Increasing gene editing and site-specific integration events using meiosis and germline promoters

Abstract

The present disclosure provides methods and compositions for increasing genomic editing and site-directed integration events using guide endonucleases as well as meiotic cell preference, egg cell preference, or embryo tissue preference promoters.

Description

[0001] This application is a divisional application of the invention patent application with application date of September 9, 2021, application number 202180061925.5, and invention name "Using meiotic and germline promoters to increase gene editing and site-specific integration events".

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 076,705, filed on September 10, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present disclosure relates to compositions and methods related to the expression of guide nucleases and guide nucleic acids in egg cells and embryonic tissues of plants.

[0005] Incorporation of Sequence Listing

[0006] The sequence listing contained in the file named "P34738WO00_SL.txt" is 175,219 bytes (in Measured in) and created on September 9, 2021, the sequence listing is submitted electronically with this application and is incorporated by reference in its entirety. Background Art

[0007] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) nucleases (e.g., Cas12a, CasX, Cas9) are proteins that are guided to a target nucleic acid molecule by a guide RNA, where the nuclease can cleave one or both strands of the target nucleic acid molecule. Summary of the invention

[0008] In one aspect, the present disclosure provides a plant comprising (a) a first nucleic acid sequence encoding a guide nuclease, which is operably linked to a heterologous egg cell-preferred or embryonic tissue-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid, which is operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of forming a complex with the guide nuclease and hybridizing to a target sequence within the plant genome and wherein the complex induces modification of the target sequence. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, the modification is an interlaced cut within a double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgene. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, at least one nuclear localization signal comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the egg cell-preferred promoter is selected from the group consisting of an EA1 promoter and an ES4 promoter. In some embodiments, the egg cell-preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82, or a functional fragment thereof. In some embodiments, the embryonic tissue-preferred promoter is selected from the group consisting of a DSUL1 promoter, an EA1 promoter, an ES4 promoter, and an EAL1 promoter. In some embodiments, the embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88, or a functional fragment thereof. In some embodiments, the second promoter is a Pol III promoter. In some embodiments, the second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the second promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of: DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter.In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence or a functional fragment thereof selected from the group consisting of SEQ ID NOs: 1-6, 21-45 and 65-88. In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of CaMV 35S promoter, actin promoter, Rab15 promoter and ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence or both are stably integrated into the genome of the plant. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence or both are stably integrated into the genome of the haploid induction line. Several embodiments relate to seeds produced by plants. In some embodiments, the seeds comprise at least one mutation in a target gene comprising a target sequence compared to seeds from a control plant of the same variety lacking the first nucleic acid sequence or the second nucleic acid sequence.

[0009] In one aspect, the present disclosure provides a plant comprising (a) a first nucleic acid sequence encoding a guide nuclease, which is operably linked to a heterologous meiotic cell-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid, which is operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of forming a complex with the guide nuclease and hybridizing to a target sequence within the plant genome, and wherein the complex induces modification of the target sequence. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, the effector protein. The modification is an interlaced cut within a double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgene. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, at least one nuclear localization signal comprises a nucleic acid sequence selected from the following: SEQ ID NO: 8 and 9. In some embodiments, the meiotic cell-preferred promoter is selected from the group consisting of a DMC1 promoter, an Mps1 promoter, and an Adf1 promoter. In some embodiments, the meiotic cell-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85 or a functional fragment thereof. In some embodiments, the second promoter is a Pol III promoter. In some embodiments, the second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the second promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of a DSUL1 promoter, an EA1 promoter, an ES4 promoter, a DMC1 promoter, an Mps1 promoter, an Adf1 promoter, and an EAL1 promoter. In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88, or a functional fragment thereof. In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of a CaMV 35S promoter, an actin promoter, a Rabl5 promoter, and a ubiquitin promoter. In some embodiments, the at least one guide nucleic acid comprises at least one guide RNA.In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the plant. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the haploid inducing line.

[0010] In one aspect, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease, which is operably linked to a heterologous egg cell-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid, which is operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of forming a complex with the guide nuclease and hybridizing with a target sequence within the plant genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one egg cell of the plant, and wherein the ribonucleoprotein produces at least one modification in the target sequence in at least one egg cell. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, an effector protein. The modification is an interleaved cut within a double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgene. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, the at least one nuclear localization signal comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:8 and 9. In some embodiments, the egg cell-preferred promoter is selected from the group consisting of EA1 promoter and ES4 promoter. In some embodiments, the egg cell-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:2-3, 21-38, 41-45, and 65-82, or a functional fragment thereof. In some embodiments, the second promoter is a Pol III promoter. In some embodiments, the second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the second promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88, or a functional fragment thereof.In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of a CaMV 35S promoter, an actin promoter, a Rab15 promoter, and a ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the plant. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the haploid induction line.

[0011] In one aspect, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease, which is operably linked to a heterologous meiotic cell-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid, which is operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of forming a complex with the guide nuclease and hybridizing with a target sequence within the plant genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one meiotic cell of the plant, and wherein the ribonucleoprotein produces at least one modification within the target sequence of at least one meiotic cell. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, an effector protein. The modification is an interleaved cut within a double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgene. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, the at least one nuclear localization signal comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:8 and 9. In some embodiments, the meiotic cell-preferred promoter is selected from the group consisting of DMC1 promoter, Mps1 promoter, and Adf1 promoter. In some embodiments, the meiotic cell-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence or a functional fragment thereof selected from the group consisting of SEQ ID NOs:4-6 and 83-85. In some embodiments, the second promoter is a Pol III promoter. In some embodiments, the second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the second promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88, or a functional fragment thereof.In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of a CaMV 35S promoter, an actin promoter, a Rab15 promoter, and a ubiquitin promoter. In some embodiments, the at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding the at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the plant. In some embodiments, the meiotic cells are derived from a haploid induction line.

[0012] In one aspect, the present disclosure provides a method for editing a plant cell genome, comprising: (a) hybridizing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding a guide nuclease, which is operably linked to a heterologous embryonic tissue-preferred promoter, and wherein the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid, which is operably linked to a heterologous second promoter, wherein at least one guide nucleic acid is capable of forming a complex with the guide nuclease and hybridizing with a target sequence within the plant genome; and (b) obtaining at least one embryo from the hybridization of step (a), wherein the guide nuclease and at least one guide nucleic acid form a ribonucleoprotein in at least one embryo, and wherein the ribonucleoprotein produces at least one modification within the target sequence in at least one embryo. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, an effector protein. The modification is an interlaced cut within a double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgenic. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, the at least one nuclear localization signal comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:8 and 9. In some embodiments, the embryonic tissue-preferred promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, and EAL1 promoter. In some embodiments, the embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence or a functional fragment thereof selected from the group consisting of SEQ ID NOs:1-3, 29-40, 43-45, and 86-88. In some embodiments, the second promoter is a Pol III promoter. In some embodiments, the second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the second promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88, or a functional fragment thereof.In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of a CaMV 35S promoter, an actin promoter, a Rab15 promoter, and a ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the first plant. In some embodiments, the first plant is a hybrid induction line.

[0013] In one aspect, the present disclosure provides a method for editing a plant cell genome, comprising: (a) hybridizing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding a guide nuclease, which is operably linked to a heterologous meiotic cell-preferred promoter, and wherein the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid, which is operably linked to a heterologous second promoter, wherein at least one guide nucleic acid is capable of forming a complex with the guide nuclease and hybridizing with a target sequence within the plant genome; and (b) obtaining at least one embryo from the hybridization of step (a), wherein the guide nuclease and at least one guide nucleic acid form a ribonucleoprotein in at least one meiotic cell, and wherein the ribonucleoprotein produces at least one modification within the target sequence in at least one meiotic cell. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, an effector protein. The modification is an interleaved cut within a double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgenic. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, the at least one nuclear localization signal comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:8 and 9. In some embodiments, the meiotic cell-preferred promoter is selected from the group consisting of DMC1 promoter, Mps1 promoter, and Adf1 promoter. In some embodiments, the meiotic cell-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence or a functional fragment thereof selected from the group consisting of SEQ ID NOs:4-6 and 83-85. In some embodiments, the second promoter is a Pol III promoter. In some embodiments, the second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the second promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88, or a functional fragment thereof.In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of a CaMV 35S promoter, an actin promoter, a Rab15 promoter, and a ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the plant. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the haploid induction line.

