domains were synthesized and cloned into a gRNA expression cassette as described in Example 1 above. Each guide RNA expression cassette consists of the maize polymerase III U6 promoter operably linked to one of the DNA -versions of the guide RNA (Table 10) and then the related U6 polymerase III terminator sequence. The DNA version of the guide RNA consists of an appropriate nucleotide variable targeting domain followed by a polynucleotide sequence capable of interacting with a double-strand break-inducing endonuclease.

- 26 041676

Table 10. Guide RNA expression cassettes for NLB18 allele exchange

Name DNA version of the guide RNA SEQID NO: NLB18-CR1 SEQ ID NO:33 NLB18-CR8 SEQ ID NO:34

NLB18-CR4 SEQ ID NO:35

Vector design of a substitution matrix.

NLB18-CR1/CR4 substitution/replacement templates contain the resistance-associated ZM-NLB18 allele (from PH26N) and homology sequences flanking 5' NLB18-TS1 (SEQ ID NO:67) and 3' NLB18-TS4 (SEQ ID NO: 68) in RN184C; matrices for NLB18-CR1/CR4 substitution contain the same resistance-associated ZM-NLB18 allele (from PH2 6N) and homology sequences flanking 5' NLB18-TS8 (SEQ ID NO:69) and 3' NLB18-TS4 (SEQ ID NO :68) in PH184C. SEQ ID NO:66 is the nucleotide sequence of NLB18 from PH184C, including 5' NLB18-CR8 to 3' NLB18-CR4. Homology arm sequences were synthesized with an additional sequence containing restriction sites; after restriction digestion, they were assembled together with the desired resistance-associated NLB18 allele (from PH26N) into a yeast backbone using standard in vivo yeast assembly protocols. Plasmids from pooled yeast transformants of the assembly reaction were recovered in E. coli and quality controlled plasmids were used as co-bombardment templates.

Delivery of the DNA guide RNA/Cas9 endonuclease system to maize.

Plasmids containing expression cassettes for Cas9, guide RNA, and substitution templates described above were bombarded with plasmids containing the selectable transformation marker NPTII and developmental-enhancing ODP2 genes (ODP2 transcription factor with AP2 domains (responsible for ovule development protein 2)) and Wuschel (20151030-6752 USPSP) into the genomes of elite maize lines. Transformation of immature maize germ can be performed using any method known in the art or using the method described in Example 1.

Screening of T0 plants and characterization of the transformant.

Screening will be carried out similarly to the experiments described earlier.

Example 4

Movement of resistance-associated HT1 and NLB18 alleles into a complex trait locus.

Selection of target sites.

A maize genomic window extending from ZMO1:13.7MM to ZMO1:16.4MM on chromosome 1 was identified and designed to evolve into complex trait locus (CTL) 1 (WO2016040030). Three sites CTL1, TS8, TS10 and TS45 were selected to translocate the NLB resistance genes NLB18PH26N (SEQ ID NO:70), Ht1-PH4GP (SEQ ID NO:65) and NLB18-PH26N (SEQ ID NO:70) respectively . In table. 11 shows the positions of the genetic map for target sites for the Cas endonuclease (TS8, TS45, TS10) and FIG. 6 schematically shows the location of these sites.

Table 11 Target sites for Cas endonuclease in the complex trait locus (CTL1) on maize chromosome 1

Target site Target site sequence Subsequence PAM Genetic position of PH B73v2 (cM) CTL1-TS8 SEQ ID NO:36 TGG 52.56 CTL1-TS45 SEQ ID NO :37 AGG 53.66 CTL1-TS10 SEQ ID NO:38 GGG 54.56

Vector construct of Cas9, guide RNA and donor template The Cas9 gene from Streptococcus pyogenes M1 GAS (SF370) was codon optimized for maize using standard techniques known in the art, and the potato ST-LS1 intron was introduced to abolish its expression in E. coli and Agrobacterium. To facilitate the intranuclear localization of the Cas9 protein in maize (SV40) cells, a single amino-terminal signal of the intranuclear localization of Simian virus 40 (SEQ ID NO:5) and a two-part signal sequence of the intranuclear localization signal of the carboxyl end of the VirD2 endonuclease for the T-DNA border regions of Agrobacterium tumefaciens (SEQ ID NO:80) were included at the amino and carboxyl ends of the Cas9 open reading frame, respectively. SEQ ID NO:73 is the nucleotide sequence containing Cas9 used in Example 4; SEQ ID NO:73 contains exon 1 (SP) cas9, intron 2 ST-LS1, exon 2 (SP) Cas9 and intranuclear localization signal

- 27 041676

VirD2. (The SP version of Cas9 differs from the ST version used in previous examples with respect to codon usage; however, the SP version and the ST version encoded by SEQ ID NO:4 are identical.) The optimized maize Cas9 gene was operably linked to the maize ubiquitin promoter using standard techniques. molecular biology.

