Methods and compositions for corn disease resistance to multiple diseases

Abstract

Provided herein are cisgenic maize plants comprising a first and / or a second cisgenic block that encode(s) disease resistance polypeptides. Also provided are methods of making the cisgenic plants, biological samples derived therefrom, DNA constructs encoding the first and second cisgenic blocks, and other related methods and compositions.

Description

Atty Docket No: 212644-WO-SEC-1METHODS AND COMPOSITIONS FOR CORN DISEASE RESISTANCE TOMULTIPLE DISEASES

[0001] The present disclosure is related to methods and compositions for corn disease resistance to multiple diseases, including resistance to southern rust, northern leaf blight, gray leaf spot, and anthracnose stalk rot.REFERENCE TO THE SEQUENCE LISTING

[0002] The official copy of the sequence listing is submitted electronically as an XML file named “212644-WO-SEC-1_Sequence_Listing” created on September 25, 2025. The XML file has a size of 210,427 bytes and is filed concurrently with this specification. The sequence listing contained in this XML file document is part of the present specification and is incorporated herein by reference in its entirety.BACKGROUND

[0003] Southern rust (SR) is a disease of maize plants caused by the fungal pathogen Puccinia polysora. P. polysora does not survive the winter in many corn-growing areas, but, under proper weather conditions, the pathogen can be transported from warmer, more humid regions to regions with colder winters. SR is a concern to farmers and grain producers due to its potential for reducing yields and / or quality of maize crops. Due to its potential for rapid development and spread under favorable conditions, SR can be especially damaging to corn.

[0004] Gray leaf spot (GLS) is a disease of maize plants caused by the fungal pathogens Cercospora zeae-maydis and Cercospora zeina. GLS is one of the most significant diseases of maize worldwide. GLS can cause significant reductions in yield, grain weight and quality. Yield losses occur from premature plant death that interrupts filling of the grain and from stalk breakage and lodging that causes ears to be lost in the field. GLS occurs in all corn growing areas and can result in 10% to 20% losses.

[0005] Anthracnose stalk rot (ANTROT) caused by the fungal pathogen Glomerella graminicola (also known as Colletotrichum graminicola') is one of the major stalk rot diseases in maize. ANTROT is a major concern due to significant reduction in yield, grain weight and quality. Yield losses occur from premature plant death that interrupts filling of the grain and from stalk breakage and lodging that causes ears to be lost in the field. ANTROT occurs in all corn growing areas and can result in 10 to 20% losses.

[0006] Northern leaf blight (NLB), induced by the fungal pathogen Exserohilum turcicum (previously called Helminthosporium turcicum), is a serious foliar wilt disease of maize in many tropical and temperate environments. Symptoms can range from cigar-shaped lesionsAtty Docket No: 212644-WO-SEC-1 on the lower leaves to complete destruction of the foliage, thereby reducing the amount of leaf surface area available for photosynthesis. A reduction in photosynthetic capability leads to a lack of carbohydrates needed for grain fill, which impacts grain yield. Mid-altitude regions of the tropics, about 900-1600 m above sea level, have a particularly favorable climate for northern leaf blight, as dew periods are long and temperatures moderate. However, NLB can also result in losses of 30-50% during wet seasons in temperate environments such as in the United States, particularly if the infection is established on the upper leaves of the plant by the silking stage.

[0007] While techniques for reducing the impact of SR, GLS, ANTROT, and NLB on maize crops exist, there remains a need for development of new techniques for managing and minimizing the potential impact of these diseases. In particular, there remains a need for plants that combine resistance against multiple pathogens that cause SR, GLS, ANTROT, and NLB and methods for introducing such combined resistance in agronomically desirable elite maize lines.SUMMARY

[0008] Plants contain a variety of genes and allelic variations thereof in their chromosomes, many of which may be desired for inclusion into new varieties. However, those genes and alleles are often not linked in a manner to facilitate efficient breeding in combination with the many other traits that may be desired in any given variety. For example, resistance against multiple diseases is an essential component of crop improvement, especially as disease pressure and patterns are quickly evolving under a changing climate. Resistance against a specific disease is typically achieved by introgressing a genomic region from a resistant source to an elite line. This process is time consuming and often leads to yield drag and other deleterious effects. In addition, introgressing loci conferring resistance against multiple diseases becomes impractical (in the context of time and resources) because of the number of loci involved and difficult in the case of genetically linked loci. This disclosure provides various methods and compositions to overcome difficulties in breeding with multiple loci and provides a platform for chromosomal engineering of gene stacks (e.g., the cisgenic blocks disclosed herein), such as for example, disease resistant genes.

[0009] The present disclosure provides maize plants that comprise cisgenic construct(s) that provide increased resistance to one or more of SR, GLS, ANTROT, and NLB. Resistance is provided via the resistance genes encoded by the cisgenic construct(s). Breeding and / or genetic engineering techniques can be used to produce plants comprising the resistance provided via the cisgenic construct(s). The plants provided herein can improve agriculturalAtty Docket No: 212644-WO-SEC-1 yields and / or reduce or eliminate the need for fungicide application when SR, GLS, ANTROT, and / or NLB is a concern.

[0010] Provided herein is a corn plant comprising a first cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12. In some examples, the corn comprising the first cisgenic block also comprises a second cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15.

[0011] Provided herein is a different corn plant comprising the second cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15.

[0012] Also provided herein is a first cisgenic DNA construct comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12. For example, the first cisgenic construct can comprise a first block comprising the sequence set forth in SEQ ID NO: 1 . Further provided is a plant comprising the first cisgenic construct.

[0013] Also provided herein is a second cisgenic DNA construct comprising a nucleic acid sequence encoding four individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15. For example, the construct can comprise a first block comprising the sequence set forth in SEQ ID NO: 2. Further provided is a plant comprising the second cisgenic construct..

[0014] Also provided herein is a method of producing hybrid corn seeds comprising a) crossing a first corn line with a second corn line; b) growing the progeny plants; and c) harvesting hybrid seed produced by the progeny plants to produce progeny plants. The first corn line comprises a first cisgenic block comprising the sequence set forth in SEQ ID NO: 1 (or encoding polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12) and a second cisgenic block comprising the sequence set forth in SEQ ID NO: 2 (or encodingAtty Docket No: 212644-WO-SEC-1 polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15).

[0015] Also provided herein is a method for producing a corn plant having increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot. The method comprises crossing a first parent corn plant with a second parent corn plant. The first parent corn plant comprises a first cisgenic block comprising the sequence set forth in SEQ ID NO: 1 (or encoding polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12) and a second cisgenic block comprising the sequence set forth in SEQ ID NO: 2 (or encoding polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15). The second parent corn plant is susceptible to southern corn rust, gray leaf spot, northern leaf blight, anthracnose stalk rot, or combinations thereof. The method produces a plurality of first generation progeny plants. The method comprises selecting a first generation progeny plant that comprises the first cisgenic block and second cisgenic block. The selected progeny plant has increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot as compared to the second parent plant.

[0016] In one aspect, provided herein is a method of detecting, in a biological sample derived from a corn plant, a cisgenic block comprising SEQ ID NO: 1 . The block can be detected by any suitable method for genotyping, including, for example, methods that employ nucleotide sequencing, polynucleotide amplification, and the like. In one example, the method comprises isolating or providing a sample comprising nucleic acid from the corn plant, contacting the sample with at least one pair of nucleic acid primers, one primer capable of binding a first portion of SEQ ID NO: 1 and the second primer capable of binding to either a second portion of SEQ ID NO: 1 or to genomic sequence from site CR3. The method comprises performing a nucleic acid amplification reaction and detecting an amplification product produced via the at least one pair of primers. Detection of the amplification product indicates that said sample is derived from a plant comprising the cisgenic block. In another example, the method comprises isolating or providing a sample comprising nucleic acid from the corn plant, and using nucleotide sequencing to detect the cisgenic block comprising SEQ ID NO: 1 . Detection by sequencing can include detecting the junction sequence between SEQID NO: 1 and its insertion site or by detecting one or more junction sequences between SEQ ID NOs: 3, 4, or 5.Atty Docket No: 212644-WO-SEC-1

[0017] In another aspect, provided herein is a method of detecting, in a biological sample derived from a corn plant, a cisgenic block (e.g. a cisgenic genomic bock) comprising SEQ ID NO: 2. The block can be detected by any suitable method for genotyping, including, for example, methods that employ nucleotide sequencing, polynucleotide amplification, and the like. In one example, the method comprises contacting the sample with at least one pair of nucleic acid primers, one primer capable of binding a first portion of SEQ ID NO: 2 and a second primer capable of binding either a second portion of SEQ ID NO: 2 or a genomic sequence from site CR18. The method comprises performing a nucleic acid amplification reaction and detecting an amplification product produced via the at least one pair of primers. Detection of the amplification product indicates that said sample is derived from a plant comprising the cisgenic block. In another example, the method comprises isolating or providing a sample comprising nucleic acid from the corn plant, and using nucleotide sequencing to detect the cisgenic block comprising SEQ ID NO: 2. Detection by sequencing can include detecting the junction sequence between SEQID NO: 2 and its insertion site (e.g., a homology arm sequence) or by detecting one or more junction sequences between SEQ ID NOs: 6, 7, 8, or 9.

[0018] Also provided herein is a method of detecting, in a biological sample derived from a corn plant, two cisgenic blocks, one block comprising SEQ ID NO: 1 and another block comprising SEQ ID NO: 2. The method comprises contacting the sample with nucleic acid primer(s) and sequencing the one or more cisgenic blocks and comparing the sequencing results to one or both of SEQ ID NO: 1 and SEQ ID NO: 2.

[0019] Also provided herein is a method for producing a corn plant comprising a first cisgenic block. The method comprises providing one or more initial corn plants or plant cells that are susceptible to one or both of northern leaf blight and southern corn rust. The method comprises introducing a first cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11, and the polypeptide sequence of SEQ ID NO: 12 into the genome of one or more of the plants or plant cells, thereby producing one or more modified plants or plant cells comprising the first cisgenic block in their genome. The method comprises selecting at least one of the modified corn plants or corn plant cells, which preferably have improved resistance to one or both of northern leaf blight and southern corn rust relative to the initial corn plants or plant cells.

[0020] Also provided herein is a method for producing a corn plant comprising a second cisgenic block, the method comprising providing one or more initial corn plants or plant cells that are susceptible to one or more of northern leaf blight, gray leaf spot, and anthracnose stalk rot.Atty Docket No: 212644-WO-SEC-1The method comprises introducing a cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15 into the genome of one or more of the corn plants or plant cells, thereby producing one or more modified corn plants or plant cells comprising the second cisgenic block in their genome. The method comprises selecting at least one of the modified corn plants or corn plant cells, which preferably have improved resistance to one or both of northern leaf blight and southern corn rust relative to the initial corn plants or plant cells.

[0021] Also provided herein is a method for producing a cisgenic corn plant. The method comprises 1) providing one or more corn plants or plant cells, that are susceptible to one or more of northern leaf blight, gray leaf spot, and anthracnose stalk rot; 2) introducing: a first cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12 or a second cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15 into the genome of one or more of the plants or plant cells, thereby producing one or more modified plant cells comprising the first or second cisgenic block in their genome; 3) introducing the second or first cisgenic block, respectively, not used in step 2 into the genome of one or more of the modified plant cells, thereby producing one or more doubly modified plants or plant cells comprising both of the first and second cisgenic blocks; and 4) selecting at least one of the doubly modified corn plant cells.

[0022] Also provided herein is a method of protecting a corn plant against one or both of northern leaf blight or southern corn rust. The method comprises expressing, in the corn plant, one or more polypeptides from a first cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12.

[0023] Additionally, provided herein is a method of protecting a corn plant against one or more of northern leaf blight, gray leaf spot, or anthracnose stalk rot. The method comprises expressing, in the corn plant, one or more polypeptides from a second cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising,Atty Docket No: 212644-WO-SEC-1 respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15.

[0024] Further provided is a method of protecting a corn plant against one or more of northern leaf blight, southern corn rust, gray leaf spot, or anthracnose stalk rot. The method comprises expressing nucleic acid sequences encoding SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , the polypeptide sequence of SEQ ID NO: 12, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15. The nucleic acid sequences can be expressed from a first cisgenic block comprising a nucleic acid sequence encoding the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12 and from a second cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15.BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0025] The disclosure can be more fully understood from the following detailed description and the sequence listing which forms a part of this application.

[0026] The sequence descriptions and sequence listing attached hereto comply with the rules governing nucleotide and / or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §§1.831-1.834 and in the WIPO ST.26 Standard. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1 .832. Unless stated otherwise, the case of individual letters representing amino acid or nucleotide residues does not have any significance (e.g., “atgc” is the same as “ATGC”, and “MRYG” is the same as “mryg”. Although only one strand is shown, each disclosed nucleotide sequence should be understood to encompass its complementary strand (e.g., its reverse complement sequence).

[0027] SEQ ID NO: 1 is the DNA sequence of cisgenic block 1 .

[0028] SEQ ID NO: 2 is the DNA sequence of cisgenic block 2.

[0029] SEQ ID NO: 3 is the DNA sequence of the NLB18 portion of cisgenic block 1 .

[0030] SEQ ID NO: 4 is the DNA sequence of the RppK portion of cisgenic block 1 .

[0031] SEQ ID NO: 5 is the DNA sequence of the Ht1 portion of cisgenic block 1 .

[0032] SEQ ID NO: 6 is the DNA sequence of the Rcg2 portion of cisgenic block 2.

[0033] SEQ ID NO: 7 is the DNA sequence of the Rcz1 portion of cisgenic block 2.Atty Docket No: 212644-WO-SEC-1

[0034] SEQ ID NO: 8 is the DNA sequence of the Rcz2 portion of cisgenic block 2.

[0035] SEQ ID NO: 9 is the DNA sequence of the NLB18 portion of cisgenic block 2.

[0036] SEQ ID NO: 10 is the NLB18 polypeptide encoded by SEQ ID NOs: 3 and 9 used in cisgenic blocks 1 and 2, respectively.

[0037] SEQ ID NO: 11 is the RppK polypeptide sequence encoded by SEQ ID NO: 4.

[0038] SEQ ID NO: 12 is the Ht1 polypeptide sequence encoded by SEQ ID NO: 5.

[0039] SEQ ID NO: 13 is the Rcg2 polypeptide sequence encoded by SEQ ID NO: 6.

[0040] SEQ ID NO: 14 is the Rcz1 polypeptide sequence encoded by SEQ ID NO: 7.

[0041] SEQ ID NO: 15 is the Rcz2 polypeptide sequence encoded by SEQ ID NO: 8.

[0042] SEQ ID NOs: 16 and 17 represent a pair of primers capable of priming a PCR reaction using a template comprising a junction between SEQ ID NO: 1 and the CR3 target site.

[0043] SEQ ID NOs: 18 and 19 represent a pair of primers capable of priming a PCR reaction using a template comprising a junction between SEQ ID NO: 1 and the CR3 target site.