[0014] In one aspect, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease, which is operably linked to a heterologous embryonic tissue-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid, which is operably linked to a heterologous second promoter, wherein at least one guide nucleic acid is capable of forming a complex with the guide nucleic acid and hybridizing with a target sequence within the plant genome; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing the at least one plant to produce at least one embryo, wherein the guide nucleic acid and at least one guide nucleic acid form a ribonucleoprotein in at least one embryo from step (c), and wherein the ribonucleoprotein produces at least one modification in a target sequence in at least one embryo. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, an effector protein. The modification is an interlaced cut within a double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgenic. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, the at least one nuclear localization signal comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:8 and 9. In some embodiments, the embryonic tissue-preferred promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, and EAL1 promoter. In some embodiments, the embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-3, 29-40, 43-45, and 86-88, or a functional fragment thereof. In some embodiments, the second promoter is a Pol III promoter. In some embodiments, the second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the second promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88, or a functional fragment thereof.In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of a CaMV 35S promoter, an actin promoter, a Rab15 promoter, and a ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the first plant. In some embodiments, the first plant is a hybrid induction line.

[0015] In one aspect, the present disclosure provides a method for producing site-directed integration in a plant, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease, which is operably linked to a heterologous egg cell-preferred promoter; (ii) a second nucleic acid sequence encoding one or more guide nucleic acids, which is operably linked to a heterologous second promoter, wherein the one or more guide nucleic acids (A) are capable of forming a complex with the guide nuclease and hybridizing with a target sequence within the plant genome; and (B) are capable of hybridizing with a first site and a second site flanking a nucleic acid sequence encoding a target gene; and (iii) a third nucleic acid sequence encoding a target gene; and (b) regenerating at least one plant from the plant cell of step (a); wherein the guide nuclease and at least one guide RNA form a ribonucleoprotein in at least one egg cell of the plant, wherein the ribonucleoprotein generates a double-stranded break in the target sequence, the first site and the second site, and wherein the target gene is integrated into the target site of at least one egg cell. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, an effector protein. The modification is a staggered cut within a double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgene. In some embodiments, the guide nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, at least one nuclear localization signal comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the egg cell-preferred promoter is selected from the group consisting of EA1 promoter and ES4 promoter. In some embodiments, the egg cell-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence or a functional fragment thereof selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82. In some embodiments, the second promoter is a Pol III promoter. In some embodiments, the second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the second promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of: DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter.In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88, or a functional fragment thereof. In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of a CaMV 35S promoter, an actin promoter, a Rab15 promoter, and a ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the plant. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the haploid induction line.

[0016] In one aspect, the present disclosure provides a method for producing site-directed integration in a plant, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease, which is operably linked to a heterologous embryonic tissue-preferred promoter; (ii) a second nucleic acid sequence encoding one or more guide nucleic acids, which is operably linked to a heterologous second promoter, wherein the one or more guide nucleic acids (A) are capable of forming a complex with the guide nuclease and hybridizing with a target sequence within the plant genome; and (B) are capable of hybridizing with a first site and a second site flanking a nucleic acid sequence encoding a target gene; (iii) a third nucleic acid sequence encoding a target gene; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing at least one plant of step (b) to produce at least one embryo; wherein the guide nuclease and at least one guide RNA form a ribonucleoprotein in at least one embryo, wherein the ribonucleoprotein produces a double-stranded break in the target sequence, the first site, and the second site, and wherein the target gene is integrated into the target site in at least one embryo. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, an effector protein. The modification is a staggered cut within a double-stranded DNA molecule of the genome. In some embodiments, the modification is the insertion of a transgene. In some embodiments, the guide nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, at least one nuclear localization signal comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the embryonic tissue-preferred promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, and EAL1 promoter. In some embodiments, the embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88, or a functional fragment thereof. In some embodiments, the second promoter is a Pol III promoter. In some embodiments, the second promoter is selected from the following: a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the second promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter.In some embodiments, the second promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88 or a functional fragment thereof. In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of CaMV 35S promoter, actin promoter, Rabl5 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the first plant. In some embodiments, the first plant is a hybrid induction line.

[0017] In one aspect, the present disclosure provides a recombinant DNA construct comprising: (a) a first nucleic acid sequence encoding a guide nuclease, which is operably linked to a heterologous egg cell-preferred promoter, a meiotic cell-preferred promoter, or an embryonic tissue-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid, which is operably linked to a heterologous second promoter, wherein at least one guide nucleic acid is capable of hybridizing with a target sequence within a plant genome. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, at least one nuclear localization signal comprises a nucleic acid sequence selected from the following: SEQ ID NO: 8 and 9. In some embodiments, the egg cell-preferred promoter is selected from the following: EA1 promoter and ES4 promoter. In some embodiments, the egg cell preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence or a functional fragment thereof selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82. In some embodiments, the embryonic tissue preferred promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, and EAL1 promoter. In some embodiments, the embryonic tissue preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence or a functional fragment thereof selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88. In some embodiments, the meiotic cell preferred promoter is selected from the group consisting of DMC1 promoter, Mps1 promoter, and Adf1 promoter. In some embodiments, the meiotic cell preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence or a functional fragment thereof selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the second promoter is a Pol III promoter. In some embodiments, the second promoter is selected from the following: a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the second promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the following: a DSUL1 promoter, an EA1 promoter, an ES4 promoter, a DMC1 promoter, an Mps1 promoter, an Adf1 promoter, and an EAL1 promoter.In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88 or a functional fragment thereof. In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of a CaMV 35S promoter, an actin promoter, a Rab15 promoter, and a ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the plant. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the haploid inducing line. In some embodiments, the recombinant DNA construct is integrated into the genome of the haploid inducing line.

[0018] Several embodiments relate to a recombinant DNA construct comprising: (a) a first nucleic acid sequence encoding a DNA modification enzyme, which is operably linked to one or more TALE binding sites and a minimal promoter; and (b) a second nucleic acid sequence encoding a TALE, which is operably linked to an egg cell-preferred promoter, a meiotic cell-preferred promoter, or an embryonic tissue-preferred promoter, wherein the minimal promoter does not drive the expression of the DNA modification enzyme in the absence of TALE binding to one or more TALE binding sites. In some embodiments, the recombinant DNA construct further comprises a third nucleic acid sequence encoding a guide nucleic acid, which is operably linked to a third promoter. In some embodiments, the DNA modification enzyme is a guide nuclease. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, the first nucleic acid sequence encoding the DNA modification enzyme and the minimal promoter is operably linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TALE binding sites. In some embodiments, the CRISPR effector protein is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is codon optimized for expression in plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, the at least one nuclear localization signal comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NOs:8 and 9. In some embodiments, the egg cell-preferred promoter is selected from the group consisting of: EA1 promoter and ES4 promoter. In some embodiments, the egg cell-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence or a functional fragment thereof selected from the group consisting of: SEQ ID NOs:2-3, 21-38, 41-45 and 65-82. In some embodiments, the embryonic tissue-preferred promoter is selected from the group consisting of: DSUL1 promoter, EA1 promoter, ES4 promoter, and EAL1 promoter. In some embodiments, the embryonic tissue preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88, or a functional fragment thereof. In some embodiments, the meiotic cell preferred promoter is selected from the group consisting of DMC1 promoter, Mps1 promoter, and Adf1 promoter. In some embodiments, the meiotic cell preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85, or a functional fragment thereof. In some embodiments, the third promoter is a PolIII promoter.In some embodiments, the third promoter is selected from the following: a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the third promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the third promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter. In some embodiments, the third promoter is selected from the following: DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the third promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence or a functional fragment thereof selected from the following: SEQ ID NO: 1-6, 21-45, and 65-88. In some embodiments, the third promoter is a constitutive promoter selected from the following: CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, a third nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence and / or the third nucleic acid sequence are stably integrated into the genome of the plant. In some embodiments, the plant is from a haploid inducible line. In some embodiments, the guide nucleic acid is provided to the plant by bombardment. Several embodiments relate to a plant comprising a recombinant DNA construct. Several embodiments relate to seeds produced by a plant comprising a recombinant DNA construct. In some embodiments, the recombinant DNA construct is integrated into the genome of a haploid inducible line.