The maize polymerase III U6 promoter (SEQ ID NO:6; see WO2015026885, WO20158026887, WO2015026883 and WO2015026886) was used to express guide RNAs that direct Cas9 nuclease to designated genomic sites. The guide RNA coding sequence was 77 bp. in length and contained a 12-30 bp variable targeting domain. from the selected target site of the maize genome at the 5' end of the U6 terminator of maize polymerase III.

In order for Cas9 endonuclease and guide RNA to form a protein/RNA complex to allow site-specific cleavage of double-stranded DNA, Cas9 endonuclease and guide RNA must be present simultaneously. To improve their co-expression and the presence of Cas9 endonuclease expression cassettes and guide RNA were combined into one DNA construct. A 480-490 bp sequence was synthesized containing a guide RNA coding sequence, a 12-30 bp variable targeting domain. from a selected target site in the maize genome and a portion of the U6 promoter. The sequence was then cloned into a backbone already having the cas9 expression cassette and the rest of the gRNA expression cassette via BstBI/HindIII restriction sites.

The transposition template for CTL1-8CR1 contains the resistance-associated ZM-NLB18 allele (from PH26N) (SEQ ID NO:70) and homology sequences flanking 5' CTL1-TS8 (SEQ ID NO:85) and 3' CTL1-TS8 (SEQ ID NO:86) in PH184C. The transposition template for CTL1-45CR1 contains the resistance-associated ZM-NLB18 allele (from PH26N) (SEQ ID NO:70) and homology sequences flanking 5' CTL1-TS45 (SEQ ID NO:87) and 3' CTL1-TS45 (SEQ ID NO:88) in PH184C. The transposition template for CTL1-10CR3 contains the resistance-associated ZM-HT1 allele (from PH4GP) (SEQ ID NO:65) and homology sequences flanking 5' CTL1-TS10 (SEQ ID NO:89) and 3' CTL1-TS10 (SEQ ID NO:90) in PH184C. Sequences of homology arms of 300-500 bp were synthesized. and then cloned with the desired resistance-associated allele sequence using the standard Gibson seamless cloning method.

A plasmid containing the codon-optimized maize endonuclease Cas9 expression cassette and guide RNA expression cassette was co-delivered with a plasmid containing a DNA template containing NLB18-PH2 6N (Fig. 7 or Fig. 8) or ZM-HT1 (PH4GP) (Fig. 9), which, after gene integration by homologous recombination (homology-guided repair), will be integrated into the indicated site when the sites are cleaved by Cas9.

RNA-DNA targeting constructs targeting various maize genomic sites and template DNA constructs that were designed to introduce resistance genes into Cas endonuclease target sites via homologous recombination (homology-driven repair) are shown in Table 1. 12. These guide RNAs, Cas9 DNA constructs, and DNA repair templates were co-delivered into the elite maize (eg, PH184C) genome using the stable transformation procedure described in Example 1.

Table 12. RNA/Cas9 target and DNA repair template used in stable maize transformation to insert Ht1-PH4GP or NLB18-PH26N into ZM01 CTL1

Experiment guide RNA SEQ ID NO: Moving matrix DNA SEQ ID NO: CTL1-TS8 ZM-U6:08CR1 74 08CR1HR1-NLB18(PH26N) genomic subsequence- 8CR1HR2 77 CTL1-TS45 ZM-U6:45CR1 75 45CR1HR1-NLB18(PH26N) genomic subsequence- 45CR1HR2 78 CTL1-TS10 ZM-U6:10CR3 76 10CR3HR1-HT1 (PH4GP) genomic sequence10CR3HR2 79

Delivery of DNA Guide RNA/Cas9 Endonuclease System to Maize See Example 1.

Screening of T0 plants and characterization of the transformant.

In order to identify transformants with displacement (positive transformants with

- 28 041676 inset), genomic DNA was isolated from leaf tissue of T0 plants, and PCR was performed on the splicing boundary region using the prepared Sigma Extract-N-Amp PCR mixture.

The location of the primers shown in figures 7, 8 and 9, and the sequence of primers are shown in table. 13. PCR screening of splicing margin regions for insert transformants showed three T0 plants with both HR1 and HR2 splicing margins for the CTL1-TS8 experiment, four T0 plants with both HR1 and HR2 splicing margins for the CTL1-TS10 experiment, and four T0 plants with both parts of the splicing boundary HR1 and HR2 for the CTL1-TS45 experiment. The identified T0 plants will be further characterized in the next generation.