[0044] SEQ ID NOs: 20 and 21 represent a pair of primers capable of priming a PCR reaction using a template comprising a junction between SEQ ID NO: 2 and the CR18 target site.

[0045] SEQ ID NOs: 22 and 23 represent a pair of primers capable of priming a PCR reaction using a template comprising a junction between SEQ ID NO: 2 and the CR18 target site.

[0046] SEQ ID NOs: 24-44 represent groups of 3 oligonucleotides. The first of each group is forward primer. The second of each group is a reverse primer. The third of each group is a fluorescent probe oligonucleotide, each of which comprises a 5’ FAM fluorescent tag and 3’ MGB quencher tag. The characteristics of these oligonucleotides are described in more detail in Table 2 and the Examples.

[0047] SEQ ID NO: 45 is a 5’ homology arm used to direct insertion of cisgenic block 1 into the CR3 site via homologous recombination.

[0048] SEQ ID NO: 46 is a 3’ homology arm used to direct insertion of cisgenic block 1 into the CR3 site via homologous recombination.

[0049] SEQ ID NO: 47 is a 5’ homology arm used to direct insertion of cisgenic block 2 into the CR18 site via homologous recombination.

[0050] SEQ ID NO: 48 is a 3’ homology arm used to direct insertion of cisgenic block 2 into the CR18 site via homologous recombination.

[0051] SEQ ID NO: 49 is DNA encoding a guide RNA protospacer sequence capable of directing cutting at site CR3 (not including the PAM sequence).

[0052] SEQ ID NO: 50 is DNA encoding a guide RNA protospacer sequence capable of directing cutting at site CR18 (not including the PAM sequence).Atty Docket No: 212644-WO-SEC-1DETAILED DESCRIPTION

[0053] Definitions

[0054] The following definitions are provided to aid in understanding the present disclosure.

[0055] The disclosure is not limited to particular examples, which can, of course, vary. The terminology and examples used herein are for the purpose of describing particular aspects of the disclosure only and are not intended to be limiting. As used herein, terms in the singular and the singular forms “a”, “an” and “the”, for example, include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “plant”, “the plant” or “a plant” also includes a plurality of plants; also, depending on the context, use of the term “plant” can also include genetically similar or identical progeny of that plant; use of the term “a nucleic acid” optionally includes, as a practical matter, many copies of that nucleic acid molecule; similarly, the term “probe” optionally (and typically) encompasses many similar or identical probe molecules.

[0056] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. In describing and claiming the subject matter of the current disclosure, the following terminology will be used in accordance with the definitions set out below and throughout the specification.

[0057] The term “allele” refers to one of two or more different nucleotide sequences that occur at a specific locus.

[0058] The term “amplifying” in the context of nucleic acid amplification is any process whereby additional copies of a selected nucleic acid (or a transcribed form thereof) are produced. Typical amplification methods include various polymerase based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods such as the ligase chain reaction (LCR) and RNA polymerase based amplification (e.g., by transcription) methods.

[0059] As used herein, the term “chromosomal interval” designates a contiguous linear span of genomic DNA that resides in planta on a single chromosome. The genetic elements or genes located on a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly limited. In some aspects, the genetic elements located within a single chromosomal interval are genetically linked, typically with a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. That is,Atty Docket No: 212644-WO-SEC-1 two genetic elements within a single chromosomal interval undergo recombination at a frequency of less than or equal to 20% or 10%.

[0060] A “chromosome” is a single piece of coiled DNA containing many genes that act and move as a unity during cell division and therefore can be said to be linked. It can also be referred to as a “linkage group”.

[0061] The term “contiguous DNA” refers to an uninterrupted stretch of genomic DNA represented by partially overlapping pieces or contigs.

[0062] The term “control” or “control plant” is used herein to refer to a first plant, cell, seed, or germplasm thereof in the context of a comparison to a second plant, cell, seed, or germplasm thereof having cisgenic block 1 , cisgenic block 2 or both cisgenic blocks 1 and 2 disclosed herein. The control or control plant material lacks the cisgenic block 1 , cisgenic block 2 or both cisgenic blocks 1 and 2, respectively, but is otherwise isogenic or near-isogenic to the second plant, cell, seed, or germplasm thereof having the disease resistance QTL or trait gene.

[0063] The term “crossed” or “cross” refers to a sexual cross and involved the fusion of two haploid gametes via pollination to produce diploid progeny (e.g., cells, seeds or plants). The term encompasses both the pollination of one plant by another and selfing (or self-pollination, e.g., when the pollen and ovule are from the same plant).

[0064] An “elite line” is any line that has resulted from breeding and selection for superior agronomic performance.

[0065] A “favorable allele” is the allele at a particular locus that confers, or contributes to, an agronomically desirable phenotype, e.g., SR resistance. A favorable allele of a marker is a marker allele that segregates with (e.g., is linked to) the favorable phenotype.

[0066] “Fragment” is intended to mean a portion of a nucleotide sequence. Fragments can be used as hybridization probes or PCR primers / probes using methods disclosed herein. Fragments can be generated during preparation of a biological sample.

[0067] “Genetic recombination frequency” is the frequency of a crossing over event (recombination) between two genetic loci. Recombination frequency can be observed by following the segregation of markers and / or traits following meiosis.

[0068] “Genome” refers to the total DNA, or the entire set of genes, carried by an individual.

[0069] The term “genotype” is the genetic constitution of an individual (or group of individuals) at one or more genetic loci. Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to an individual’s genetic constitution at a single locus, at multiple loci, or, more generally, the term genotype can be used to refer to an individual’s genetic make-up for all the genes in its genome.Atty Docket No: 212644-WO-SEC-1

[0070] “Germplasm” refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture, or more generally, all individuals within a species or for several species (e.g., maize germplasm collection or Andean germplasm collection). The germplasm can be part of an organism or cell or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed, or tissues from which new plants may be grown, or plant parts, such as leaves, stems, pollen, or cells, that can be cultured into a whole plant.

[0071] A plant referred to as “haploid” has a single set (genome) of chromosomes.

[0072] A “haplotype” is the genotype of an individual at a plurality of genetic loci, i.e. , a combination of alleles. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome segment.

[0073] The term “heterogeneity” is used to indicate that individuals within the group differ in genotype at one or more specific loci.

[0074] The heterotic response of material, or “heterosis”, can be defined by performance which exceeds the average of the parents (or high parent) when crossed to other dissimilar or unrelated groups.

[0075] An individual is “heterozygous” if more than one allele type is present at a given locus (e.g., a diploid individual with one copy each of two different alleles).

[0076] The term “homogeneity” indicates that members of a group have the same genotype at one or more specific loci.

[0077] An individual is “homozygous” if the individual has only one type of allele at a given locus (e.g., a diploid individual has a copy of the same allele at a locus for each of two homologous chromosomes).

[0078] The term “hybrid” refers to the progeny obtained between the crossing of at least two genetically dissimilar parents.

[0079] “Hybridization” or “nucleic acid hybridization” refers to the pairing of complementary RNA and DNA strands as well as the pairing of complementary DNA single strands.

[0080] The term “hybridize” means to form base pairs between complementary regions of nucleic acid strands.

[0081] The term “inbred” refers to a line that has been bred for genetic homogeneity.

[0082] “Introducing”, as used herein, means presenting to the plant or plant cell the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior ofAtty Docket No: 212644-WO-SEC-1 a cell. The methods of the present disclosure do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide(s) or polypeptide(s) gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide(s) or polypeptide(s) into plants include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0083] “Stable transformation” as used herein means that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” as used herein means that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant, or a polypeptide is introduced into a plant.

[0084] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (US Patent Numbers 5,563,055 and 5,981 ,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722) and ballistic (biolistic) particle acceleration (see, for example, US Patent Numbers 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin) and McCabe et al. (1988) Biotechnology 6:923-926) and Led transformation (WO 00 / 28058). For potato transformation see, Tu et al. (1998) Plant Molecular Biology 37 :829-838 and Chong et al. (2000) Transgenic Research 9:71-78. Additional transformation procedures can be found in Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); US Patent Numbers 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91 :440- 444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311 :763-764; US Patent Number 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp.Atty Docket No: 212644-WO-SEC-1197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appt. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495- 1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens).

[0085] The term “introgression” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The presence of a desired allele, e.g., in a cis block or transgenic construct disclosed herein, can be detected by any method disclosed herein, including by nucleotide sequencing, DNA amplification, or a marker that is associated with the cis block or construct, or the like. In any case, offspring comprising the desired allele can be repeatedly backcrossed to a line comprising a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background.

[0086] The process of “introgressing” is often referred to as “backcrossing” when the sexual cross process is repeated two or more times.

[0087] “Backcrossing” refers to the process whereby hybrid progeny are crossed back to one of the parents. In a backcrossing scheme, the “donor” parent refers to the parental plant with the desired gene / genes, locus / loci, or specific phenotype to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being backcrossed. Repeated backcrossing can result in “introgression” of the allele into the recipient or recurrent parent line. For example, see Ragot, M. et al. (1995) Marker-assisted backcrossing: a practical example, in Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56, and Openshaw et al., (1994) Marker-assisted Selection in Backcross Breeding, Analysis of Molecular Marker Data, pp. 41- 43. The initial cross gives rise to the Fi generation; the term “BCi” then refers to the second use of the recurrent parent, “BC2” refers to the third use of the recurrent parent, and so on. Similarly, “BC5F2” refers to the second generation produced by the sixth use of the recurrent parent.

[0088] A “line” or “strain” is a group of individuals of identical parentage that are generally inbred to some degree and that are generally homozygous and homogeneous at most lociAtty Docket No: 212644-WO-SEC-1(isogenic or near isogenic). A “subline” refers to an inbred subset of descendants that are genetically distinct from other similarly inbred subsets descended from the same progenitor.

[0089] “Maize” refers to a plant of the Zea mays genus and species and is also known as corn .

[0090] The term “maize plant” or “plant” includes whole maize plants and portions of maize plants. Such portions include, for example, maize plant cells, maize plant protoplasts, maize plant cell culture or maize tissue culture from which maize plants can be regenerated, maize plant calli, maize plant clumps or maize plant cells that are part of larger plant structures, maize seeds, maize cobs, maize flowers, maize cotyledons, maize leaves, maize stems, maize buds, maize roots, maize root tips and the like.

[0091] The term “progeny” refers to the offspring generated from a cross. A “progeny plant” is a plant generated from a cross between two plants.

[0092] As used herein, a "recombinant" plant, plant cell, or nucleic acid is a plant, plant cell, or nucleic acid comprising a heterologous nucleic acid sequence (which can encode a heterologous protein / peptide or a noncoding RNA).

[0093] As used herein “heterologous” in reference to a nucleotide or amino acid sequence means that the sequence originates from a foreign species, or, if the sequence is from the same species, it is (i) substantially modified from its native form in composition and / or (ii) located at a genomic position that differs from its native genomic locus by deliberate human intervention. For example, a promoter operably linked to heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same species, one or both are substantially modified from their original form or genetic locus, or the promoter is not the native promoter of the operably linked polynucleotide. In another example, a heterologous polynucleotide sequence of the present disclosure may be “heterologous” in that the sequence is located at a genomic locus that is different than its native or naturally occurring genomic locus. For example, a heterologous nucleic acid sequence may be located on a different chromosome or at a different chromosome location or locus compared to the chromosome location of a corresponding native sequence, or the heterologous nucleic acid sequence may be located such that it is linked to (in close proximity to or between) different genes compared to the native sequence. A heterologous gene can be inserted into a genome via, for example, transformation and / or site-specific nuclease-based methods. In a further example, use of genome editing techniques to place disease resistance gene alleles at a different chromosome location or locus is described in International Publication WO 2022 / 040134.Atty Docket No: 212644-WO-SEC-1

[0094] After generating a heterologous gene by deliberate human intervention, the heterologous gene can be transferred into the genome of another plant line by breeding methods such as introgression. A nucleotide construct is “heterologous to” a plant or plant cell if the construct contains nucleotide sequence(s) that, when incorporated into the genome, would be heterologous to the plant / plant cell. See, e.g., WO 2022 / 040134.

[0095] As used herein, “cisgenic” or “cis” in reference to a nucleotide or amino acid sequence (e.g., a “cisgenic construct” or “cis” block) means that the sequence originates (e.g., is transferred) from the same species (or a closely related species that can be conventionally bred with the same species) as the organism harboring the sequence. However, the sequence is substantially modified from its native genomic locus by deliberate human intervention. Thus, an organism is said to harbor a cisgenic or cis sequence if the cisgenic sequence originates (e.g., has been transferred) from a different organism of the same species (or a closely related species that can interbreed with) as the organism that harbors the cisgenic sequence. For example, a cisgenic construct is “cisgenic” relative to the organism harboring it when all sequences in the construct are native to a different organism of the same species as the harboring organism. Prior to transferring or introducing one or more cisgenic sequences to a harboring organism, the sequences are not present in the organism’s specific variety or lineage. Additionally or alternatively, cisgenic sequence(s) may be native to the organism but are said to be “cisgenic” when they are introduced (e.g., by gene editing or other genomic modification) at a different, nonnatural genomic locus. As discussed herein, introducing cisgenic sequences can provide benefits when multiple copies of such sequences, or several different sequences, are desired in a single individual or variety.

[0096] As used herein, “cisgenic”, in reference to a plant or seed, refers to a plant or seed comprising a cisgenic nucleotide sequence in the genome of the plant or seed.

[0097] As used herein, “site CR3”, “CR3”, “target site CR3”, and the like refer to a location in the corn genome at which cutting is directed by a guide RNA comprising the protospacer sequence encoded by SEQ ID NO: 49. Target site CR3 was described in W02022040134A1 (e.g., Table 2 of W02022040134A1), which is incorporated herein by reference in its entirety.

[0098] As used herein, “site CR18”, “CR18”, “target site CR18”, and the like refer to a location in the corn genome at which cutting is directed by a guide RNA comprising the protospacer sequence encoded by SEQ ID NO: 50. Target site CR18 was described in WQ2022040134A1 (e.g., Table 2 of W02022040134A1).

[0099] CR3 and CR18 target sites are found in a variety of publicly available lines, including for example commercially released Pioneer hybrid P1197. CR3 and CR18 target sites are alsoAtty Docket No: 212644-WO-SEC-1 found in maize inbred line PH1V5T and lines derived from PH1V5T. US Patent No. 8,907,160 (referring to seed deposit under ATCC accession number PTA-121499); see also, e.g., US Patent Nos.12,225,870; 12,279,580; 10,201,146; 9,398,751 ; 9,867,358; 9,282,702; and 9,326,475.

[0100] The term “yield” refers to the productivity per unit area of a particular plant product of commercial value. For example, yield of maize is commonly measured in bushels of seed per acre or metric tons of seed per hectare per season. Yield is affected by both genetic and environmental factors. “Agronomics”, “agronomic traits”, and “agronomic performance” refer to the traits (and underlying genetic elements) of a given plant variety that contribute to yield over the course of growing season. Individual agronomic traits include emergence vigor, vegetative vigor, stress tolerance, disease resistance or tolerance, herbicide resistance, branching, flowering, seed set, seed size, seed density, standability, threshability and the like. Yield is, therefore, the final culmination of all agronomic traits.