[0019] Several embodiments relate to a recombinant DNA construct comprising: (a) a first nucleic acid sequence encoding at least one guide nucleic acid, which is operably linked to one or more TALE binding sites and a minimal promoter; and (b) a second nucleic acid sequence encoding a TALE, which is operably linked to an egg cell-preferred promoter, a meiotic cell-preferred promoter, or an embryonic tissue-preferred promoter, wherein the minimal promoter does not drive the expression of the guide nucleic acid in the absence of TALE binding to one or more TALE binding sites. In some embodiments, the recombinant DNA construct further comprises a third nucleic acid sequence encoding a guide nuclease, which is operably linked to a third promoter. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, the first nucleic acid sequence encoding the guide nucleic acid and the minimal promoter is operably linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TALE binding sites. In some embodiments, the CRISPR effector protein is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the third nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the third nucleic acid sequence is codon optimized for expression in plants. In some embodiments, the third nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, the at least one nuclear localization signal comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NOs:8 and 9. In some embodiments, the egg cell preferred promoter is selected from the group consisting of: EA1 promoter and ES4 promoter. In some embodiments, the egg cell preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence or a functional fragment thereof selected from the group consisting of: SEQ ID NOs:2-3, 21-38, 41-45 and 65-82. In some embodiments, the embryonic tissue preferred promoter is selected from the group consisting of: DSUL1 promoter, EA1 promoter, ES4 promoter, and EAL1 promoter. In some embodiments, the embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88, or a functional fragment thereof. In some embodiments, the meiotic cell-preferred promoter is selected from the group consisting of DMC1 promoter, Mps1 promoter, and Adf1 promoter. In some embodiments, the meiotic cell-preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85, or a functional fragment thereof. In some embodiments, the third promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the third promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter.In some embodiments, the third promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter. In some embodiments, the third promoter is selected from the following: DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the third promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence or a functional fragment thereof selected from the following: SEQ ID NO: 1-6, 21-45, and 65-88. In some embodiments, the third promoter is a constitutive promoter selected from the following: CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the first nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, and / or the third nucleic acid sequence are stably integrated into the genome of the plant. In some embodiments, the plant is from a haploid induction line. Several embodiments relate to plants comprising a recombinant DNA construct. Several embodiments relate to seeds produced by a plant comprising a recombinant DNA construct. In some embodiments, the recombinant DNA construct is integrated into the genome of the haploid inducing line.

[0020] Several embodiments relate to a recombinant DNA construct comprising: (a) a first nucleic acid sequence encoding a guide nuclease; (b) a second nucleic acid sequence encoding a first promoter; and (c) a third nucleic acid sequence encoding a DNA modification enzyme, which is operably linked to an egg cell-preferred promoter, a meiotic cell-preferred promoter, or an embryonic tissue-preferred promoter, wherein the third nucleic acid is located between the first nucleic acid and the second nucleic acid, and wherein the third nucleic acid comprises a first target site for the DNA modification enzyme at the 5' end and a second target site for the DNA modification enzyme at the 5' end. In some embodiments, the recombinant DNA construct further comprises a fourth nucleic acid sequence encoding one or more guide nucleic acids, which is operably linked to the third promoter. In some embodiments, the DNA modification enzyme is a recombinase. In some embodiments, the first and second target sites are Lox sites. In some embodiments, the DNA modification enzyme is an endonuclease. In some embodiments, the DNA modification enzyme is a CRISPR effector protein. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, the at least one nuclear localization signal comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:8 and 9. In some embodiments, the first promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the first promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the first promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-6, 21-45, and 65-88, or a functional fragment thereof. In some embodiments, the first promoter is a constitutive promoter selected from the group consisting of CaMV 35S promoter, actin promoter, Rabl5 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the egg cell-preferred promoter is selected from the group consisting of an EA1 promoter and an ES4 promoter. In some embodiments, the egg cell-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82, or a functional fragment thereof.In some embodiments, the embryonic tissue-preferred promoter is selected from the group consisting of: DSUL1 promoter, EA1 promoter, ES4 promoter, and EAL1 promoter. In some embodiments, the embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88, or a functional fragment thereof. In some embodiments, the meiotic cell-preferred promoter is selected from the group consisting of: DMC1 promoter, Mps1 promoter, and Adf1 promoter. In some embodiments, the meiotic cell-preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 4-6 and 83-85, or a functional fragment thereof. In some embodiments, the third promoter is a Pol III promoter. Several embodiments relate to plants comprising a recombinant DNA construct. Several embodiments relate to seeds produced by plants comprising a recombinant DNA construct. In some embodiments, the plant is from a haploid inducing line. In some embodiments, the recombinant DNA construct is integrated into the genome of the haploid inducing line.

[0021] In several embodiments, high levels of egg, embryo and / or meiotic tissue-specific expression of DNA modifying enzymes such as guide nucleases (e.g., CRISPR / Cas systems) are achieved by providing plant cells with: 1) an expression construct comprising a promoter described in Table 1, which is operably linked to a sequence encoding a CRISPR effector protein, such as dCas12a or dCas9 fused to a transcriptional activator; 2) an expression construct comprising one or more target sites operably linked to a minimal promoter and a sequence encoding the DNA modifying enzyme; and 3) an expression construct encoding a guide RNA that hybridizes to one or more target sites; and thereby producing plants. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target sites are operably linked to a minimal promoter. In some embodiments, the plant cells are from a haploid induction line. In some embodiments, the female plants produced express high levels of DNA modifying enzymes in eggs, embryos and / or meiotic tissues. In some embodiments, the female plants are outcrossed, and a population of R1 plants containing unique edits is identified.

[0022] In several embodiments, high levels of egg, embryo and / or meiotic tissue-specific expression of DNA modification enzymes such as guide nucleic acid enzymes (e.g., CRISPR / Cas systems) are achieved by providing plant cells with: 1) an expression construct comprising a promoter described in Table 1, which is operably linked to a sequence encoding a TALE and 2) an expression construct comprising one or more TALE binding sites (TBs) operably linked to a minimal promoter and a sequence encoding a DNA modification enzyme and thereby producing a plant. In some embodiments, an expression construct encoding one or more guide nucleic acids is also provided. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TBs are operably linked to a minimal promoter. In some embodiments, plant cells are from haploid induction lines. In some embodiments, the female plants produced express high levels of DNA modification enzymes in eggs, embryos and / or meiotic tissues. In some embodiments, female plants are outcrossed, and R1 plant populations containing unique editing are identified.