Table 13. Primers used to screen for insertion (transfer) in transformant

Experiments Primer name Primer orientation Primer sequence NLB18-PH26N up to CTL1 TS8 8HRlfl Straight SEQ ID NO:39 PH26NPr Back SEQ ID NO:40 PH26NTf Straight SEQ ID NO:41 8HR2rl Back SEQ ID NO:42 Htl-PH4GP to CTL1 TS10 loHRlf Straight SEQ ID NO:43 HtlPr Back SEQ ID NO:44 HtlTf Straight SEQ ID NO:45 10HR2r Back SEQ ID NO:46 NLB18-PH26N up to CTL1 TS45 45hrlfl Straight SEQ ID NO:47 PH26NPr Back SEQ ID NO:48 PH26NTf Straight SEQ ID NO:49 45hr2rl Back SEQ ID NO:50

T2 Plant Screening and Transformant Characterization T0 plants containing the NLB18 (BC26N) and HT1 (ED4GP) transposition were backcrossed to wild-type recurrent parents to produce BC0 (T1) seeds. BC0 seedlings were molecularly characterized for PCR splicing boundary regions to confirm insertion in CTL1-TS45 with NLB18 (BC26) and insertion in CTL1-TS10 with HT1 (ED4GP) inherited for the next generation. qPCR for helper genes used during transformation was also performed to ensure they were separated from moving plants, null segregants were also confirmed by SbS (Southern sequencing). Seeds from own plants (BC0F2) were planted in the field, zygosity analysis was performed on leaf samples, a 1:2:1 homozygous:hemizygote:null insertion ratio was observed for both CTL1-TS45 with BC26N and CTL1-TS10 with HT1 (ED4GP) insert. Plants were also analyzed for RNA expression using qRT-PCR after NLB inoculation. Compared to null, both hemizygotes and homozygotes showed resistance to infection, and expression of both NLB18 and HT1 was upregulated (FIGS. 10 and 11).

The transformants were tested in the greenhouse for efficacy against the northern leaf spot pathogen (Exserohilum turcicum). First, all transformants were infected with a pathogen against which the Ht1 and NLB18 genes are known to confer resistance. All positive transformants determined by qPCR were resistant to the pathogen. The results are shown in tables 14 and 15. The number of plants for each transformant is indicated in column N, representing the number of plants tested. The persistence score for NLNB ranges from 1 to 9, with 9 being the most robust.

Table 14. TS10 Ht1-ED4GP

Zygosity Average NLFBLT Standard error NLFBLT N homozygous 7.3 0, 1 16 Hemizygosity 6, 3 oh 1 28 Zero 4.9 oh 1 12

-

Claims (10)

Table 15. TS45 NLB18-BC26N Zygosity Average NLFBLT Standard error NLFBLT N homozygous 8.5 0, 1 53.0 Hemizygosity 6, 2 0, 1 91.0 Zero 4.7 oh 1 55.0 CLAIM 1. A method for producing a plant cell with a modified NLB18 nucleotide sequence, wherein the plant cell exhibits resistance to northern leaf spot, the method comprising: (a) introducing, with a nuclease selected from the group consisting of TALEN, a meganuclease, a zinc finger nuclease, and a CRISPR-associated nuclease, a site-specific modification into at least one target site of an endogenous genomic locus encoding NLB18 in a plant cell; (b) obtaining a plant cell having a modified NLB18 nucleotide sequence; and (c) introducing an NLB18 replacement template into a plant cell, wherein said NLB18 replacement template contains at least one nucleic acid change compared to an endogenous genomic locus encoding NLB18, and where said NLB18 replacement template is incorporated into the endogenous genomic locus, encoding NLB18; where the specified NLB18 replacement matrix includes: (A) any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:64; (B) any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:62. 2. The method of claim 1, wherein said modified NLB18 nucleotide sequence comprises a modification in the promoter of the endogenous NLB18 coding sequence. 3. The method of claim 1, wherein said method further comprises growing a plant from a plant cell having a modified NLB18 nucleotide sequence. 4. The method of claim 3, wherein said plant exhibits increased resistance to northern leaf spot. 5. The method according to claim 1, where the nucleotide sequence contains SEQ ID NO:70. 6. The method of claim 1, wherein said site-specific modification is induced with Cas9 endonuclease. 7. The method of claim 6 wherein the Cas9 endonuclease is directed by at least one guide RNA. 8. Plant cell obtained using the method according to any one of claims 1 to 7, where the specified plant cell contains: (A) any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 64; (B) any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 62. 9. A northern leaf spot resistant plant comprising the plant cell of claim 8, wherein said plant comprises: (A) any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 64; (B) any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 62. 10. The seed produced by the plant according to claim 9, where the specified seed contains: (A) any nucleotide sequence that encodes a polypeptide having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 64; -