[0101] Sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MEGALIGN® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wl). Unless stated otherwise, multiple alignment of the sequences provided herein were performed using the CLUSTAL V method of alignment (Higgins and Sharp, CABIOS. 5:151 153 (1989)) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the CLUSTAL V method are KTUPLE=1 , GAP PENALTY=3, WIND0W=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WIND0W=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the CLUSTAL V program, it is possible to obtain “percent identity” and “divergence” values by viewing the “sequence distances” table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.

[0102] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Sambrook”).

[0103] Corn plants comprising cisgenic blocks

[0104] Provided herein are cisgenic corn plants comprising one or more cisgenic blocks. The blocks comprise nucleic acid constructs that encode polypeptides. The polypeptides canAtty Docket No: 212644-WO-SEC-1 comprise disease resistance polypeptides. The corn plants can comprise a first cisgenic block, a second cisgenic block, or both the first and second cisgenic blocks.

[0105] A first cisgenic block (cisgenic block 1) comprises a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, a polypeptide at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO: 10; a polypeptide at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of sequence of SEQ ID NO: 11 ; and a polypeptide at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO: 12.

[0106] In some examples, the first cisgenic block can comprise a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, a polypeptide comprising the polypeptide sequence of SEQ ID NO: 10; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 11 , and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 12.

[0107] A second cisgenic block (cisgenic block 2) comprises a nucleic acid sequence encoding four individual polypeptides, the polypeptides comprising, respectively, a polypeptide at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO: 10; a polypeptide at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of sequence of SEQ ID NO: 13; a polypeptide at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO: 14; and a polypeptide at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO: 15.

[0108] The first cisgenic block can comprise a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, a polypeptide comprising the polypeptide sequence of SEQ ID NO: 10; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 13; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 14; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 15.

[0109] In some examples, the cisgenic corn plant comprises the first cisgenic block.

[0110] In some examples, the cisgenic corn plant comprises the second cisgenic block.

[0111] In some examples, the cisgenic corn plants comprises both the first and second cisgenic blocks.

[0112] In some examples, the first cisgenic block is integrated into the corn genome. The integration site can be near enough to a pre-existing molecular stack such that both the preexisting stack and the cis block can be consistently inherited together, without being disrupted and separated into different siblings by crossing over. The pre-existing genomic stack canAtty Docket No: 212644-WO-SEC-1 comprise insect control genes. In some examples, the first cisgenic block is integrated into genomic site CR3.

[0113] In some examples, the second cisgenic block is integrated into the corn genome. The integration site can be near enough to a pre-existing molecular stack such that both the preexisting stack and the second cis block can be consistently inherited together, without being disrupted and separated into different sibling offspring by crossing over. The pre-existing genomic stack can comprise insect control genes. In some examples, the second cisgenic block is integrated into genomic site CR18.

[0114] If both the first and the second cisgenic blocks are present, the two blocks can be colocated such that both blocks can be consistently inherited together, without being disrupted and separated into different sibling offspring by crossing over.

[0115] In some examples, a chromosomal region comprising from 5’ to 3’: (a) flanking genomic sequence, (b) the first and second cisgenic blocks, and (c) flanking genomic sequence can be subjected to a chromosomal inversion, thereby reversing the orientation of the entire chromosomal region. This can be accomplished, for example via induction of double-strand breaks at both ends of the chromosomal region via, for example, genome editing. See, e.g., International Publication Nos. WO 202 / 143115 and WO 2023 / 164550. In particular examples, the genomic flanking sequence of (a) or (c) can include one or more desirable trait gene sequences (e.g., a trait gene that confers insect resistance or herbicide tolerance).

[0116] The cisgenic corn plant can comprise increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, anthracnose stalk rot. The increased resistance can be relative to a control plant lacking the first cisgenic block and second cisgenic block (e.g., an otherwise isogenic plant).

[0117] In some examples, the first cisgenic block can be encoded by a nucleic acid comprising at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. The first cisgenic block can be encoded by a nucleic acid comprising SEQ ID NO: 1.

[0118] In some examples, the first cisgenic block can be encoded by a nucleic acid comprising at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2. The first cisgenic block can be encoded by a nucleic acid comprising SEQ ID NO: 2.

[0119] DNA constructs comprising cisgenic blocks

[0120] The cisgenic blocks disclosed herein can be present in DNA constructs.

[0121] Provided herein is a first DNA construct comprising the first cisgenic block.

[0122] Provided herein is a second DNA construct comprising the second cisgenic block.Atty Docket No: 212644-WO-SEC-1

[0123] Also provided herein are corn plants comprising the first, second, or both the first and the second DNA constructs. The corn plants can be used for breeding, production of commercial seed, and / or agronomic production of corn.

[0124] Biological Samples and Testing

[0125] Provided herein is a biological sample derived from the plant or seed of any of the plants or seed disclosed herein. The biological sample can comprise the polypeptides encoded by the first and / or second cisgenic blocks. The sample can comprise polynucleotides encoding the first and / or second cisgenic blocks. The polypeptides encoded by the first and / or second cisgenic blocks and the polynucleotides encoding the first and / or second cisgenic blocks are detectable in the sample using mass spectrometry, a nucleic acid amplification method, a nucleic acid hybridization method, a nucleic acid sequencing method, or any other method suitable to detect such polypeptides or polynucleotides.

[0126] The polynucleotides encoding the first and / or second cisgenic blocks can comprise SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0127] The polynucleotides encoding the first and / or second cisgenic blocks can be fragmented during the sample preparation process but can nonetheless be detectable. The sample can comprise plant tissue derived from the cisgenic plants or cisgenic seeds of the present disclosure or a nucleic acid sample extracted therefrom.

[0128] In some examples, the biological sample is chosen from or extracted from any of corn flour, corn meal, corn syrup, corn oil, corn starch, or cereals manufactured in whole or in part to contain corn by-products.

[0129] Also provided herein is a method of detecting, in a biological sample derived from a cisgenic corn plant, a cisgenic block comprising SEQ ID NO: 1. The method comprises contacting the sample with at least one pair of nucleic acid primers capable of binding a first portion of SEQ ID NO: 1 with a first primer of the pair and binding either a second portion of SEQ ID NO: 1 or a genomic sequence from site CR3 with a second primer of the pair. The genomic sequence selected from site CR3 can be a portion of SEQ ID NO: 45 or 46. The method comprises performing a nucleic acid amplification reaction and detecting an amplification product produced via the at least one pair of primers. The detection of the amplification product indicates that said sample is derived from a plant comprising the cisgenic block. The detection of the amplification product can be via any appropriate method that can determine either (a) the presence or absence of the cisgenic block (e.g., imaging of an amplification product in an agarose gel) or (b) the relative abundance of the cisgenic block (e.g., real-time PCR).Atty Docket No: 212644-WO-SEC-1

[0130] In some examples, the at least one pair of nucleic acid primers comprises a first primer comprising the sequence set forth in SEQ ID NO: 16 and a second primer comprising the sequence set forth in SEQ ID NO: 17.

[0131] In some examples, the at least one pair of nucleic acid primers comprises a first primer comprising the sequence set forth in SEQ ID NO: 18 and a second primer comprising the sequence set forth in SEQ ID NO: 19.

[0132] In some examples, two pairs of nucleic acid primers are employed. For example, the junction between the cisgenic block and the genomic DNA can be interrogated on both the 5’ and 3’ sides. In some examples, a first pair of nucleic acid primers comprises a first primer comprising the sequence set forth in SEQ ID NO: 16 and a second primer comprising the sequence set forth in SEQ ID NO: 17. In some examples, a second pair of nucleic acid primers comprises a first primer comprising the sequence set forth in SEQ ID NO: 18 and a second primer comprising the sequence set forth in SEQ ID NO: 19.

[0133] In some examples, the cisgenic block comprising SEQ ID NO: 1 can be sequenced in part or in its entirety to further confirm the integrity of the cisgenic block. The sequencing can comprise Sanger sequencing, next generation sequencing, or any other suitable method. In some examples, the Southern-by-sequencing (SbS; doi: 10.3835 / plantgenome2014.08.0037. PMID: 33228291) method can be employed.

[0134] Also provided herein is a method of detecting, in a biological sample derived from a cisgenic corn plant, a cisgenic block comprising SEQ ID NO: 2. The method comprises contacting the sample with at least one pair of nucleic acid primers capable of binding a first portion of SEQ ID NO: 2 with a first primer of the pair and binding either a second portion of SEQ ID NO: 2 or a genomic sequence from site CR18 with a second primer of the pair. The genomic sequence selected from site CR18 can be a portion of SEQ ID NO: 47 or 48. The method comprises performing a nucleic acid amplification reaction and detecting an amplification product produced via the at least one pair of primers. The detection of the amplification product indicates that said sample is derived from a plant comprising the cisgenic block.

[0135] In some examples, the at least one pair of nucleic acid primers comprises a first primer comprising the sequence set forth in SEQ ID NO: 20 and a second primer comprising the sequence set forth in SEQ ID NO: 21 .

[0136] In some examples, the at least one pair of nucleic acid primers comprises a first primer comprising the sequence set forth in SEQ ID NO: 22 and a second primer comprising the sequence set forth in SEQ ID NO: 23.Atty Docket No: 212644-WO-SEC-1

[0137] Any primer sequence that binds the cisgenic block (e.g., SEQ ID NO: 2) can be paired with another such primer or a primer that binds to adjacent genomic DNA. When the second primer binds to adjacent genomic DNA an amplification product can suggest the both the presence of the cisgenic block and the blocks location in the genome.

[0138] In some examples, two pairs of nucleic acid primers are employed. For example, the junction between the cisgenic block and the genomic DNA can be interrogated on both the 5’ and 3’ sides. In some examples, a first pair of nucleic acid primers comprises a first primer comprising the sequence set forth in SEQ ID NO: 20 and a second primer comprising the sequence set forth in SEQ ID NO: 21 . In some examples, a second pair of nucleic acid primers comprises a first primer comprising the sequence set forth in SEQ ID NO: 22 and a second primer comprising the sequence set forth in SEQ ID NO: 23.

[0139] Any primer sequence that binds the cisgenic block (e.g., SEQ ID NO: 1 and / or SEQ ID NO: 2) can be paired with another such primer or a primer that binds to adjacent genomic DNA. When the second primer binds to adjacent genomic DNA an amplification product can suggest both the presence of the cisgenic block and the blocks location in the genome.

[0140] In some examples, the cisgenic block comprising SEQ ID NO: 2 can be sequenced in part or in its entirety to further confirm the integrity of the cisgenic block. The sequencing can comprise Sanger sequencing, next generation sequencing, or any other suitable method. In some examples, the Southern-by-sequencing (SbS; doi: 10.3835 / plantgenome2014.08.0037. PMID: 33228291) method can be employed.

[0141] In some examples, both cisgenic block 1 (comprising SEQ ID NO: 1) and cisgenic block 2 (comprising SEQ ID NO: 2) can be detected by the above methods in the same sample. In such situations, at least two pairs of probes are used (at least one for each block). The probes can bind the same locations, as described above.

[0142] In some examples, other amplification-based assays can be used to detect, in a biological sample derived from a cisgenic corn plant, a cisgenic block comprising SEQ ID NO: 1 and / or a cisgenic block comprising SEQ ID NO: 2. These other methods can include the FRET- based assay described in Example 3 and Table 2. For example, a primer comprising the sequence of SEQ ID NO: 24 can be paired with a primer comprising the sequence of SEQ ID NO: 25 and a fluorescent probe comprising the sequence of SEQ ID NO: 26. For example, a primer comprising the sequence of SEQ ID NO: 27 can be paired with a primer comprising the sequence of SEQ ID NO: 28 and a fluorescent probe comprising the sequence of SEQ ID NO: 29. For example, a primer comprising the sequence of SEQ ID NO: 30 can be paired with a primer comprising the sequence of SEQ ID NO: 31 and a fluorescent probe comprising theAtty Docket No: 212644-WO-SEC-1 sequence of SEQ ID NO: 32. For example, a primer comprising the sequence of SEQ ID NO: 33 can be paired with a primer comprising the sequence of SEQ ID NO: 34 and a fluorescent probe comprising the sequence of SEQ ID NO: 35. For example, a primer comprising the sequence of SEQ ID NO: 36 can be paired with a primer comprising the sequence of SEQ ID NO: 37 and a fluorescent probe comprising the sequence of SEQ ID NO: 38. For example, a primer comprising the sequence of SEQ ID NO: 39 can be paired with a primer comprising the sequence of SEQ ID NO: 40 and a fluorescent probe comprising the sequence of SEQ ID NO: 41. For example, a primer comprising the sequence of SEQ ID NO: 42 can be paired with a primer comprising the sequence of SEQ ID NO: 43 and a fluorescent probe comprising the sequence of SEQ ID NO: 44.

[0143] Also provided herein is a method of sequencing, in a biological sample derived from a corn plant, one or more cisgenic blocks comprising SEQ ID NO: 1 or SEQ ID NO: 2. The method comprises contacting the sample with nucleic acid primer(s) and sequencing the one or more cisgenic blocks. The sequencing can be performed by any method suitable to determine the nucleotide sequence of the cisgenic block(s) (e.g., with random primers or specific primers, with Sanger sequencing, or with next generation sequencing). The method comprises comparing the sequencing results to one or both of SEQ ID NO: 1 and SEQ ID NO: 2.

[0144] In some examples, a cisgenic block comprising SEQ ID NO: 1 is sequenced and the results are compared to SEQ ID NO: 1.

[0145] In some examples, a cisgenic block comprising SEQ ID NO: 2 is sequenced and the results are compared to SEQ ID NO: 2.

[0146] In some examples, a cisgenic block comprising SEQ ID NO: 1 is sequenced and the results are compared to SEQ ID NO: 1. Furthermore, a cisgenic block comprising SEQ ID NO: 2 is sequenced and the results are compared to SEQ ID NO: 2. Thus, plants comprising one or both of SEQ ID NO: 1 and SEQ ID NO: 2 can be sequenced to, for example, determine the integrity of the cisgenic blocks and / or the copy number thereof (e.g., by SbS or FRET-based real-time PCR assays).

[0147] Methods of producing cisgenic corn plants

[0148] Provided herein is a method for producing a cisgenic corn plant. The method comprises providing one or more corn plant or plant cells that are susceptible to one or both of northern leaf blight and southern corn rust. The plant or plant cell(s) are susceptible to one or both of northern leaf blight and southern corn rust. The cells can be derived from a plant or plant tissue appropriate for transformation. For example, the plant or plant tissue can be appropriate for bacterium-mediated transformation or biolistic particle transformation. For example, embryos orAtty Docket No: 212644-WO-SEC-1 leaves can be a source of plant tissue. The method comprises introducing a cisgenic block (e.g, cisgenic block 1) comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12 into the genome of one or more of the cells, thereby producing one or more modified plant cells comprising the cisgenic block in their genome.