[0023] Several embodiments relate to a method of producing two or more offspring plants with unique edits from a single transformed plant cell, the method comprising: (a) introducing into a plant cell: a first nucleic acid sequence encoding a guide nuclease operably linked to a heterologous meiosis-preferred promoter; and a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) regenerating a first plant from the plant cell of step (a), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within at least one meiotic cell of the first plant, and wherein the ribonucleoprotein produces at least one modification within the target sequence in the at least one meiotic cell; (c) pollinating the first plant of step (b); (d) germinating the two or more seeds produced in step (c) to produce two or more offspring plants with unique edits. In some embodiments, the modification is a staggered cut within a double-stranded DNA molecule of the genome. In some embodiments, the target sequence comprises genomic DNA. In some embodiments, the target sequence comprises intergenic DNA. In some embodiments, the target sequence is within a gene of interest. In some embodiments, the target gene encodes a protein or a non-protein coding RNA. In some embodiments, the target gene encodes a non-protein coding RNA selected from the following: microRNA, small interfering RNA (siRNA), trans-acting siRNA or a precursor thereof. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, at least one nuclear localization signal comprises a nucleic acid sequence selected from the following: SEQ ID NO:8 and 9. In some embodiments, the meiotic cell preferred promoter is selected from the following: DMC1 promoter, Mps1 promoter, and Adf1 promoter. In some embodiments, the meiotic cell-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85 or a functional fragment thereof. In some embodiments, the second promoter is a PolIII promoter. In some embodiments, the second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter.In some embodiments, the second promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence or a functional fragment thereof selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88. In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first plant is self-pollinated. In some embodiments, the first plant is outcrossed. In some embodiments, the plant cell is from a haploid induction line. In some embodiments, the progeny plant is haploid. In some embodiments, the method further comprises screening the haploid progeny for modification at the target site. In some embodiments, the method further comprises inducing genome doubling of the haploid plant.

[0024] Several embodiments relate to a method of producing two or more progeny plants with unique edits from a single transformed plant cell, the method comprising: (a) introducing into a plant cell: a first nucleic acid sequence encoding a guide nuclease operably linked to a heterologous first promoter; and a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous meiosis-preferred promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) regenerating a first plant from the plant cell of step (a), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within at least one meiotic cell of the first plant, and wherein the ribonucleoprotein produces at least one modification within a target cell in the at least one meiotic cell; (c) pollinating the first plant of step (b); (d) germinating the two or more seeds produced in step (c) to produce two or more progeny plants with unique edits. In some embodiments, the effector protein. The modification is a staggered cut within a double-stranded DNA molecule of the genome. In some embodiments, the target sequence comprises genomic DNA. In some embodiments, the target sequence comprises intergenic DNA. In some embodiments, the target sequence is within a gene of interest. In some embodiments, the target gene encodes a protein or a non-protein coding RNA. In some embodiments, the target gene encodes a non-protein coding RNA selected from the following: microRNA, small interfering RNA (siRNA), trans-acting siRNA or a precursor thereof. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, at least one nuclear localization signal comprises a nucleic acid sequence selected from the following: SEQ ID NO:8 and 9. In some embodiments, the meiotic cell preferred promoter is selected from the following: DMC1 promoter, Mps1 promoter, and Adf1 promoter. In some embodiments, the meiotic cell-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85 or a functional fragment thereof. In some embodiments, the first promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the first promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the first promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter.In some embodiments, the first promoter is selected from the group consisting of: DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the first promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1-6, 21-45, and 65-88, or a functional fragment thereof. In some embodiments, the first promoter is a constitutive promoter selected from the group consisting of: CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first plant is self-pollinated. In some embodiments, the first plant is outcrossed. In some embodiments, the plant cell is from a haploid induction line. In some embodiments, the progeny plant is haploid. In some embodiments, the method further comprises screening the haploid progeny for modification at the target site. In some embodiments, the method further comprises inducing genome doubling of the haploid plant.

[0025] A method of producing two or more offspring plants with unique editing from a single transformed plant cell, the method comprising: (a) introducing into a plant cell: a first nucleic acid sequence encoding a guide nuclease operably linked to a heterologous egg cell preferred promoter; and a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within a genome; and (b) regenerating a first plant from the plant cell of step (a), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within at least one egg cell of the first plant, and wherein the ribonucleoprotein produces at least one modification within a target sequence in at least one egg cell; (c) pollinating the first plant of step (b); (d) germinating the two or more seeds produced in step (c) to produce two or more offspring plants with unique editing. In some embodiments, the effector protein. The modification is a staggered cut within a double-stranded DNA molecule of the genome. In some embodiments, the target sequence comprises genomic DNA. In some embodiments, the target sequence comprises intergenic DNA. In some embodiments, the target sequence is within a target gene. In some embodiments, the target gene encodes a protein or a non-protein coding RNA. In some embodiments, the target gene encodes a non-protein coding RNA selected from the following: microRNA, small interfering RNA (siRNA), trans-acting siRNA or a precursor thereof. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, at least one nuclear localization signal comprises a nucleic acid sequence selected from the following: SEQ ID NO:8 and 9. In some embodiments, the egg cell preferred promoter is selected from the following: EA1 promoter and ES4 promoter. In some embodiments, the egg cell preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82, or a functional fragment thereof. In some embodiments, the second promoter is a Pol III promoter. In some embodiments, the second promoter is selected from the group consisting of a tissue preferred promoter, a tissue specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell preferred, egg cell preferred, or embryonic tissue preferred promoter. In some embodiments, the second promoter is a meiotic cell specific, egg cell specific, or embryonic tissue specific promoter.In some embodiments, the second promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88 or a functional fragment thereof. In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first plant is self-pollinated. In some embodiments, the first plant is outcrossed. In some embodiments, the plant cell is from a haploid induction line. In some embodiments, the progeny plant is haploid. In some embodiments, the method further comprises screening the haploid progeny for modification at the target site. In some embodiments, the method further comprises inducing genome doubling of the haploid plant.

[0026] A method of producing two or more offspring plants with unique editing from a single transformed plant cell, the method comprising: (a) introducing into a plant cell: a first nucleic acid sequence encoding a guide nuclease operably linked to a heterologous first promoter; and a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous egg cell preferred promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) regenerating a first plant from the plant cell of step (a), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within at least one egg cell of the first plant, and wherein the ribonucleoprotein produces at least one modification within the target sequence in the at least one egg cell; (c) pollinating the first plant of step (b); (d) germinating the two or more seeds produced in step (c) to produce two or more offspring plants with unique editing. In some embodiments, the effector protein. The modification is a staggered cut within a double-stranded DNA molecule of the genome. In some embodiments, the target sequence comprises genomic DNA. In some embodiments, the target sequence comprises intergenic DNA. In some embodiments, the target sequence is within a target gene. In some embodiments, the target gene encodes a protein or a non-protein coding RNA. In some embodiments, the target gene encodes a non-protein coding RNA selected from the following: microRNA, small interfering RNA (siRNA), trans-acting siRNA or a precursor thereof. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, at least one nuclear localization signal comprises a nucleic acid sequence selected from the following: SEQ ID NO:8 and 9. In some embodiments, the egg cell preferred promoter is selected from the following: EA1 promoter and ES4 promoter. In some embodiments, the egg cell preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82, or a functional fragment thereof. In some embodiments, the first promoter is selected from the group consisting of a tissue preferred promoter, a tissue specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the first promoter is a meiotic cell preferred, egg cell preferred, or embryonic tissue preferred promoter. In some embodiments, the first promoter is a meiotic cell specific, egg cell specific, or embryonic tissue specific promoter.In some embodiments, the first promoter is selected from the group consisting of: DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the first promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1-6, 21-45, and 65-88, or a functional fragment thereof. In some embodiments, the first promoter is a constitutive promoter selected from the group consisting of: CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first plant is self-pollinated. In some embodiments, the first plant is outcrossed. In some embodiments, the plant cell is from a haploid induction line. In some embodiments, the progeny plant is haploid. In some embodiments, the method further comprises screening the haploid progeny for modification at the target site. In some embodiments, the method further comprises inducing genome doubling of the haploid plant.