[0149] In another method, The method comprises providing one or more corn plant or plant cells that are susceptible to one or both of northern leaf blight and southern corn rust. The plant or plant cell(s) are susceptible to one or more of anthracnose, gray leaf spot, and northern leaf blight. The cells can be derived from a plant or plant tissue appropriate for transformation. For example, the plant or plant tissue can be appropriate for bacterium-mediated transformation or biolistic particle transformation. For example, embryos or leaves can be a source of plant tissue. The method comprises introducing a cisgenic block (e.g, cisgenic block 2) comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15 into the genome of one or more of the cells. Cisgenic blocks comprising the sequence variations described in the “Corn plants comprising cisgenic blocks” section above can also be employed.

[0150] In some examples, the introduction of the cisgenic block(s) can be accomplished via bacterium-mediated transformation or biolistic particle transformation. In some examples, the introduction can be accomplished by methods that do not include conventional breeding or essential biological process(es).

[0151] The method comprises selecting at least one of the modified corn plant cells. The selection can comprise use of a molecular marker and / or sequencing or any other suitable technique to discriminate between modified and unmodified cells.

[0152] In some examples (e.g., when cisgenic block 1 is employed), the method comprises effecting a site-specific modification of the CR3 target site in the genome of the corn plant cell(s). In some examples, the method comprises introducing a polynucleotide modification template comprising the cisgenic block (e.g., cisgenic block 1) into the corn plant cell(s) and thereby producing the modified corn plant cells.

[0153] In some examples (e.g., when cisgenic block 2 is employed), the method comprises effecting a site-specific modification of the CR18 target site in the genome of the corn plant cell(s). In some examples, the method comprises introducing a polynucleotide modificationAtty Docket No: 212644-WO-SEC-1 template comprising the cisgenic block (e.g., cisgenic block 2) into the corn plant cell(s) and thereby producing the modified corn plant cells.

[0154] In some examples, the site-specific modification is induced by a CRISPR-associated endonuclease.

[0155] In some examples, the cisgenic block comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0156] In some examples (e.g., when cisgenic block 1 is employed), the method comprises producing one or more modified corn plant cells and further comprises growing a modified corn plant from the selected corn plant cell, wherein the modified plant exhibits increased resistance to one or both of northern leaf blight and southern corn rust relative to the unmodified plant of the same variety from which the one or more corn plant cells were obtained. In some examples, the CRISPR-associated endonuclease is guided by a guide polynucleotide comprising a protospacer sequence encoded by the sequence set forth in SEQ ID NO: 49.

[0157] In some examples (e.g., when cisgenic block 2 is employed), the method comprises producing one or more modified corn plant cells and further comprises growing a modified corn plant from the selected corn plant cell, wherein the modified plant exhibits increased resistance to one or more of northern leaf blight, gray leaf spot, and anthracnose stalk rot relative to the unmodified plant of the same variety from which the one or more corn plant cells were obtained. In some examples, the CRISPR-associated endonuclease is guided by a guide polynucleotide comprising a protospacer sequence encoded by the sequence set forth in SEQ ID NO: 50.

[0158] In some examples, regardless of whether cisgenic block 1 , cisgenic block 2, or both are employed, the site-specific modification comprises a single strand break or double strand break (DSB) that is made by a site-specific endonuclease such as, for example, TALENs, meganucleases, zinc finger nucleases, and CRISPR-associated (Cas) protein / guide polynucleotide complexes. In some examples, the introduction of a site-specific modification can be combined with the introduction of a polynucleotide modification template (e.g., a construct comprising cisgenic block 1 or 2) intended to be inserted via homologous recombination at the site of the site-specific modification.

[0159] In some examples, a polynucleotide modification template is introduced into a cell by any method suitable to deliver the template into the nucleus of a cell, such as, but not limited to, transient introduction methods, transfection, electroporation, microinjection, particle mediated delivery, topical application, whiskers mediated delivery, delivery via cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct delivery.

[0160] In some examples, the polynucleotide modification template may be introduced into a cell as a single stranded polynucleotide molecule, a double stranded polynucleotide molecule,Atty Docket No: 212644-WO-SEC-1 or as part of a circular DNA (vector DNA). The polynucleotide modification template may also be tethered to the guide polynucleotide and / or the Cas endonuclease. Tethered templates can allow for co-localizing target and template DNA, useful in genome editing and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous homologous recombination HR machinery is expected to be highly diminished (Mali et al. 2013 Nature Methods Vol. 10 : 957-963.) The polynucleotide modification template may be present transiently in the cell or it can be introduced via a viral replicon.

[0161] The polynucleotide modification template, as used herein, refers to a polynucleotide that comprises at least one nucleotide modification when compared to the target nucleotide sequence to be edited. The polynucleotide modification template can comprise substantially more than one nucleotide modification (e.g., a cisgenic block). A nucleotide modification is at least one nucleotide substitution, addition or deletion. In some examples, the polynucleotide modification template can further comprise homologous nucleotide sequences flanking the at least one nucleotide modification (e.g., homology arms), wherein the flanking homologous nucleotide sequences provide sufficient homology to support the incorporation of the polynucleotide modification template into the genome of the recipient plant cell.

[0162] The process for editing a genomic sequence employing both site-specific modifications and modification templates generally comprises providing to a host cell a site-specific endonuclease (or a nucleic acid encoding the site-specific endonuclease) that recognizes a target sequence in the chromosomal sequence and induction by the site-specific endonuclease of a site-specific modification (e.g., a DSB) in the genomic sequence. The process also comprises providing at least one polynucleotide modification template. The endonuclease can be provided to a cell by any suitable method (e.g., transient introduction methods, transfection, microinjection, topical application, and / or indirectly via recombination constructs). The endonuclease may be provided as a protein or as a guided polynucleotide complex directly to a cell or indirectly via recombination constructs. The endonuclease may be introduced into a cell transiently or can be incorporated into the genome of the host cell. In the case of a CRISPR- Cas system, uptake of the endonuclease and / or the guided polynucleotide into the cell can be facilitated with a Cell Penetrating Peptide (CPP) as described in WO2016073433.

[0163] TAL effector nucleases (TALEN) are a class of sequence-specific endonucleases that can be used to make 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).

[0164] Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Endonucleases include restriction endonucleases, which cleave DNA atAtty Docket No: 212644-WO-SEC-1 specific sites without damaging the bases, and meganucleases, also known as homing endonucleases (HEases), which like restriction endonucleases, bind and cut at a specific recognition site, however the recognition sites for meganucleases are typically longer, about 18 bp or more (patent application PCT / US 12 / 30061 , filed on March 22, 2012). Meganucleases have been classified into four families based on conserved sequence motifs, the families are the LAGLIDADG, GIY-YIG, H-N-H, and His-Cys box families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. The naming convention for meganuclease is similar to the convention for other restriction endonuclease. Meganucleases are also characterized by prefix F-, I-, or PI- for enzymes encoded by free-standing ORFs, introns, and inteins, respectively. One step in the recombination process involves polynucleotide cleavage at or near the recognition site. The cleaving activity can be used to produce a double-strand break. For reviews of site-specific recombinases and their recognition sites, see, Sauer (1994) Curr Op Biotechnol 5:521-7; and Sadowski (1993) FASEB 7:760-7. In some examples the recombinase is from the Integrase or Resolvase families.

[0165] Zinc finger nucleases (ZFNs) are engineered double-strand break inducing agents comprised of a zinc finger DNA binding domain and a double-strand-break-inducing agent domain. Recognition site specificity is conferred by the zinc finger domain, which typically comprising two, three, or four zinc fingers, for example comprising a C2H2 structure, however other zinc finger structures are known and have been engineered. Zinc finger domains are amenable for designing polypeptides which specifically bind a selected polynucleotide recognition sequence. ZFNs include an engineered DNA-binding zinc finger domain linked to a non-specific endonuclease domain, for example nuclease domain from a Type Ils endonuclease such as Fokl. Additional functionalities can be fused to the zinc-finger binding domain, including transcriptional activator domains, transcription repressor domains, and methylases. In some examples, dimerization of 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 recognized a sequence of 9 contiguous nucleotides, with a dimerization requirement of the nuclease, two sets of zinc finger triplets are used to bind an 18 nucleotide recognition sequence.

[0166] Methods of producing hybrid, cisgenic corn seeds and plants via breeding

[0167] Also provided herein is a method of producing hybrid, cisgenic corn seeds. The method comprises crossing a first corn line with a second corn line. The first corn line is cisgenic and comprises a first cisgenic block comprising the sequence set forth in SEQ ID NO: 1 and aAtty Docket No: 212644-WO-SEC-1 second cisgenic block comprising the sequence set forth in SEQ ID NO: 2. The cross produces progeny plant(s). Alternatively, the first corn line comprises the first and second cisgenic blocks or the sequence variations thereof described herein. The second corn line can be an elite line.

[0168] The method comprises growing the progeny plant(s) and harvesting hybrid seed produced by the progeny plants. The progeny plants can comprise the first and second cisgenic blocks and the sequence variations described herein.

[0169] In some examples, the hybrid seed comprises the first cisgenic block and the second cisgenic block, or the sequence variations described herein.

[0170] In some examples, the first corn line comprises increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot. In some examples, the increased resistance is relative to a control corn plant line lacking the first cisgenic block and second cisgenic block.

[0171] In some examples, a plant grown from the hybrid seed displays greater disease resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot, compared to the second corn line. The second corn line may lack one or both of cisgenic blocks 1 and 2.

[0172] In some examples, the first cisgenic block and the second cisgenic block are integrated at site CR3 and at site CR18, respectively in the first corn line and the progeny.

[0173] Also provided herein is a method for producing a corn plant having increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot. The method comprises crossing a first parent corn plant with a second parent corn plant. The first parent corn plant comprises a first cisgenic block comprising the sequence set forth in SEQ ID NO: 1 and a second cisgenic block comprising the sequence set forth in SEQ ID NO: 2. The second parent corn plant is susceptible to southern corn rust, gray leaf spot, northern leaf blight, anthracnose stalk rot, or combinations thereof, thereby producing a plurality of first generation progeny plants. The second corn plant can lack one or both of the first and second cisgenic blocks. The second corn plant can be an elite line.

[0174] The method comprises selecting a first generation progeny plant that comprises the first cisgenic block and second cisgenic block, wherein the selected progeny plant has increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot as compared to the second parent plant.

[0175] In some examples, the method comprises backcrossing the first generation progeny plant with the second parent plant, thereby producing a plurality of backcross progeny plants and selecting from the backcross progeny plants, a plant that comprises the first cisgenic blockAtty Docket No: 212644-WO-SEC-1 and second cisgenic block. The selected backcross progeny plant has increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot as compared to the second parent plant.

[0176] Methods of protecting corn plants

[0177] Provided herein is a method of protecting a corn plant against one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot. The method comprises expressing in the corn plant one or more polypeptides in the corn plant. The one or polypeptides can be expressed from a first cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12. Alternatively or additionally, the one or polypeptides can be expressed from a second cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15. The cisgenic blocks comprising sequence variations disclosed herein may also be employed.

[0178] In some examples, the method comprises expressing polypeptides from both the first cisgenic block and the second cisgenic block.

[0179] In some examples, protection is provided against two or more of the following diseases: northern leaf blight, southern corn rust, gray leaf spot, or anthracnose stalk rot.

[0180] In some examples, protection is provided against three or more of the following diseases: northern leaf blight, southern corn rust, gray leaf spot, or anthracnose stalk rot.

[0181] In some examples, protection is provided against northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot.

[0182] In some examples, the first cisgenic block comprises a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 1 .

[0183] In some examples, the second cisgenic block comprises a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 2.

[0184] Modification of plants or plant cells comprising cisgenic blocks

[0185] In some examples, it may be desired to modify DNA encoding cisgenic blocks (e.g., cisgenic block 1 or 2) disclosed herein. Such modifications can edit or change the encoded polypeptides such that they are changed to either naturally occurring alleles or non-naturally alleles. Such alleles may provide enhanced or different functionality (e.g., disease resistance). In some examples, regulatory elements (e.g., introns, splice sites, promoters, terminators,Atty Docket No: 212644-WO-SEC-1 enhancers) may be edited or switched to natural or non-natural alternatives or removed or added to the cisgenic block. In some examples, flanking sequences can be modified. In some examples, one gene or an entire cisgenic block can be removed or added. In some examples, heterologous genes or stacks thereof may be added.

[0186] For example, genome engineering technology can be used to modify a corn plant genome comprising cisgenic block 1 (e.g., SEQ ID NO: 1), cisgenic block 2 (e.g., SEQ IDNO: 2) event, or both cisgenic blocks 1 and 2 to modify the DNA of said corn plant. The modified DNA can be modified to comprise (i) at least 90% or 95% sequence identity to cisgenic block 1 or cisgenic block 2; (ii) duplicate one or more sequences within cisgenic block 1 or block 2, (iii) duplicate one or more of cisgenic block 1 and block 2, (iv) excise one or more sequences within cisgenic block 1 or block 2, (v) excise cisgenic block 1 , cisgenic block 2 or both cisgenic block 1 and cisgenic block 2, (vi) a chromosomal inversion, translocation, duplication of the region comprising cisgenic block 1, cisgenic block 2, or both cisgenic block 1 and cisgenic block 2 (see e.g., International Publication Nos. WO 202 / 143115 and WO 2023 / 164550).

[0187] Cas Polypeptide

[0188] In some examples, a site-specific modification (e.g., at CR3 and / or CR18) can be introduced using genome editing technology, including an endonuclease (e.g., a site-specific endonuclease). Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Examples of site-specific endonucleases include restriction endonucleases, meganucleases, TAL effector nucleases (TALENs), zinc finger nucleases, and Cas (CRISPR- associated) effector endonucleases.

[0189] CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats) (also known as SPIDRs-SPacer Interspersed Direct Repeats) constitute a family of recently described DNA loci. CRISPR loci consist of short and highly conserved DNA repeats (typically 24 to 40 bp, repeated from 1 to 140 times-also referred to as CRISPR-repeats) which are partially palindromic. The repeated sequences (usually specific to a species) are interspaced by variable sequences of constant length (typically 20 to 58 by depending on the CRISPR locus (International Publication W02007 / 025097 published March 1 , 2007).

[0190] Cas endonucleases, either as single effector proteins or in an effector complex with other components, unwind the DNA duplex at a target sequence and, optionally, cleave at least one DNA strand, as mediated by recognition of the target DNA sequence by a guide polynucleotide (such as, but not limited to, a CRISPR RNA (crRNA) or guide RNA) that is complexed with a Cas endonuclease. Such recognition and cutting of a target DNA sequence by a Cas endonuclease typically occurs if the correct protospacer-adjacent motif (PAM) is located at or adjacent to the 3'Atty Docket No: 212644-WO-SEC-1 end of the target DNA sequence. Alternatively, a Cas endonuclease can lack DNA cleavage or nicking activity but can still specifically bind to a target DNA sequence when complexed with a suitable guide polynucleotide. (See also U.S. Patent Application US20150082478 published 19 March 2015 and US20150059010 published 26 February 2015).

[0191] Cas endonucleases that have been described include, but are not limited to, for example: Cas9, Cas12f (Cas-alpha, Cas14), Cas12l (Cas-beta), Cas12a (Cpf1), Cas12b (a C2c1 protein), Cas13 (a C2c2 protein), Cas12c (a C2c3 protein), Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas3, Cas3-HD, Cas 5, Cas6, Cas7, Cas8, Casio, or combinations or complexes of these. In some aspects, the methods and compositions described herein can utilize transposon-associated TnpB, a programmable RNA-guided DNA endonuclease.

[0192] Cas endonucleases and effector proteins can be used for targeted genome editing (via simplex and multiplex double-strand breaks and nicks) and targeted genome regulation (via tethering of epigenetic effector domains to either the Cas protein or sgRNA. A Cas endonuclease can also be engineered to function as an RNA-guided recombinase, and via RNA tethers could serve as a scaffold for the assembly of multiprotein and nucleic acid complexes (Mali etal., 2013, Nature Methods Vol. 10: 957-963).

[0193] A Cas endonuclease, effector protein, or functional fragment thereof, for use in the disclosed methods, can be isolated from a native source, or from a recombinant source where the genetically modified host cell is modified to express the nucleic acid sequence encoding the protein. Alternatively, the Cas endonuclease protein can be produced using cell free protein expression systems or be synthetically produced. Cas endonucleases may be isolated and introduced into a heterologous cell or may be modified from its native form to exhibit a different type or magnitude of activity than what it would exhibit in its native source. Such modifications include but are not limited to: fragments, variants, substitutions, deletions, and insertions.

[0194] Fragments and variants of Cas endonucleases can be obtained via methods such as site- directed mutagenesis and synthetic construction. Methods for measuring endonuclease activity are well known in the art such as, but not limited to, International Publication WO2013 / 166113 published 07 November 2013, International Publication WO2016 / 186953 published 24 November 2016, and International Publication WO2016 / 186946 published 24 November 2016.

[0195] The Cas endonuclease can comprise a modified form of the Cas polypeptide. The modified form of the Cas polypeptide can include an amino acid change (e.g., deletion, insertion, or substitution) that reduces the naturally-occurring nuclease activity of the Cas protein. For example, in some instances, the modified form of the Cas 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 nucleaseAtty Docket No: 212644-WO-SEC-1 activity of the corresponding wild-type Cas polypeptide (US Application Publication 2014 / 0068797 published 06 March 2014). In some cases, the modified form of the Cas polypeptide has no substantial nuclease activity and is referred to as catalytically “inactivated Cas” or “deactivated Cas (dCas).” An inactivated Cas / deactivated Cas includes a deactivated Cas endonuclease (dCas). A catalytically inactive Cas endonuclease can be fused to a heterologous sequence to induce or modify activity.

[0196] A Cas endonuclease can be part of a fusion protein comprising one or more heterologous protein domains (e.g., 1 , 2, 3, or more domains in addition to the Cas protein). Suitable fusion partners include, but are not limited to, a polypeptide that provides an activity that indirectly increases transcription by acting directly on the target DNA or on a polypeptide (e.g., a histone or other DNA-binding protein) associated with the target DNA. Additional suitable fusion partners include, but are not limited to, a polypeptide that provides for methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity. Further suitable fusion partners include, but are not limited to, a polypeptide that directly provides for increased transcription of the target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule / drug-responsive transcription regulator, etc.). A catalytically inactive Cas can also be fused to a Fokl nuclease to generate double-strand breaks (Guilinger et al. Nature Biotechnology, volume 32, number 6, June 2014). In some aspects, the Cas endonuclease is a fusion protein further comprising a nuclease domain, a transcriptional activator domain, a transcriptional repressor domain, an epigenetic modification domain, a cleavage domain, a nuclear localization signal, a cell-penetrating domain, a translocation domain, a marker, or a transgene that is heterologous to the target polynucleotide sequence or to the cell from which said target polynucleotide sequence is obtained or derived. In some aspects, the nuclease fusion protein comprises Clo51 or Fokl.

[0197] The Cas endonucleases described herein can be expressed and purified by methods known in the art, for example as described in International Publication WO2016 / 186953.

[0198] A Cas endonuclease can comprise a heterologous nuclear localization sequence (NLS). A heterologous NLS amino acid sequence herein may be of sufficient strength to drive accumulation of a Cas protein in a detectable amount in the nucleus of a yeast cell herein, for example.

[0199] Cas9 PolypeptideAtty Docket No: 212644-WO-SEC-1

[0200] In some aspects of the methods disclosed herein, a genome editing system comprises a Cas9 endonuclease and one or more guide polynucleotides that introduce one or more sitespecific modifications in a target polynucleotide sequence. In some aspects, a genome editing system comprises a Cas9 endonuclease, one or more guide polynucleotides, and a polynucleotide modification template. Some exemplary Cas9 endonucleases are described, for example, in International Publication WO2019165168.

[0201] Cas9 (formerly referred to as Cas5, Csn1, or Csx12) is a Cas endonuclease that forms a complex with a crNucleotide and a tracrNucleotide, or with a single guide polynucleotide, for specifically recognizing and cleaving all or part of a DNA target sequence. The canonical Cas9 recognizes a 3’ GC-rich PAM sequence on the target dsDNA, typically comprising an NGG motif. The Cas endonucleases described herein may recognize additional PAM sequences and can be used to modify target sites with different recognition sequence specificity.

[0202] A Cas9 polypeptide comprises a RuvC nuclease with an HNH (H-N-H) nuclease adjacent to the RuvC-ll domain. The RuvC nuclease and HNH nuclease each can cleave a single DNA strand at a target sequence (the concerted action of both domains leads to DNA double-strand cleavage, whereas activity of one domain leads to a nick). In general, the RuvC domain comprises subdomains I, II and III, where domain I is located near the N-terminus of Cas9 and subdomains II and III are located in the middle of the protein, flanking the HNH domain (Hsu et al., 2013, Cell 157:1262-1278). Cas9 endonucleases are typically derived from a type II CRISPR system, which includes a DNA cleavage system utilizing a Cas9 endonuclease in complex with at least one polynucleotide component. For example, a Cas9 can be in complex with a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In another example, a Cas9 can be in complex with a single guide RNA (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15).

[0203] The type II CRISPR / Cas system from bacteria employs a crRNA and tracrRNA to guide the Cas endonuclease to its DNA target. The crRNA (CRISPR RNA) contains the region complementary to one strand of the double strand DNA target and base pairs with the tracrRNA (trans-activating CRISPR RNA) forming an RNA duplex that directs the Cas endonuclease to cleave the DNA target. In some aspects, a guide polynucleotide comprises a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variable targeting domain, and a tracrRNA. In some aspects, a guide polynucleotide comprises a variable targeting domain of 12 to 30 nucleotides and an RNA fragment that interacts with a Cas9 endonuclease.

[0204] Cas-alpha Polypeptide

[0205] In some aspects of the methods disclosed herein, a genome editing system comprises a Cas-alpha (e.g., Cas12f) endonuclease and one or more guide polynucleotides that introduce oneAtty Docket No: 212644-WO-SEC-1 or more site-specific modifications in a target polynucleotide sequence. In some aspects, a genome editing system comprises a Cas-alpha endonuclease, one or more guide polynucleotides, and a polynucleotide modification template. Some exemplary Cas-alpha endonucleases are described, for example, in US Patent 10,934,536 and International Publication WO2022 / 082179.

[0206] A Cas-alpha endonuclease is a functional RNA-guided, PAM-dependent dsDNA cleavage protein of fewer than 800 amino acids, comprising: a C-terminal RuvC catalytic domain split into three subdomains and further comprising bridge-helix and one or more Zinc finger motif(s); and an N-terminal Rec subunit with a helical bundle, WED wedge-like (or “Oligonucleotide Binding Domain”, OBD) domain, and, optionally, a Zinc finger motif.

[0207] Cas-alpha endonucleases comprise one or more Zinc Finger (ZFN) coordination motif(s) that may form a Zinc binding domain. Zinc Finger-like motifs can aid in target and non-target strand separation and loading of the guide polynucleotide into the DNA target. Cas-alpha endonucleases comprising one or more Zinc Finger motifs can provide additional stability to a ribonucleoprotein complex on a target polynucleotide. Cas-alpha endonucleases comprise C4 or C3H zinc binding domains.

[0208] A Cas-alpha endonuclease can function as a double-strand-break-inducing agent, a single-strand-break inducing agent, or as a nickase. In some aspects, a catalytically inactive Cas- alpha endonuclease can be used to target or recruit to a target DNA sequence but not induce cleavage. In some aspects, a catalytically inactive Cas-alpha protein can be combined with a base editing molecule, such as a cytidine deaminase or an adenine deaminase.

[0209] NHEJ and HDR

[0210] In some aspects of the methods and compositions described herein, a genome editing system comprises a Cas endonuclease, one or more guide polynucleotides, and optionally polynucleotide modification template, and editing a target polynucleotide sequence comprises nonhomologous end-joining (NHEJ) or homologous recombination (HR) following a Cas endonuclease-mediated double-strand break. Once a double-strand break is induced in the DNA, the cell's DNA repair mechanism is activated to repair the break. The most common repair mechanism to bring the broken ends together is the nonhomologous end-joining pathway (Bleuyard et al., (2006) DNA Repair 5:1-12). The structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements are possible. Alternatively, the double-strand break can be repaired by homologous recombination between homologous DNA sequences. Once the sequence around the double-strand break is altered, for example, by exonuclease activities involved in the maturation of double-strand breaks, geneAtty Docket No: 212644-WO-SEC-1 conversion pathways can restore the original structure if a homologous sequence is available, such as a homologous chromosome in non-dividing somatic cells, or a sister chromatid after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and / or epigenic DNA sequences may also serve as a DNA repair template for homologous recombination (Puchta, (1999) Genetics 152:1173-81).

[0211] In some aspects of the methods and compositions described herein, the genome editing system comprises a Cas endonuclease, one or more guide polynucleotides, and a polynucleotide modification template. As used herein, “polynucleotide modification template” is a DNA construct that comprises a polynucleotide of interest (e.g., a cisgenic block of the present disclosure such as cisgenic block 1 or 2)) to be inserted into the target site of a site-specific nuclease such as a Cas endonuclease. Once a site-specific modification (e.g., a double-strand break) is introduced in the target site by the endonuclease, the first and second regions of homology of the polynucleotide modification template (e.g., homology arms) can undergo homologous recombination with their corresponding genomic regions of homology resulting in exchange of DNA between the donor and the target genome. As such, the provided methods result in the integration of the polynucleotide of interest of the polynucleotide modification template into the double-strand break in the target site in the plant genome, thereby altering the original target site and producing an altered genomic target site.

[0212] Base Editing

[0213] In some aspects of the methods and compositions described herein, a genome editing system comprises a base editing agent and one or more guide polynucleotides and editing a target polynucleotide sequence comprises introducing a one or more nucleobase edits in the target polynucleotide sequence resulting in a disease resistant allele.

[0214] One or more nucleobases of a target polynucleotide can be chemically altered, in some cases to change the base from one type to another, for example from a Cytosine to a Thymine, or an Adenine to a Guanine. In some aspects, a plurality of bases, for example 2 or more, 5 or more, 10 or more, etc. of bases may be modified or altered, to produce a plant with a plurality of modified bases.

[0215] Any base editing complex, such as a base editing agent associated with an RNA-guided protein, may be used to target and bind to a desired locus in the genome of an organism and chemically modify one or more components of a target polynucleotide.

[0216] Site-specific base conversions can be achieved to engineer one or more nucleotide changes to create one or more edits into the genome. These include for example, a site-specific base edit mediated by a C«G to T«A or an A*T to G«C base editing deaminase enzymes (GaudelliAtty Docket No: 212644-WO-SEC-1 et al., Programmable base editing of A«T to G«C in genomic DNA without DNA cleavage." Nature (2017); Nishida et al. “Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.” Science 353 (6305) (2016); Komor et al. “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.” Nature 533 (7603) (2016):420-4. A catalytically “dead” or inactive Cas9 (dCas9), for example a catalytically inactive “dead” version of a Cas endonuclease disclosed herein, fused to a cytidine deaminase or an adenine deaminase protein becomes a specific base editor that can alter DNA bases without inducing a DNA break. Base editors convert C->T (or G->A on the opposite strand) or an adenine base editor that would convert adenine to inosine, resulting in an A->G change within an editing window specified by the gRNA. Any molecule that effects a change in a nucleobase is a “base editing agent”.

[0217] For many traits of interest, the creation of single double-strand breaks and the subsequent repair via HDR or NHEJ is not ideal for quantitative traits. An observed phenotype includes both genotype effects and environmental effects. The genotype effects further comprise additive effects, dominance effects, and epistatic effects. The probability of no effect per any single edit can be greater than zero, and any single phenotypic effect can be small, depending on the method used and site selected. Double-stranded break repair can additionally be “noisy” and have low repeatability.

[0218] One approach to ameliorate the probability of no effect per edit or small phenotypic effect outcome is to multiplex genome modification, such that a plurality of target sites are modified. Methods to modify a genomic sequence that do not introduce double-strand breaks would allow for single base substitutions. Combining these approaches, multiplexed base editing is beneficial for creating large numbers of genotype edits that can produce observable phenotype modifications. In some cases, dozens or hundreds or thousands of sites can be edited within one or a few generations of an organism.

[0219] A multiplexed approach to base editing in an organism, has the potential to create a plurality of significant phenotypic variations in one or a few generations, with a positive directional bias to the effects. In some aspects, the organism is a plant. A plant or a population of plants with a plurality of edits can be cross-bred to produce progeny plants, some of which will comprise multiple pluralities of edits from the parental lines. In this way, accelerated breeding of desired traits can be accomplished in parallel in one or a few generations, replacing time-consuming traditional sequential crossing and breeding across multiple generations.

[0220] A base editing deaminase, such as a cytidine deaminase or an adenine deaminase, may be fused to an RNA-guided endonuclease that can be deactivated (“dCas”, such as a deactivatedAtty Docket No: 212644-WO-SEC-1Cas9) or partially active (“nCas”, such as a Cas9 nickase) so that it does not cleave a target site to which it is guided. The dCas forms a functional complex with a guide polynucleotide that shares homology with a polynucleotide sequence at the target site and is further complex with the deaminase molecule. The guided Cas endonuclease recognizes and binds to a double-stranded target sequence, opening the double-strand to expose individual bases. In the case of a cytidine deaminase, the deaminase deaminates the cytosine base and creates an uracil. Uracil glycosylase inhibitor (UGI) is provided to prevent the conversion of U back to C. DNA replication or repair mechanisms then convert the Uracil to a thymine (U to T), and subsequent repair of the opposing base (formerly G in the original G-C pair) to an Adenine, creating a T-A pair. For example, see Komor et al. Nature Volume 533, Pages 420-424, 19 May 2016.