[0027] A method for producing two or more offspring plants with unique editing from a single transformed plant cell, the method comprising: (a) introducing into a plant cell: a first nucleic acid sequence encoding a guide nuclease, which is operably linked to a heterologous embryonic cell-preferred promoter; and a second nucleic acid sequence encoding at least one guide nucleic acid, which is operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) regenerating a first plant from the plant cell of step (a), (c) pollinating the first plant of step (b), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within the embryonic cell, and wherein the ribonucleoprotein produces at least one modification within the target sequence in the embryonic cell; (d) germinating the two or more seeds produced in step (c) to produce two or more offspring plants with unique editing. In some embodiments, the effector protein. The modification is an interlaced cut within a double-stranded DNA molecule of the genome. In some embodiments, the target sequence comprises gene DNA. In some embodiments, the target sequence comprises intergenic DNA. In some embodiments, the target sequence is within a target gene. In some embodiments, the target gene encodes a protein or a non-protein encoding RNA. In some embodiments, the target gene encodes a non-protein coding RNA selected from the following: microRNA, small interfering RNA (siRNA), trans-acting siRNA or a precursor thereof. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, at least one nuclear localization signal comprises a nucleic acid sequence selected from the following: SEQ ID NO:8 and 9. In some embodiments, the embryonic tissue preferred promoter is selected from the following: DSUL1 promoter, EA1 promoter, ES4 promoter, and EAL1 promoter. In some embodiments, the embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88, or a functional fragment thereof. In some embodiments, the second promoter is a Pol III promoter. In some embodiments, the second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the second promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter.In some embodiments, the second promoter is selected from the group consisting of DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88 or a functional fragment thereof. In some embodiments, the second promoter is a constitutive promoter selected from the group consisting of CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first plant is self-pollinated. In some embodiments, the first plant is outcrossed. In some embodiments, the plant cell is from a haploid induction line. In some embodiments, the progeny plant is haploid. In some embodiments, the method further comprises screening the haploid progeny for modification at the target site. In some embodiments, the method further comprises inducing genome doubling of the haploid plant.

[0028] A method for producing two or more offspring plants with unique editing from a single transformed plant cell, the method comprising: (a) introducing into a plant cell: a first nucleic acid sequence encoding a guide nuclease, which is operably linked to a heterologous first promoter; and a second nucleic acid sequence encoding at least one guide nucleic acid, which is operably linked to a heterologous embryo cell-preferred promoter, wherein at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) regenerating a first plant from the plant cell of step (a), (c) pollinating the first plant of step (b), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within the embryo cell, and wherein the ribonucleoprotein produces at least one modification within the target sequence in the embryo cell; (d) germinating the two or more seeds produced in step (c) to produce two or more offspring plants with unique editing. In some embodiments, the effector protein. The modification is an interlaced cut within a double-stranded DNA molecule of the genome. In some embodiments, the target sequence comprises gene DNA. In some embodiments, the target sequence comprises intergenic DNA. In some embodiments, the target sequence is within a target gene. In some embodiments, the target gene encodes a protein or a non-protein encoding RNA. In some embodiments, the target gene encodes a non-protein coding RNA selected from the following: microRNA, small interfering RNA (siRNA), trans-acting siRNA or a precursor thereof. In some embodiments, the guide nuclease is a CRISPR effector protein. In some embodiments, the guide nuclease is selected from the following: Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:7. In some embodiments, the first nucleic acid sequence is a codon optimized for plants. In some embodiments, the first nucleic acid sequence encodes at least one nuclear localization signal. In some embodiments, at least one nuclear localization signal comprises a nucleic acid sequence selected from the following: SEQ ID NO:8 and 9. In some embodiments, the embryonic tissue preferred promoter is selected from the following: DSUL1 promoter, EA1 promoter, ES4 promoter, and EAL1 promoter. In some embodiments, the embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88, or a functional fragment thereof. In some embodiments, the first promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. In some embodiments, the first promoter is a meiotic cell-preferred, egg cell-preferred, or embryonic tissue-preferred promoter. In some embodiments, the first promoter is a meiotic cell-specific, egg cell-specific, or embryonic tissue-specific promoter.In some embodiments, the first promoter is selected from the group consisting of: DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the first promoter comprises a nucleic acid sequence having at least 90% identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1-6, 21-45, and 65-88, or a functional fragment thereof. In some embodiments, the first promoter is a constitutive promoter selected from the group consisting of: CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably linked to one or more self-cleaving ribozymes. In some embodiments, the first plant is self-pollinated. In some embodiments, the first plant is outcrossed. In some embodiments, the plant cell is from a haploid induction line. In some embodiments, the progeny plant is haploid. In some embodiments, the method further comprises screening the haploid progeny for modification at the target site. In some embodiments, the method further comprises inducing genome doubling of the haploid plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Editing in reciprocal F1 plants with reproductive promoters. Transgenic R1 lines were used as female or male plants to generate reciprocal F1. Black bars indicate the percentage of events with active LbCas12a when provided by the female or male parent, as indicated by new edits appearing in F1 plants. Gray bars are the percentage of F1 individuals containing new edits. Light gray bars are the number of unique edits appearing in F1.

[0030] Figure 2 : A schematic diagram of a vector designed for TALE-induced LbCas12a expression is depicted. 35S(-46) is the 35S minimal promoter. TB indicates the TALE binding site.

[0031] Figure 3 : RNA expression of Cas12a and TALE in maize leaf protoplasts.

[0032] Figure 4 : Schematic diagram of a T-DNA vector designed for TALE-induced LbCas12a preferential expression in meiocytes / embryos / oocytes in plants. LB represents the left frame. RB represents the right frame. P represents the promoter. 35S(-46) is the 35S minimal promoter. TB is the TALE binding site.

[0033] Figure 5: Schematic diagram of a T-DNA vector designed for meiocyte / embryo / oocyte-preferential expression of Cas12a driven by a strong constitutive promoter. P represents the promoter. Cre represents the Cre recombinase. The arrow indicates directionality. DETAILED DESCRIPTION

[0034] Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by ordinary technicians in the field to which the present disclosure belongs. Where a term is provided in the singular, the inventors also contemplate the various aspects of the present disclosure described in the plural form of the term. If there are differences in the terms and definitions used in the references incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meanings in the fields in which they are used, as exemplified by the field-specific dictionaries, for example, "The American Science Dictionary" (American Heritage Dictionaries ed., 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to be limited to the mechanism or mode of action. References thereto are provided for illustrative purposes only.

[0035] Unless otherwise indicated, the practice of the present disclosure will involve conventional techniques of biochemistry, chemistry, molecular biology, microbiology, cell biology, plant biology, genomics, biotechnology, and genetics, which are within the skill of the art. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition (2012); Current Protocols In Molecular Biology (FM Ausubel et al., ed., (1987)); Plant Breeding Methodology (NF Jensen, Wiley-Interscience 21988)); Methods In Enzymology Series (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, ed., (1995)); Harlow and Lane, ed., (1988) Antibodies, A Laboratory Manual; Animal Cell Culture (RI Freshney, ed., (1987)); Recombinant Protein Purification: Principles And Methods, 18-1142-75, GE Healthcare Life Sciences. Sciences; CN Stewart, A. Touraev, V. Citovsky, T. Tzfira, editors, (2011) Plant Transformation Technologies (Wiley-Blackwell); and RH Smith (2013) Plant Tissue Culture: Techniques and Experiments (Academic Press, Inc.).

[0036] Any references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are hereby incorporated by reference in their entirety.

[0037] When a group of alternatives is presented, any and all combinations of the members that make up the group are specifically contemplated. For example, if the item is selected from the group consisting of A, B, C, and D, the inventor specifically contemplates each alternative (e.g., A alone, B alone, etc.), as well as combinations such as A, B, and D; A and C; B and C, etc.