[0221] Prime Editing

[0222] In some aspects of the methods and compositions described herein, a genome editing system comprises a prime editing agent and a guide polynucleotide and editing a target nucleotide sequence comprises introducing one or more insertions, deletions, or nucleobase swaps in a target nucleotide sequence without generating a double-stranded DNA break.

[0223] In some aspects, the prime editing agent is a Cas polypeptide fused to a reverse transcriptase, wherein the Cas polypeptide is modified to nick DNA rather than generating doublestrand break. This Cas-polypeptide-reverse transcriptase fusion can also be referred to as a “prime editor” or “PE”. In some aspects, the guide polynucleotide comprises a prime editing guide polynucleotide (pegRNA) and is larger than standard sgRNAs commonly used for CRISPR gene editing (e.g., >100 nucleobases). The pegRNA comprises a primer binding sequence (PBS) and a template containing the desired or target RNA sequence at its 3’ end.

[0224] During prime editing, the PE:pegRNA complex binds to a target DNA sequence and the modified Cas polypeptide nicks one target DNA strand resulting in a flap. The PBS on the pegRNA binds to the DNA flap and the target RNA sequence is reverse transcribed using the reverse transcriptase. The edited strand is incorporated into the target DNA at the end of the nicked flap, and the target DNA sequence is repaired with the new reverse transcribed DNA.

[0225] Guide RNA Design for Genome Editing

[0226] For a validated gene target or for gene-function discovery purposes, a number of guide RNA (gRNA) tools are available based on target sequences and the CRISPR-Cas polypeptide of choice (e.g., Cas9, Cas12a, Cas12f and others). For a general overview of some of the editing techniques using targeted nucleases, base editors, transposases, and polymerase-based Prime editing, see e.g., Anzalone et al., (2020) Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol 38:824-844. gRNA design software toolsAtty Docket No: 212644-WO-SEC-1 are available from several resources and commercial vendors. See for example, DeepSpCas9 (https: / / deepcrispr.info / DeepSpCas9); for base editors DeepBE (https: / / deepcrispr.info / DeepBE / ) ; DeepPrime and DeepPrime-FT (Yu et al., Prediction of efficiencies for diverse prime editing systems in multiple cell types. Cell. 2023 May 11 ;186(10):2256-2272.); CRISPOR (Haeussler et al., Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 2016 Jul 5; 17(1):148); CRISPR MultiTargeter (https: / / www.multicrispr.net / ).

[0227] For gene knockouts, the error-prone non-homologous end joining (NHEJ) process often creates indels, leading to frameshifts that disrupt protein coding. For example, using S. pyogenes Cas9 (SpCas9), target sites can be [5’-20nt-NGG] or [5’-CCN-20nt], targeting either DNA strand. To avoid alternative start codons, target sites near the N-terminus are generally avoided and, to ensure non-functional alleles, sites near the C-terminus are also avoided. For a 1 kilobase gene, potential target sites appear roughly every 8 nucleotides. Limiting guide RNAs (gRNAs) to 5-65% of the coding region still offers many options, enabling one of skill in the art to select an optimized gRNA sequence.

[0228] For specific edits like inserting a heterologous regulatory element, a sequence encoding a mutation, or any other genetic sequence of interest, homology directed repair (HDR) is typically used, requiring an external DNA template. HDR is more inefficient compared to NHEJ and may need more screening. When using HDR, the target site is typically close to the desired edit location, as efficiency may drop if the cut site is more than about ~30-50 nucleotides from the repair template’s ends. This parameter helps design more optimal guide RNAs (gRNAs). While SpCas9, which prefers the NGG PAM sequence, is the most common enzyme used, other enzymes like SaCas9, Cas12a, Cas12f and engineered versions of these provide more PAM options, increasing gRNA choices.

[0229] Two other technologies provide alternatives to NHEJ and HDR for making edits. Base editors, like Cas-deaminase fusion proteins, can change DNA without creating doublestrand breaks. For C>T and A>G base editors, the edit is generally preferred to be within a 5-10 nucleotide window relative to the PAM, and unintended edits may occur if another target C or A is present. Prime editing, another method, is not limited to single nucleotide changes but still needs a nearby PAM.

[0230] For modulating gene expression, CRISPR activation and CRISPR inhibition use a nuclease-dead Cas polypeptide (dCas) associated with an activator domain or a repressor element, directed near the gene's promoter. CRISPR activation works best in a ~100nt window upstream of the transcription start site (TSS), while CRISPR inhibition is most effective in a ~1 OOntAtty Docket No: 212644-WO-SEC-1 window downstream of the TSS. Accurate TSS location helps in designing the gRNA. Databases such as TSSFinder (Oliveira et al., TSSFinder — fast and accurate ab initio prediction of the core promoter in eukaryotic genomes, Briefings in Bioinformatics, Volume 22, Issue 6, November 2021), Smar2C2 (Murray et al., Simple and accurate transcriptional start site identification using Smar2C2 and examination of conserved promoter features. Plant J. 2022 Oct; 112(2): 583-596) and FANTOM database for mammalian genomes are suitable for predicting or estimating TSS locations. The narrow target window limits the number of optimal guide RNAs (gRNAs), making both location and sequence important in design.

[0231] Analyzing Genome Edits A number of genome sequence-based analysis tools are available for evaluating specificity of genome edits. For example, CRISPResso (Pinello et al., Analyzing CRISPR genome-editing experiments with CRISPResso. Nat Biotechnol. 2016 Jul 12;34(7):695-7.); MAGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout).

[0232] Clauses of the Disclosure

[0233] Various aspects of the present disclosure will be understood by reference to the following clauses.1. A corn plant comprising: a first cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11, and the polypeptide sequence of SEQ ID NO: 12 and / or a second cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15.2. The corn plant of clause 1 , wherein the corn plant comprises increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot, wherein the increased resistance is relative to a control plant lacking the first cisgenic block and second cisgenic block.3. The corn plant of any one of clauses 1 or 2, wherein the first cisgenic block comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1 , and the second cisgenic block comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 2.4. The corn plant of any one of clauses 1, 2, or 3, wherein the first cisgenic block is integrated at site CR3, and the second cisgenic block is integrated at site CR18.5. A seed derived from the corn plant of any one of clauses 1-4 and comprising the first cisgenic block and second cisgenic block.Atty Docket No: 212644-WO-SEC-16. A DNA construct comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11, and the polypeptide sequence of SEQ ID NO: 12.7. The DNA construct of clause 6, wherein the nucleic acid sequence encoding the polypeptides comprises the nucleic acid sequence set forth in SEQ ID NO: 1.8. A DNA construct comprising a nucleic acid sequence encoding four individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15.9. The DNA construct of clause 8, wherein the nucleic acid sequence encoding the polypeptides comprises the nucleic acid sequence set forth in SEQ ID NO: 2.10. A corn plant comprising the DNA construct of any one of clauses 6-9, wherein the construct is cisgenic to the corn plant.11. A seed derived from the corn plant of clause 10, wherein the seed comprises the DNA construct.12. A biological sample derived from the plant or seed of any one of clauses 1-5, 10, or 11 , wherein the sample comprises the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2; wherein the nucleotide sequence is detectable in the sample using a nucleic acid amplification method, a nucleic acid hybridization method, or a nucleic acid sequencing method; and wherein the polynucleotide(s) comprising the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2 are or are not fragmented.13. The biological sample of clause 12, wherein the biological sample comprises plant tissue derived from the plant or seed of any one of clauses 1-5, 10, or 11.14. The biological sample of clause 12, wherein the biological sample is a nucleic acid sample extracted from plant or seed material derived from the plant or seed of any one of clauses 1-4, 9, or 10.15. The biological sample of clause 12, wherein the biological sample is chosen from any of corn flour, corn meal, corn syrup, corn oil, corn starch, or cereals manufactured in whole or in part to contain corn by-products.16. A method of producing hybrid corn seeds comprising: a) crossing a first corn line with a second corn line, wherein the first corn line comprises a first cisgenic block comprising the sequence set forth in SEQ ID NO: 1 and a second cisgenic block comprising the sequence set forth in SEQ ID NO: 2 to produce progeny plants; b) growing the progeny plants ; and c) harvesting hybrid seed produced by the progeny plants.Atty Docket No: 212644-WO-SEC-117. The method of clause 16, wherein the hybrid seed comprises the first cisgenic block and the second cisgenic block.18. The method of any one of clauses 16-17, wherein the first corn line comprises increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot, wherein the increased resistance is relative to a control corn plant line lacking the first cisgenic block and second cisgenic block.19. The method of any one of clauses 16-18, wherein a plant grown from the hybrid seed displays greater disease resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot, compared to the second corn line.20. The method of any one of clauses 16-19, wherein the first cisgenic block and the second cisgenic block are integrated at site CR3 and at site CR18, respectively in the first corn line.21. A method for producing a corn plant having increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot, the method comprising: a) crossing a first parent corn plant with a second parent corn plant, wherein the first parent corn plant comprises a first cisgenic block comprising the sequence set forth in SEQ ID NO: 1 and a second cisgenic block comprising the sequence set forth in SEQ ID NO: 2 and wherein the second parent corn plant is susceptible to southern corn rust, gray leaf spot, northern leaf blight, anthracnose stalk rot, or combinations thereof, thereby producing a plurality of first generation progeny plants; and b) selecting a first generation progeny plant that comprises the first cisgenic block and second cisgenic block, wherein the selected progeny plant has increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot as compared to the second parent plant.22. The method of clause 21 , wherein the method further comprises backcrossing the first generation progeny plant of step (b) with the second parent plant, thereby producing a plurality of backcross progeny plants; and c) selecting from the backcross progeny plants, a plant that comprises the first cisgenic block and second cisgenic block, wherein the selected backcross progeny plant has increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot as compared to the second parent plant.23. Seed produced by the selected plants of clause 21 or clause 22.24. A method of detecting, in a biological sample derived from a corn plant, a cisgenic block comprising SEQ ID NO: 1 , the method comprising: a) contacting the sample with at least one pair of nucleic acid primers capable of binding a first portion of SEQ ID NO: 1 with a first primer of the pair and binding either a second portion of SEQ ID NO: 1 or a genomic sequence from site CR3 with a second primer of the pair; b) performing a nucleic acid amplification reaction; andAtty Docket No: 212644-WO-SEC-1 c) detecting an amplification product produced via the at least one pair of primers, wherein detection of the amplification product indicates that said sample is derived from a plant comprising the cisgenic block.25. The method of clause 24, wherein the at least one pair of nucleic acid primers comprises a first primer comprising the sequence set forth in SEQ ID NO: 16 and a second primer comprising the sequence set forth in SEQ ID NO: 17.26. The method of clause 25, wherein the at least one pair of nucleic acid primers comprises a first primer comprising the sequence set forth in SEQ ID NO: 18 and a second primer comprising the sequence set forth in SEQ ID NO: 19.27. The method of clause 26, wherein two pairs of nucleic acid primers are used, wherein the first pair comprises: a first primer comprising the sequence set forth in SEQ ID NO: 16 and a second primer comprising the sequence set forth in SEQ ID NO: 17 and the second pair comprises: a first primer comprising the sequence set forth in SEQ ID NO: 18 and a second primer comprising the sequence set forth in SEQ ID NO: 19.28. The method of any one of clauses 24-27, further comprising sequencing the cisgenic block, wherein the cisgenic block comprises a sequence matching that set forth in SEQ ID NO: 1.29. A method of detecting, in a biological sample derived from a corn plant, a cisgenic block comprising SEQ ID NO: 2, the method comprising: a) contacting the sample with at least one pair of nucleic acid primers capable of binding a first portion of SEQ ID NO: 2 with a first primer of the pair and binding either a second portion of SEQ ID NO: 2 or a genomic sequence from site CR18 with a second primer of the pair; b) performing a nucleic acid amplification reaction; and c) detecting an amplification product produced via the at least one pair of primers, wherein detection of the amplification product indicates that said sample is derived from a plant comprising the cisgenic block.30. The method of clause 29, wherein the at least one pair of nucleic acid primers comprises a first primer comprising the sequence set forth in SEQ ID NO: 20 and a second primer comprising the sequence set forth in SEQ ID NO: 21.31. The method of clause 29, wherein the at least one pair of nucleic acid primers comprises a first primer comprising the sequence set forth in SEQ ID NO: 22 and a second primer comprising the sequence set forth in SEQ ID NO: 23.32. The method of clause 29, wherein two pairs of nucleic acid primers are used, wherein the first pair comprises:Atty Docket No: 212644-WO-SEC-1 a first primer comprising the sequence set forth in SEQ ID NO: 20 and a second primer comprising the sequence set forth in SEQ ID NO: 21 and the second pair comprises: a first primer comprising the sequence set forth in SEQ ID NO: 22 and a second primer comprising the sequence set forth in SEQ ID NO: 23.33. The method of any one of clauses 29-32, further comprising sequencing the cisgenic block, wherein the cisgenic block comprises a sequence matching that set forth in SEQ ID NO: 2.34. The method of any one of clauses 24, 28, 29, or 33, wherein the at least one pair of primers comprises:(i) a primer comprising the sequence set forth in SEQ ID NO: 24 and a primer comprising the sequence set forth in SEQ ID NO: 25;(ii) a primer comprising the sequence set forth in SEQ ID NO: 27 and a primer comprising the sequence set forth in SEQ ID NO: 28;(iii) a primer comprising the sequence set forth in SEQ ID NO: 30 and a primer comprising the sequence set forth in SEQ ID NO: 31 ;(iv) a primer comprising the sequence set forth in SEQ ID NO: 33 and a primer comprising the sequence set forth in SEQ ID NO: 34;(v) a primer comprising the sequence set forth in SEQ ID NO: 36 and a primer comprising the sequence set forth in SEQ ID NO: 37;(vi) a primer comprising the sequence set forth in SEQ ID NO: 39 and a primer comprising the sequence set forth in SEQ ID NO: 40; or(vii) a primer comprising the sequence set forth in SEQ ID NO: 42 and a primer comprising the sequence set forth in SEQ ID NO: 43.35. The method of clause 35, further comprising employing a fluorescent probe to detect the amplification product, wherein the fluorescent probe comprises the sequence set forth in any one of SEQ ID NOs: 26, 29, 32, 35, 38, 41 , or 44.36. A method of sequencing, in a biological sample derived from a corn plant, one or more cisgenic blocks comprising SEQ ID NO: 1 or SEQ ID NO: 2, the method comprising: a) contacting the sample with nucleic acid primer(s) and sequencing the one or more cisgenic blocks and b) comparing the sequencing results to one or both of SEQ ID NO: 1 and SEQ ID NO: 2.37. The method of clause 36, wherein a cisgenic block comprising SEQ ID NO: 1 is sequenced and the results are compared to SEQ ID NO: 1.38. The method of clause 36, wherein a cisgenic block comprising SEQ ID NO: 2 is sequenced and the results are compared to SEQ ID NO: 2.Atty Docket No: 212644-WO-SEC-139. The method of clause 36, wherein i) a cisgenic block comprising SEQ ID NO: 1 is sequenced and the results are compared to SEQ ID NO: 1 and ii) a cisgenic block comprising SEQ ID NO: 2 is sequenced and the results are compared to SEQ ID NO: 2.40. A method for producing a cisgenic corn plant, the method comprising: providing one or more corn plant cells that are susceptible to one or both of northern leaf blight and southern corn rust; introducing a cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12 into the genome of one or more of the cells, thereby producing one or more modified plant cells comprising the cisgenic block in their genome; and selecting at least one of the modified corn plant cells.41. The method of clause 40, wherein the method comprises: a) effecting a site-specific modification of the CR3 target site in the genome of the corn plant cell(s); and b) introducing a polynucleotide modification template comprising the cisgenic block into the corn plant cell(s) and thereby producing the modified corn plant cells.42. The method of clause 41 , wherein the site-specific modification is induced by a CRISPR- associated endonuclease.43. The method of any one of clauses 40-42, wherein the cisgenic block comprises SEQ ID NO: 1.44. The method of any one of clauses 40-43, wherein the method comprises producing one or more modified corn plant cells and further comprises growing a modified corn plant from the selected corn plant cell, wherein the modified plant exhibits increased resistance to one or both of northern leaf blight and southern corn rust relative to the unmodified plant variety from which the one or more corn plant cells were obtained.45. The method of clause 42, wherein the CRISPR-associated endonuclease is guided by a guide polynucleotide comprising a protospacer sequence encoded by the sequence set forth in SEQ ID NO: 49.46. The method of any one of clauses 41-45, wherein a cisgenic block comprising the nucleotide sequence of SEQ ID NO: 2 is also introduced into the genome of one or more of the cells, optionally at site CR18, and wherein the selected modified corn cell comprises both of the cisgenic blocks.47. A method for producing a cisgenic corn plant, the method comprising:Atty Docket No: 212644-WO-SEC-1 providing one or more corn plant cells, that are susceptible to one or more of northern leaf blight, gray leaf spot, and anthracnose stalk rot; introducing a cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15 into the genome of one or more of the cells, thereby producing one or more modified plant cells comprising the cisgenic block in their genome; and selecting at least one of the modified corn plant cells.48. The method of clause 47, wherein the method comprises: a) effecting a site-specific modification of the CR18 target site in the genome of the corn plant cell(s); and b) introducing a polynucleotide modification template comprising the cisgenic block into the corn plant cell(s) and thereby producing the modified corn plant cells.49. The method of clause 48, wherein the site-specific modification is induced by a CRISPR- associated endonuclease.50. The method of any one of clauses 47-49, wherein the cisgenic block comprises SEQ ID NO: 2.51. The method of any one of clauses 47-50, wherein the method comprises producing one or more modified corn plant cells and further comprises growing a modified corn plant from the selected corn plant cell, wherein the modified plant exhibits increased resistance to one or more of northern leaf blight, gray leaf spot, and anthracnose stalk rot relative to the unmodified plant variety from which the one or more corn plant cells were obtained.52. The method of clause 49, wherein the CRISPR-associated endonuclease is guided by a guide polynucleotide comprising a protospacer sequence encoded by the sequence set forth in SEQ ID NO: 50.53. The method of any one of clauses 47-52, wherein a cisgenic block comprising the nucleotide sequence of SEQ ID NO: 1 is also introduced into the genome of one or more of the cells, optionally at site CR3, and wherein the selected modified corn cell comprises both of the cisgenic blocks.54. A method for producing a cisgenic corn plant, the method comprising: a) providing one or more corn plants or plant cells, that are susceptible to one or more of northern leaf blight, gray leaf spot, and anthracnose stalk rot; b) introducing:(i) a cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12,Atty Docket No: 212644-WO-SEC-1(ii) a cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15 into the genome of one or more of the cells, thereby producing one or more modified plant cells comprising the cisgenic block in their genome; and c) introducing the cisgenic block not used in step b into the genome of one or more of the modified plant cells, thereby producing one or more doubly modified plant cells comprising both of the cisgenic blocks; and d) selecting at least one of the doubly modified corn plants or plant cells.55. The method of clause 54, wherein the method comprises selecting one or more of the doubly modified plant cells and further comprises regenerating one or more plants from the one or more modified plant cells and obtaining the one or more modified plant cells used in step 3 from the regenerated plant(s) or progeny thereof.56. The method of clause 55, further comprising removing one or more nucleic acid sequences from the regenerated plant(s) via breeding and / or excision.57. The method of any one of clauses 54-56, further comprising regenerating a cisgenic corn plant from the selected doubly modified corn plant cell(s).58. The method of clause 57, further comprising removing one or more nucleic acid sequences from the plant(s) regenerated from the doubly modified cells via breeding and / or excision.59. The method of any one of clauses 54-58, wherein the cisgenic block encoding the three individual polypeptides comprises the nucleotide sequence of SEQ ID NO: 1 and is optionally introduced at site CR3.60. The method of any one of clauses 54-59, wherein the cisgenic block encoding the three individual polypeptides comprises the nucleotide sequence of SEQ ID NO: 2 is optionally introduced at site CR18.61. The method of clause 57, wherein the plant regenerated from the doubly modified corn plant cell(s) exhibits increased resistance to one or more of northern leaf blight, gray leaf spot, and anthracnose stalk rot relative to the unmodified plant variety from which the one or more corn plant cells were obtained.62. A method of protecting a corn plant against one or more of northern leaf blight, southern corn rust, gray leaf spot, or anthracnose stalk rot, the method comprising expressing, in the corn plant, one or more polypeptides from a first cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11, and the polypeptide sequence of SEQ ID NO: 12 orAtty Docket No: 212644-WO-SEC-1 from a second cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15.63. The method of clause 62, wherein the method comprises expressing polypeptides from both the first cisgenic block and the second cisgenic block.64. The method of clause 62 or 63, wherein protection is provided against two or more of the following diseases: northern leaf blight, southern corn rust, gray leaf spot, or anthracnose stalk rot.65. The method of clause 62 or 63, wherein protection is provided against three or more of the following diseases: northern leaf blight, southern corn rust, gray leaf spot, or anthracnose stalk rot.66. The method of clause 62 or 63, wherein protection is provided against northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot.67. The method of any one of clauses 62-66, wherein the first cisgenic block comprises a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 1.68. The method of any one of clauses 62-67, wherein the second cisgenic block comprises a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 2.

[0234] EXAMPLES

[0235] The present disclosure will be more completely understood with reference to the following examples. The examples are presented to aid in understanding of the present disclosure and are not intended to limit the scope of the present disclosure.

[0236] Example 1 - DNA construct production

[0237] Two cisgenic DNA blocks were created for the production of a DSL variety (“DSL Maize”) from a maize inbred (Inbred A). The first cisgenic block (“cisgenic block 1”) comprised three polynucleotides encoding native maize disease resistance genes. All three polynucleotides comprised promoter regions, coding regions (both introns and exons), and terminator regions that were identical to corresponding Zea mays genomic DNA from various varieties. No nonnatural DNA sequences were included. SEQ ID NO: 1 provides the DNA sequence of cisgenic block 1. Homology arms (SEQ ID NO: 45 and 46) were included in DNA construct 1 comprising cisgenic block 1 , along with DNA sequences encoding components of a CRISPR-Cas system for targeted insertion of cisgenic block 1 into the maize genome. Cisgenic construct 1 comprised, from 5’ to 3’, polynucleotides encoding each of NLB18 (SEQ ID NOs: 3 and 10), RppK (SEQ ID NOs: 4 and 11), and Ht1 (SEQ ID NOs: 5 and 12), which are all native maize genes.Atty Docket No: 212644-WO-SEC-1

[0238] The second cisgenic block (“cisgenic block 2”) comprised four polynucleotides encoding maize disease resistance genes. All four polynucleotides comprised promoter regions, coding regions (both introns and exons), and terminator regions that were identical to corresponding Zea mays genomic DNA from various varieties. No nonnatural DNA sequences were included. SEQ ID NO: 2 provides the DNA sequence of cisgenic block 2 (e.g., the template strand). Homology arms (SEQ ID NO: 47 and 48) were included in DNA construct 2 comprising cisgenic block 1 , along with DNA sequences encoding components of a CRISPR-Cas system for targeted insertion of cisgenic block 2 into maize genome. Cisgenic blocks 1 and 2 were designed to be inserted into the genome in opposite (e.g., complementary) orientations. That is, the coding strands and template strands for each construct are reversed. Cisgenic construct 2 comprised, from 5’ to 3’, polynucleotides encoding each of NLB18 (SEQ ID NOs: 9 and 10), Rcz2 (SEQ ID NOs: 8 and 15), Rcz1 (SEQ ID NOs: 7 and 14), and Rcg2 (SEQ ID NOs: 6 and 13), which are all native maize genes.

[0239] DNA construct 1 was created without significant issues. However, DNA construct 2 was surprisingly difficult to produce. It was necessary to screen 75 E. coli clones to find a construct that was produced correctly, without missing portions. Normally, only 8 clones are needed. Additional improvements to the protocol included growing E. coli at 28C instead of 37C and selecting smaller, slower growing colonies. Furthermore, attempts were made to produce five other alternative constructs with different combinations of native maize disease resistant genes, but none of these attempts were successful. All five alternative constructs contained Rcg2, Rcz1 , and Rcz2; two of the five also contained NLB18; and one of the 5 also contained NLB18, RppK, and Ht1. Although some of the alternative constructs were shorter than DNA construct 2 and some were longer, only DNA construct 2 was successfully produced.

[0240] Example 2 - Selection of insertion sites

[0241] Candidate genomic sites for targeted insertion can exhibit variable cutting frequencies by the corresponding guide RNA. In order to determine the higher-frequency sites, a Cas endonuclease was used to cut at various candidate sites in maize cells. The genomic sites described in WQ2022040134A1 were tested, along with their corresponding guide RNAs in a transient assay. The candidate sites were labeled as CR1 , CR2, CR3, CR4, CR5, CR6, CR7, CR9, CR14, CR17, and CR18. Candidate sites were chosen as described in WQ2022040134A1.

[0242] Table 1: Cutting frequency at various candidate genomic sites.Atty Docket No: 212644-WO-SEC-1

[0243] Table 1 shows that sites CR3 and CR18 showed the highest cutting frequencies. CR3 and CR18 were selected for targeted insertion of cisgenic block 1 and cisgenic block 2, respectively via homologous recombination. It was expected that using the insertion sites with the highest cutting frequencies would increase the chances of success in obtaining Toplants with the desired insertion(s).

[0244] Example 3 - Transformation, regeneration of Toplants, and production of Ti plants

[0245] Maize embryos of Inbred A were transformed via Agrobacterium-mediated transformation with a DNA construct 1 comprising cisgenic block 1 , flanked by the homology arms (DNA sequences immediately upstream and downstream of the CR3 site), and also encoding a Cas endonuclease and a CR3-targeting guide RNA comprising a protospacer sequence encoded by SEQ ID NO: 49. DNA construct also comprised morphogenic genes to facilitate plant regeneration and the NPTII selectable marker. To plants were regenerated and screened by PCR to identify those containing a complete insertion of cisgenic block 1 via homologous recombination at site CR3. Two primer pairs, one for each end of the cisgenic block, were designed. One primer of each pair binds to the flanking genomic sequence and the other primer binds to an end of the block. If both primer pairs showed positive results, then it was indicated that the block was inserted at the CR3 site. For cisgenic block 1 , SEQ ID NO: 16 and SEQ ID NO: 17 were used as one primer pair, and SEQ ID NO: 18 and SEQ ID NO: 19 were used as a second primer pair. To plants with both positive PCR assays were then crossed with unmodified Inbred A in order to remove DNA sequences from construct 1 unintended to be inserted into the genome (e.g., Cas9, CR3 guide RNA, morphogenic genes, and the selectable marker).

[0246] The resultant Ti plants were subjected to the same PCR assay to confirm the presence of cisgenic block 1. The integrity of cisgenic block 1 was then confirmed on the DNA sequence level using targeted next-generation sequencing technology called Southern-by-sequencing (SbS). SbS also confirmed absence of unintentionally inserted DNA from construct 1 in T1 plants. The Ti plants were self-pollinated to create T2 seed. The T2 plants were segregated forAtty Docket No: 212644-WO-SEC-1 cisgenic block 1. Three FRET-based real-time PCR assays were conducted to confirm the zygosity of cisgenic block 1. Table 2 describes the assays. Briefly, the doubly labeled probe is destroyed when the Taq polymerase amplifies over the probe binding site. This separates the two labels and stops FRET between FAM and MGB. The new fluorescence from FAM is then detected during the real-time PCR.

[0247] The T2plants that were homozygous for cisgenic block 1 were used as the immature embryo source for the subsequent transformation with DNA construct 2 containing cisgenic block 2, flanked by homology arms (DNA sequences immediately upstream and downstream of the CR18 site), and also encoding a Cas endonuclease, CR18 guide RNA comprising a protospacer sequence encoded by SEQ ID NO: 50, morphogenic genes to facilitate plant regeneration, and NPTII selectable marker. Transformation, regeneration, and screening procedures, focused on confirmation of the cisgenic block 2 insertion and absence of construct 2 DNA sequences unintended for the insertion, were similar to those described above and ultimately produced transgene-free Ti plants comprising both cisgenic block 1 at CR3 and cisgenic block 2 at CR18. These Ti plants and their progeny comprising both cisgenic blocks are termed “DSL Maize variants”.

[0248] For cisgenic block 2, SEQ ID NO: 20 and SEQ ID NO: 21 were used as one primer pair, and SEQ ID NO: 22 and SEQ ID NO: 23 were used as a second primer pair for the PCR assay. The insertion of cisgenic construct 2 was accomplished using a guide RNA comprising a protospacer sequence encoded by SEQ ID NO: 50. The FRET-based real-time PCR assays used to determine zygosity for cisgenic block 2 are described in Table 2.

[0249] Table 2: FRET-based real-time PCR assaysAtty Docket No: 212644-WO-SEC-1

[0250] Example 4 - Generation of hybrid lines and initial disease resistance results

[0251] Three DSL Maize variants derived from independent To plants were used to study the efficacy of DSL Maize. The DSL Maize variants that were homozygous for cisgenic block 1 and block 2 were crossed with eight female inbreds of mid-maturity to late-maturity for the US.