[0038] As used herein, for example, the singular and the singular forms of the terms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0039] Any of the compositions, nucleic acid molecules, polypeptides, cells, plants, etc. provided herein are specifically contemplated for use in any of the methods provided herein.

[0040] Several embodiments described herein relate to compositions and methods for preferentially expressing DNA modification enzymes (e.g., guide nucleases) in egg cells, meiotic cells, and / or embryonic cells of plants. In some embodiments, the present invention provides compositions and methods for preferentially expressing components of CRISPR / Cas editing systems in egg cells, meiotic cells, and / or embryonic cells of plants. Several embodiments relate to compositions and methods for producing offspring with unique editing from parents, wherein the parents are included in egg cells, meiotic cells, and / or embryonic cells that preferentially provide DNA modification enzymes (e.g., guide nucleases) expression cassettes. In some embodiments, maternal plants that preferentially express DNA modification enzymes (e.g., guide nucleases) in egg cells, meiotic cells, and / or embryonic cells are provided. In some embodiments, paternal plants that preferentially express DNA modification enzymes (e.g., guide nucleases) in egg cells, meiotic cells, and / or embryonic cells are provided. In some embodiments, seed populations are provided, wherein two or more seeds contain unique editing, wherein the seed population is produced by parents expressing DNA modification enzymes (e.g., guide nucleases), preferably in egg cells, meiotic cells, and / or embryonic cells. Non-limiting examples of expression elements that can be used in the compositions and methods described herein are provided in Table 1.

[0041] As used herein, "egg cell" refers to the haploid egg cell produced by the female gametophyte of a plant. After fertilization of the haploid pollen cell, a diploid zygote is formed, thereby producing an embryo. As used herein, "embryonic tissue" refers to a diploid tissue, which comprises the precursor tissue of leaves, stems and root tissues, and one or more cotyledons. The embryonic tissue is eventually introduced into the seed. Once the embryo begins to germinate, a seedling or plantlet will be produced. As used herein, meiosis refers to the cell division process that produces gametes in a sexually reproducing organism. Meiosis includes two rounds of cell division, ultimately producing four haploid cells. Meiotic cells refer to cells that undergo meiosis.

[0042] In one aspect, the present disclosure provides a recombinant DNA construct comprising a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous egg cell-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within a plant genome. In one aspect, the present disclosure provides a recombinant DNA construct comprising a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous embryonic tissue-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within a plant genome. In one aspect, the present disclosure provides a recombinant DNA construct comprising a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous meiosis-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within a plant genome.

[0043] In one aspect, the present disclosure provides a recombinant DNA construct comprising a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to an egg cell-preferred promoter, wherein at least one guide nucleic acid is capable of hybridizing to a target sequence within a plant genome. In one aspect, the present disclosure provides a recombinant DNA construct comprising a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to an embryonic tissue-preferred promoter, wherein at least one guide nucleic acid is capable of hybridizing to a target sequence within a plant genome. In one aspect, the present disclosure provides a recombinant DNA construct comprising a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a meiosis-preferred promoter, wherein at least one guide nucleic acid is capable of hybridizing to a target sequence within a plant cell genome.

[0044] In one aspect, the disclosure provides a plant comprising a recombinant DNA construct comprising a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous egg cell-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome of the plant. In one aspect, the disclosure provides a plant comprising a recombinant DNA construct comprising a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous embryonic tissue-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome of the plant. In one aspect, the present disclosure provides a plant comprising a recombinant DNA construct comprising a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous meiosis-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome of the plant.

[0045] In one aspect, the present disclosure provides a plant comprising a recombinant DNA construct comprising a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to an egg cell-preferred promoter, wherein at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome of the plant. In one aspect, the present disclosure provides a plant comprising a recombinant DNA construct comprising a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to an embryonic tissue-preferred promoter, wherein at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome of the plant. In one aspect, the present disclosure provides a plant comprising a recombinant DNA construct comprising a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a meiosis-preferred promoter, wherein at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome of the plant.

[0046] In one aspect, the present disclosure provides seeds of any of the plants disclosed herein.

[0047] Nucleic acids and amino acids

[0048] The use of the term "polynucleotide" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides comprising deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also contemplated. One of ordinary skill in the art will recognize that polynucleotides and nucleic acid molecules can include deoxyribonucleotides, ribonucleotides, or a combination of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs. The polynucleotides of the present disclosure also encompass all forms of sequences, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, etc. In one aspect, the nucleic acid molecules provided herein are DNA molecules. In another aspect, the nucleic acid molecules provided herein are RNA molecules. In one aspect, the nucleic acid molecules provided herein are single-stranded. In another aspect, the nucleic acid molecules provided herein are double-stranded.

[0049] As used herein, the term "recombinant" refers to nucleic acid (DNA or RNA) molecules, proteins, constructs, vectors, etc., to nucleic acid or amino acid molecules or sequences that are man-made and not normally found in nature and / or are present in an environment not normally found in nature, including nucleic acid molecules (DNA or RNA) molecules, proteins, constructs, etc., which contain a combination of polynucleotide or protein sequences that do not naturally appear consecutively or closely together without human intervention, and / or polynucleotide molecules, proteins, constructs, etc., which contain at least two polynucleotide sequences or protein sequences that are heterologous to each other.

[0050] In one aspect, the methods and compositions provided herein comprise a vector. As used herein, the term "vector" refers to a DNA molecule used as a vehicle to carry exogenous genetic material into a cell.

[0051] In one aspect, one or more polynucleotide sequences from the vector are stably integrated into the plant genome. In one aspect, one or more polynucleotide sequences from the vector are stably integrated into the plant cell genome.

[0052] In one aspect, the first nucleic acid sequence and the second nucleic acid sequence are provided in a single vector. In another aspect, the first nucleic acid sequence is provided in a first vector, and the second nucleic acid sequence is provided in a second vector.

[0053] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide can be encoded by a polynucleotide as provided herein. An example of a polypeptide is a protein. A protein as provided herein can be encoded by a nucleic acid molecule as provided herein.

[0054] The routine techniques of the art can be used to separate nucleic acids. For example, any method can be used to separate nucleic acids, including but not limited to recombinant nucleic acid technology and / or polymerase chain reaction (PCR). General PCR technology is described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid technology includes, for example, restriction enzyme digestion and connection, which can be used to separate nucleic acids. Isolated nucleic acids can also be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides. Polypeptides can be purified from natural sources (e.g., biological samples) by known methods such as DEAE ion exchange, gel filtration and hydroxyapatite chromatography. Polypeptides can also be purified, for example, by expressing nucleic acids in expression vectors. In addition, purified polypeptides can be obtained by chemical synthesis. The degree of purity of the polypeptide can be measured using any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis or HPLC analysis.

[0055] Without limitation, nucleic acids can be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0056] Polypeptides can be detected with antibodies. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), protein blotting, immunoprecipitation, and immunofluorescence. Antibodies provided herein can be polyclonal antibodies or monoclonal antibodies. Methods well known in the art can be used to produce antibodies with specific binding affinity for polypeptides provided herein. Antibodies provided herein can be attached to solid supports such as microtiter plates using methods well known in the art.

[0057] As used herein, the term "percent identity" or "percent identity" refers to two or more nucleotide or protein sequences, which is calculated by (i) comparing two optimally aligned sequences (nucleotides or proteins) over a comparison window, (ii) determining the number of positions at which the same nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) occurs in the two sequences to produce the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the comparison window, and then (iv) multiplying this quotient by 100% to obtain the percent identity. If the "percent identity" is calculated with respect to a reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of matched positions over the aligned region by the total length of the reference sequence. Therefore, for the purposes of this application, when two sequences (query and subject) are optimally aligned (taking into account gaps in the alignment), the "percent identity" of the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or comparison window), and then multiplied by 100%. When percent sequence identity is applied to proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, in which amino acid residues are substituted with other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity), thereby not altering the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitutions. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity."

[0058] The term "sequence complementarity percentage" or "complementarity percentage" of two nucleotide sequences referred to herein is similar to the concept of identity percentage, but refers to the percentage of nucleotides in the query sequence that optimally base pair or hybridize with the nucleotides of the subject sequence when the query sequence and the subject sequence are arranged linearly and without secondary folded structures (such as loops, stems or hairpins). This complementarity percentage can be between two DNA chains, two RNA chains, or a DNA chain and an RNA chain. "Complementarity percentage" can be calculated by (i) optimally base pairing or hybridizing the two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structure) within a comparison window, (ii) determining the number of base pair positions between the two sequences on the comparison window to generate the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying this quotient by 100% to obtain the complementarity percentage of the two sequences. The optimal base pairing of the two sequences can be determined based on known nucleotide base pairings, such as GC, AT and AU, by hydrogen bonding. If the "complementarity percentage" of a reference sequence is calculated without specifying a particular comparison window, the identity percentage is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Thus, for the purposes of this application, when two sequences (query and subject) are optimally base paired (taking into account mismatches or non-base paired nucleotides), the "complementarity percentage" of the query sequence is equal to the number of base paired positions between the two sequences divided by the total number of positions in the query sequence over its length, which is then multiplied by 100%.

[0059] In order to optimize sequence alignment to calculate the percent identity, various pairwise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or the Basic Local Alignment Search Tool. Etc., can be used to compare sequence identity or similarity between two or more nucleotide or protein sequences. Although other alignment and comparison methods are well known in the art, the alignment and percent identity between two sequences (including the above percent identity ranges) can be determined by the ClustalW algorithm, see, e.g., Chenna R. et al., "Multiple sequence alignment with the Clustal series of programs," Nucleic Acids Research 31:3497-3500 (2003); Thompson JD et al., "Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice," Nucleic Acids Research 22:4673-4680 (1994); Larkin MA et al., "Clustal W and Clustal X version 2.0," Bioinformatics 23:2947-48 (2007); and Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410 (1990), the entire contents and disclosure of which are incorporated herein by reference.

[0060] As used herein, a first nucleic acid molecule can "hybridize" with a second nucleic acid molecule in a sequence-specific, antiparallel manner (i.e., nucleic acid-specific binding to complementary nucleic acids) through non-covalent interactions (e.g., Watson-Crick base pairing) under appropriate in vitro and / or in vivo temperature and solution ionic strength conditions. As known in the art, standard Watson-Crick base pairing includes: adenine (A) pairing with thymine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). In addition, for hybridization between two RNA molecules (e.g., dsRNA), guanine base pairs with uracil are also known in the art. For example, G / U base pairing is partially responsible for the degradation (i.e., redundancy) of the genetic code in the case of tRNA anticodon base pairing with codons in mRNA. In the context of the present disclosure, the guanine of the protein binding segment (dsRNA duplex) of the subject DNA targeting RNA molecule is considered to be complementary to uracil, and vice versa. Thus, when a G / U base pair can form at a given nucleotide position (the protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule), that position is not considered non-complementary, but rather is considered complementary.

[0061] Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0062] Hybridization requires that the two nucleic acids contain complementary sequences, but there may be mismatches between the bases. The conditions suitable for hybridization between the two nucleic acids depend on the length and complementarity of the nucleic acids, which are variables well known in the art. The greater the degree of complementarity between the two nucleotide sequences, the greater the melting temperature (Tm) value of the nucleic acid hybrid with these sequences. For hybridization between nucleic acids with short-length complementarity (e.g., complementarity of more than 35 or less nucleotides), the position of mismatches becomes important (see Sambrook et al.). Typically, the length of hybridizable nucleic acids is at least 10 nucleotides. The illustrative minimum length of hybridizable nucleic acids is: at least 15 nucleotides; at least 18 nucleotides; at least 20 nucleotides; at least 22 nucleotides; at least 25 nucleotides and at least 30 nucleotides. In addition, those skilled in the art will recognize that temperature and washing solution salt concentration can be adjusted as needed according to factors such as the length and complementarity of the complementary region.

[0063] It is understood in the art that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to be specifically hybridizable or hybridizable. In addition, a polynucleotide may hybridize in one or more segments so that intermediate or adjacent segments are not involved in the hybridization event (e.g., a loop structure or a hairpin structure). For example, an antisense nucleic acid, in which 18 of the 20 nucleotides of the antisense compound are complementary to the target region and will therefore specifically hybridize, would represent 90% complementarity. In this example, the remaining non-complementary nucleotides may be clustered or interspersed with the complementary nucleotides and need not be adjacent to each other or to the complementary nucleotides. The percent complementarity between specific nucleic acid sequence segments within a nucleic acid can be routinely determined using methods known in the art. Program (Basic Local Alignment Search Tool) and the PowerBLAST program (see Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489).

[0064] SpawnEdit

[0065] Several embodiments described herein relate to compositions and methods for producing heritable edits in the genome of progeny plants by preferentially expressing DNA modification enzymes (e.g., guide nucleases) in egg cells, meiotic cells, and / or embryonic cells of parental plants. In some embodiments, the parental plant expressing the DNA modification enzyme is a female plant. In some embodiments, the parental plant expressing the DNA modification enzyme is a male plant.

[0066] As used herein, the term "genome editing" or "editing" refers to any modification of a nucleotide sequence in a site-specific manner. In the present disclosure, genome editing techniques include the use of DNA modification enzymes such as endonucleases, recombinases, transposases, deaminases, methylases, helicases, and any combination thereof. In one aspect, "modification" includes hydrolysis and deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the sequence-specific editing system comprises an adenine deaminase. In one aspect, "modification" includes hydrolysis and deamination of adenine or adenosine. In one aspect, "modification" includes hydrolysis and deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In one aspect, "modification" comprises the insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides. In some embodiments, "modification" comprises the insertion of one or more transgenes. In another aspect, "modification" includes deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides. In one other aspect, "modification" includes converting at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides. In yet another aspect, "modification" includes substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides.In yet another aspect, "modification" includes duplicating at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000 or at least 10,000 nucleotides. In some embodiments, "modification" includes replacing "A" in the nucleic acid sequence with "C", "G" or "T". In some embodiments, "modification" includes replacing "C" in the nucleic acid sequence with "A", "G" or "T". In some embodiments, "modification" includes replacing "G" in the nucleic acid sequence with "A", "C" or "T". In some embodiments, the "modification" comprises replacing a "T" in a nucleic acid sequence with an "A", "C" or "G". In some embodiments, the "modification" comprises replacing a "C" in a nucleic acid sequence with a "U". In some embodiments, the "modification" comprises replacing a "G" in a nucleic acid sequence with an "A". In some embodiments, the "modification" comprises replacing an "A" in a nucleic acid sequence with a "G". In some embodiments, the "modification" comprises replacing a "T" in a nucleic acid sequence with a "C".

[0067] In one aspect, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous meiosis-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within at least one meiotic cell of the plant, and wherein the ribonucleoprotein produces at least one double-stranded break within the target sequence in the at least one meiotic cell.

[0068] In one aspect, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous meiosis-preferred promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within at least one meiotic cell of the plant, and wherein the ribonucleoprotein produces at least one double-stranded break within the target sequence in the at least one meiotic cell.

[0069] In one aspect, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous egg cell-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within at least one egg cell of the plant, and wherein the ribonucleoprotein produces at least one double-stranded break within the target sequence in the at least one egg cell.

[0070] In one aspect, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to an egg cell-preferred promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within at least one egg cell of the plant, and wherein the ribonucleoprotein produces at least one double-stranded break within the target sequence in the at least one egg cell.