[0252] These DSL Maize hybrids were planted in 2024 in seven locations across the Midwest and southern US. Two locations receive natural southern rust disease pressure from the pathogen Puccinia polysora. Two locations were inoculated with the gray leaf spot pathogen, Cercospora zeae-maydis, and three locations were inoculated with the northern leaf blight pathogen, Exserohilum turcicum. One location was inoculated with anthracnose stalk rot pathogen, Colletotrichum graminicola. Efficacy data was collected in individual experiments forAtty Docket No: 212644-WO-SEC-1 each pathogen. The wild type hybrids (not containing cisgenic blocks 1 and 2) were planted for comparison.

[0253] Table 3 shows the initial results from this test. The DSL Maize hybrids displayed improved resistance to all targeted pathogens, compared to the wild type hybrids. Table 4 shows numerical results for the same tests.

[0254] Table 3: Disease resistance levels in DSL Maize hybridsTable 4: Numerical Disease resistance levels in DSL Maize hybrids for Example 4

[0255] Example 5 - Additional disease resistance results

[0256] In 2025 the DSL Maize hybrid efficacy trial was conducted for a second year. In the 2025 experiment, four DSL Maize variants derived from independent To plants were used to study the efficacy of DSL Maize. The DSL Maize variants that were homozygous for cisgenic block 1 and block 2 were crossed with eight female inbreds of mid-maturity to late-maturity for the US.

[0257] These DSL Maize hybrids were planted in 2025 at multiple locations across the Midwest and southern US. Three locations received natural southern rust disease pressure from the pathogen Puccinia polysora. Two locations were inoculated with the gray leaf spot pathogen, Cercospora zeae-maydis, and two locations were inoculated with the northern leaf blightAtty Docket No: 212644-WO-SEC-1 pathogen, Exserohilum turcicum. One location was inoculated with anthracnose stalk rot pathogen, Colletotrichum graminicola. Efficacy data was collected in individual experiments for each pathogen. The wild type hybrids (not containing cisgenic blocks 1 and 2) were planted for comparison.

[0258] For the foliar diseases southern rust, northern leaf blight and gray leaf spot, phenotyping was performed using a 1-9 scale evaluating symptoms on the entire row with a score of 1 as most susceptible (i.e. almost all tissue covered by lesions) and 9 as most resistant (no visible lesions). For anthracnose stalk rot, plants were inoculated at the base of the stalk with Colletotrichum graminicola inoculum. Approximately 3 weeks after inoculation, the stalks were split, and plants were evaluated for the spread of the pathogen throughout the stalk.Phenotyping was performed using a 1-10 scale, with 1 as most resistant (minimal progression of the pathogen in the stalk) to 10 as most susceptible (severe spread of pathogen throughout stalk).

[0259] Table 5 shows numerical disease resistance levels in DSL Maize hybrids for Example 5

Claims

Atty Docket No: 212644-WO-SEC-1Claims:

1. A corn plant comprising: a first cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12 and / or a second cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15.

2. The corn plant of claim 1 , wherein the corn plant comprises increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot, wherein the increased resistance is relative to a control plant lacking the first cisgenic block and second cisgenic block.

3. The corn plant of any one of claims 1 or 2, wherein the first cisgenic block comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1 , and / or the second cisgenic block comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 2.

4. The corn plant of any one of claims 1 , 2, or 3, wherein the first cisgenic block is integrated at site CR3, and the second cisgenic block is integrated at site CR18.

5. A seed derived from the corn plant of any one of claims 1-4 and comprising the first cisgenic block and second cisgenic block.

6. A DNA construct comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11, and the polypeptide sequence of SEQ ID NO: 12.

7. The DNA construct of claim 6, wherein the nucleic acid sequence encoding the polypeptides comprises the nucleic acid sequence set forth in SEQ ID NO: 1.

8. A DNA construct comprising a nucleic acid sequence encoding four individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15.

9. The DNA construct of claim 8, wherein the nucleic acid sequence encoding the polypeptides comprises the nucleic acid sequence set forth in SEQ ID NO: 2.

10. A corn plant comprising the DNA construct of any one of claims 6-9, wherein the construct is cisgenic to the corn plant.

11. A seed derived from the corn plant of claim 10, wherein the seed comprises the DNA construct.Atty Docket No: 212644-WO-SEC-112. A biological sample derived from the plant or seed of any one of claims 1-5, 10, or 11 , wherein the sample comprises the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2; wherein the nucleotide sequence is detectable in the sample using a nucleic acid amplification method, a nucleic acid hybridization method, or a nucleic acid sequencing method; and wherein the polynucleotide(s) comprising the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2 are or are not fragmented.

13. The biological sample of claim 12, wherein the biological sample comprises plant tissue derived from the plant or seed of any one of claims 1-5, 10, or 11.

14. The biological sample of claim 12, wherein the biological sample is a nucleic acid sample extracted from plant or seed material derived from the plant or seed of any one of claims 1-4, 9, or 10.

15. The biological sample of claim 12, wherein the biological sample is chosen from any of corn flour, corn meal, corn syrup, corn oil, corn starch, or cereals manufactured in whole or in part to contain corn by-products.

16. A method of producing hybrid corn seeds comprising: a) crossing a first corn line with a second corn line, wherein the first corn line comprises a first cisgenic block comprising the sequence set forth in SEQ ID NO: 1 and a second cisgenic block comprising the sequence set forth in SEQ ID NO: 2 to produce progeny plants; b) growing the progeny plants ; and c) harvesting hybrid seed produced by the progeny plants.

17. The method of claim 16, wherein the hybrid seed comprises the first cisgenic block and the second cisgenic block.

18. The method of any one of claims 16-17, wherein the first corn line comprises increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot, wherein the increased resistance is relative to a control corn plant line lacking the first cisgenic block and second cisgenic block.

19. The method of any one of claims 16-18, wherein a plant grown from the hybrid seed displays greater disease resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot, compared to the second corn line.

20. The method of any one of claims 16-19, wherein the first cisgenic block and the second cisgenic block are integrated at site CR3 and at site CR18, respectively in the first corn line.

21. A method for producing a corn plant having increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot, the method comprising: a) crossing a first parent corn plant with a second parent corn plant, wherein the first parent corn plant comprises a first cisgenic block comprising the sequence set forth in SEQAtty Docket No: 212644-WO-SEC-1ID NO: 1 and a second parent plant comprises a second cisgenic block comprising the sequence set forth in SEQ ID NO: 2 and wherein the second parent corn plant is susceptible to southern corn rust, gray leaf spot, northern leaf blight, anthracnose stalk rot, or combinations thereof, thereby producing a plurality of first generation progeny plants; and b) selecting a first generation progeny plant that comprises the first cisgenic block and second cisgenic block, wherein the selected progeny plant has increased resistance to one or more of northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot as compared to the first or second parent plant.

22. The method of claim 21, wherein the method further comprises backcrossing the first generation progeny plant of step (b) with the first or second parent plant, thereby producing a plurality of backcross progeny plants; and c) selecting from the backcross progeny plants, a plant that comprises the first cisgenic block and second cisgenic block.

23. Seed produced by the selected plants of claim 21 or claim 22.

24. A method of detecting, in a biological sample derived from a corn plant, a cisgenic block comprising SEQ ID NO: 1 , the method comprising: a) contacting the sample with at least one pair of nucleic acid primers capable of binding a first portion of SEQ ID NO: 1 with a first primer of the pair and binding either a second portion of SEQ ID NO: 1 or a genomic sequence from site CR3 with a second primer of the pair; b) performing a nucleic acid amplification reaction; and c) detecting an amplification product produced via the at least one pair of primers, wherein detection of the amplification product indicates that said sample is derived from a plant comprising the cisgenic block.

25. A method of detecting, in a biological sample derived from a corn plant, a cisgenic block comprising SEQ ID NO: 2, the method comprising: a) contacting the sample with at least one pair of nucleic acid primers capable of binding a first portion of SEQ ID NO: 2 with a first primer of the pair and binding either a second portion of SEQ ID NO: 2 or a genomic sequence from site CR18 with a second primer of the pair; b) performing a nucleic acid amplification reaction; and c) detecting an amplification product produced via the at least one pair of primers, wherein detection of the amplification product indicates that said sample is derived from a plant comprising the cisgenic block.

26. The method of claim 24 or 25, further comprising employing a fluorescent probe to detect the amplification product, wherein the fluorescent probe comprises the sequence set forth in any one of SEQ ID NOs: 26, 29, 32, 35, 38, 41 , or 44.

27. The method of any one of claims 24-26, further comprising detecting both a cisgenic block comprising SEQ ID NO: 1 and a cisgenic block comprising SEQ ID NO: 2.Atty Docket No: 212644-WO-SEC-128. A method for producing a cisgenic corn plant, the method comprising: providing one or more corn plant cells from an unmodified plant variety that are susceptible to one or both of northern leaf blight and southern corn rust; introducing a cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12 into the genome of one or more of the cells, thereby producing one or more modified plant cells comprising the cisgenic block in their genome; and selecting at least one of the modified corn plant cells.

29. The method of claim 28, wherein the method comprises: a) effecting a site-specific modification of the CR3 target site in the genome of the corn plant cell(s); and b) introducing a polynucleotide modification template comprising the cisgenic block into the corn plant cell(s) and thereby producing the modified corn plant cells.

30. The method of claim 29, wherein the site-specific modification is induced by a CRISPR- associated endonuclease.

31. The method of any one of claims 28-30, wherein the cisgenic block comprises SEQ ID NO: 1.

32. The method of any one of claims 28-31 , wherein the method comprises producing one or more modified corn plant cells and further comprises growing a modified corn plant from the selected corn plant cell, wherein the modified plant exhibits increased resistance to one or both of northern leaf blight and southern corn rust relative to the unmodified plant variety from which the one or more corn plant cells were obtained.

33. The method of claim 30, wherein the CRISPR-associated endonuclease is guided by a guide polynucleotide comprising a protospacer sequence encoded by the sequence set forth in SEQ ID NO: 49.

34. The method of any one of claims 29-33, wherein a cisgenic block comprising the nucleotide sequence of SEQ ID NO: 2 is also introduced into the genome of one or more of the cells, optionally at site CR18, and wherein the selected modified corn cell comprises both of the cisgenic blocks.

35. A method for producing a cisgenic corn plant, the method comprising: providing one or more corn plant cells, that are susceptible to one or more of northern leaf blight, gray leaf spot, and anthracnose stalk rot; introducing a cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15 into the genome of one or more of the cells, thereby producing one or more modified plant cells comprising the cisgenic block in their genome; andAtty Docket No: 212644-WO-SEC-1 selecting at least one of the modified corn plant cells.

36. The method of claim 35, wherein the method comprises: a) effecting a site-specific modification of the CR18 target site in the genome of the corn plant cell(s); and b) introducing a polynucleotide modification template comprising the cisgenic block into the corn plant cell(s) and thereby producing the modified corn plant cells.

37. The method of claim 36, wherein the site-specific modification is induced by a CRISPR- associated endonuclease.

38. The method of any one of claims 35-37, wherein the cisgenic block comprises SEQ ID NO: 2.

39. The method of any one of claims 35-38, wherein the method comprises producing one or more modified corn plant cells and further comprises growing a modified corn plant from the selected corn plant cell, wherein the modified plant exhibits increased resistance to one or more of northern leaf blight, gray leaf spot, and anthracnose stalk rot relative to the unmodified plant variety from which the one or more corn plant cells were obtained.

40. The method of claim 39, wherein the CRISPR-associated endonuclease is guided by a guide polynucleotide comprising a protospacer sequence encoded by the sequence set forth in SEQ ID NO: 50.

41. The method of any one of claims 35-40, wherein a cisgenic block comprising the nucleotide sequence of SEQ ID NO: 1 is also introduced into the genome of one or more of the cells, optionally at site CR3, and wherein the selected modified corn cell comprises both of the cisgenic blocks.

42. A method for producing a cisgenic corn plant, the method comprising: a) providing one or more corn plant cells, that are susceptible to one or more of northern leaf blight, gray leaf spot, and anthracnose stalk rot; b) introducing: a cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11 , and the polypeptide sequence of SEQ ID NO: 12 or a cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15 into the genome of one or more of the cells, thereby producing one or more modified plant cells comprising the cisgenic block in their genome; andAtty Docket No: 212644-WO-SEC-1 c) introducing the cisgenic block not used in step 2 into the genome of one or more of the modified plant cells, thereby producing one or more doubly modified plant cells comprising both of the cisgenic blocks; and d) selecting at least one of the doubly modified corn plant cells.

43. The method of claim 42, further comprising regenerating one or more plants from the one or more modified plant cells and obtaining the one or more modified plant cells used in step 3 from the regenerated plant(s) or progeny thereof.

44. The method of claim 43, further comprising removing one or more nucleic acid sequences from the regenerated plant(s) via breeding and / or excision.

45. The method of any one of claims 42-44, further comprising regenerating a cisgenic corn plant from the selected doubly modified corn plant cell(s).

46. The method of claim 45, further comprising removing one or more nucleic acid sequences from the plant(s) regenerated from the doubly modified cells via breeding and / or excision.

47. The method of any one of claims 42-46, wherein the cisgenic block encoding the three individual polypeptides comprises the nucleotide sequence of SEQ ID NO: 1 and is optionally introduced at site CR3.

48. The method of any one of claims 42-47, wherein the cisgenic block encoding the three individual polypeptides comprises the nucleotide sequence of SEQ ID NO: 2 is optionally introduced at site CR18.

49. The method of claim 48, wherein the plant regenerated from the doubly modified corn plant cell(s) exhibits increased resistance to one or more of northern leaf blight, gray leaf spot, and anthracnose stalk rot relative to the unmodified plant variety from which the one or more corn plant cells were obtained.

50. A method of protecting a corn plant against one or more of northern leaf blight, southern corn rust, gray leaf spot, or anthracnose stalk rot, the method comprising expressing, in the corn plant, one or more polypeptides from a first cisgenic block comprising a nucleic acid sequence encoding three individual polypeptides, the polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 11, and the polypeptide sequence of SEQ ID NO: 12 or from a second cisgenic block comprising a nucleic acid sequence encoding four individual polypeptides comprising, respectively, the polypeptide sequence of SEQ ID NO: 10, the polypeptide sequence of SEQ ID NO: 13, the polypeptide sequence of SEQ ID NO: 14, and the polypeptide sequence of SEQ ID NO: 15.

51. The method of claim 50, wherein the method comprises expressing polypeptides from both the first cisgenic block and the second cisgenic block.Atty Docket No: 212644-WO-SEC-152. The method of claim 50 or 51 , wherein protection is provided against two or more of the following diseases: northern leaf blight, southern corn rust, gray leaf spot, or anthracnose stalk rot.

53. The method of claim 50 or 51 , wherein protection is provided against three or more of the following diseases: northern leaf blight, southern corn rust, gray leaf spot, or anthracnose stalk rot.

54. The method of claim 50 or 51 , wherein protection is provided against northern leaf blight, southern corn rust, gray leaf spot, and anthracnose stalk rot.

55. The method of any one of claims 50-54, wherein the first cisgenic block comprises a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 1.

56. The method of any one of claims 50-55, wherein the second cisgenic block comprises a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 2.

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