[0071] In one aspect, the present disclosure provides a method for editing the genome of a plant cell, comprising: (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous embryonic tissue-preferred promoter, and wherein the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) obtaining at least one embryo from the crossing of step (a), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within the at least one embryo, and wherein the ribonucleoprotein produces at least one double-stranded break within the target sequence in the at least one embryo.

[0072] In one aspect, the present disclosure provides a method for editing the genome of a plant cell, comprising: (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter, and wherein the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to an embryonic tissue-preferred promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) obtaining at least one embryo from the hybridization of step (a), wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within the at least one embryo, and wherein the ribonucleoprotein produces at least one double-stranded break within the target sequence in the at least one embryo.

[0073] In one aspect, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous embryonic tissue-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing the at least one plant to produce at least one embryo, wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within the at least one embryo from step (c), and wherein the ribonucleoprotein produces at least one double-stranded break within the target sequence in the at least one embryo.

[0074] In one aspect, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to an embryonic tissue-preferred promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing the at least one plant to produce at least one embryo, wherein the guide nuclease and the at least one guide nucleic acid form a ribonucleoprotein within the at least one embryo from step (c), and wherein the ribonucleoprotein produces at least one double-stranded break within the target sequence in the at least one embryo.

[0075] In one aspect, the present disclosure provides a method for producing site-directed integration in a plant, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous meiosis-preferred promoter; and (ii) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a heterologous second promoter, wherein the one or more guide nucleic acids (a) are capable of hybridizing to a target sequence within the genome of the plant; and (b) are capable of hybridizing to a first site and a second site flanking a nucleic acid sequence encoding a gene of interest; and (iii) a third nucleic acid sequence encoding the gene of interest; (b) regenerating at least one plant from the plant cell of step (a); wherein the guide nuclease and the at least one guide RNA form a ribonucleoprotein in at least one meiotic cell of the plant, wherein the ribonucleoprotein produces a double-stranded break within the target sequence, the first site, and the second site, and wherein the gene of interest integrates into the target site in at least one egg cell.

[0076] In one aspect, the present disclosure provides a method for producing site-directed integration in a plant, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a meiosis-preferred second promoter, wherein the one or more guide nucleic acids (a) are capable of hybridizing to a target sequence within the genome of the plant; and (b) are capable of hybridizing to a first site and a second site flanking a nucleic acid sequence encoding a gene of interest; and (iii) a third nucleic acid sequence encoding the gene of interest; (b) regenerating at least one plant from the plant cell of step (a); wherein the guide nuclease and the at least one guide RNA form a ribonucleoprotein in at least one meiotic cell of the plant, wherein the ribonucleoprotein produces a double-stranded break within the target sequence, the first site, and the second site, and wherein the gene of interest integrates into the target site in at least one meiotic cell.

[0077] In one aspect, the present disclosure provides a method for producing site-directed integration in a plant, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous egg cell-preferred promoter; and (ii) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a heterologous second promoter, wherein the one or more guide nucleic acids (a) are capable of hybridizing to a target sequence within the genome of the plant; and (b) are capable of hybridizing to a first site and a second site flanking a nucleic acid sequence encoding a target gene; and (iii) a third nucleic acid sequence encoding the target gene; (b) regenerating at least one plant from the plant cell of step (a); wherein the guide nuclease and the at least one guide RNA form a ribonucleoprotein in at least one egg cell of the plant, wherein the ribonucleoprotein produces a double-stranded break in the target sequence, the first site, and the second site, and wherein the target gene integrates into the target site in the at least one egg cell.

[0078] In one aspect, the present disclosure provides a method for producing site-directed integration in a plant, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to an egg cell-preferred second promoter, wherein the one or more guide nucleic acids (a) are capable of hybridizing to a target sequence within the genome of the plant; and (b) are capable of hybridizing to a first site and a second site flanking a nucleic acid sequence encoding a gene of interest; and (iii) a third nucleic acid sequence encoding the gene of interest; (b) regenerating at least one plant from the plant cell of step (a); wherein the guide nuclease and the at least one guide RNA form a ribonucleoprotein in at least one egg cell of the plant, wherein the ribonucleoprotein produces a double-stranded break within the target sequence, the first site, and the second site, and wherein the gene of interest integrates into the target site in the at least one egg cell.

[0079] In one aspect, the present disclosure provides a method for producing site-directed integration in a plant, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous embryonic tissue-preferred promoter; (ii) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a heterologous second promoter, wherein the one or more guide nucleic acids (a) are capable of hybridizing to a target sequence within the genome of the plant; and (b) are capable of hybridizing to a first site and a second site flanking a nucleic acid sequence encoding a gene of interest; and (iii) a third nucleic acid sequence encoding the gene of interest; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing at least one plant from step (b) to form at least one embryo; wherein the guide nuclease and the at least one guide RNA form a ribonucleoprotein within the at least one embryo, wherein the ribonucleoprotein produces a double-stranded break within the target sequence, the first site, and the second site, and wherein the gene of interest integrates into the target site in the at least one embryo.

[0080] In one aspect, the present disclosure provides a method for producing site-directed integration in a plant, comprising: (a) introducing into a plant cell: (i) a second nucleic acid sequence encoding a guide nuclease capable of producing staggered cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; (ii) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to an embryonic tissue-preferred promoter, wherein the one or more guide nucleic acids (a) are capable of hybridizing to a target sequence within the genome of the plant; and (b) are capable of hybridizing to a first site and a second site flanking a nucleic acid sequence encoding a gene of interest; and (iii) a third nucleic acid sequence encoding the gene of interest; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing at least one plant from step (b) to form at least one embryo; wherein the guide nuclease and the at least one guide RNA form a ribonucleoprotein within the at least one embryo, wherein the ribonucleoprotein produces a double-stranded break within the target sequence, the first site, and the second site, and wherein the gene of interest integrates into the target site in the at least one embryo.

[0081] Regulatory elements

[0082] Regulatory elements such as promoters, leader regions (also referred to as 5'UTR), enhancers, introns and transcription termination regions (or 3'UTR) play an indispensable role in the overall expression of genes in living cells. As used herein, the term "regulatory element" refers to a DNA molecule with gene regulatory activity. As used herein, the term "gene regulatory activity" refers to the ability to affect the expression of operably connected transcribable DNA molecules, such as by affecting the transcription and / or translation of operably connected transcribable DNA molecules. Regulatory elements that work in plants, such as promoters, leader regions, enhancers, introns and 3'UTR, can be used to change plant phenotypes by genetic engineering. Regulatory elements can be characterized by their gene expression patterns, for example, positive and / or negative effects, such as constitutive expression or time, space, development, tissue, environment, physiology, pathology, cell cycle and / or chemical responsiveness, and any combination thereof, and characterized by quantitative or qualitative indications. As used herein, "gene expression pattern" is any pattern by which an operably connected DNA molecule is transcribed into a transcription RNA molecule. The transcribed RNA molecules can be translated to produce protein molecules or can provide antisense or other regulatory RNA molecules, such as double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), etc. As used herein, the term "protein expression" is any pattern of translating transcribed RNA molecules into protein molecules. Protein expression can be characterized by its temporal, spatial, developmental or morphological characteristics and quantitative or qualitative indications.

[0083] As generally understood in the art, the term "promoter" refers to a DNA sequence that comprises an RNA polymerase binding site, a transcription start site and / or a TATA box and assists or promotes the transcription and expression of a related transcribable polynucleotide sequence and / or a gene (or transgenic). The promoter can initially be separated from the 5' untranslated region (5'UTR) of a gene genomic copy. The promoter can be synthesized, altered or derived from a known or naturally occurring promoter sequence or other promoter sequence. The promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences. Therefore, the promoter of the present application can include a variant of a promoter sequence that is similar but not identical to other promoter sequences known or provided herein in composition. Promoters can be classified according to various standards related to the expression pattern of the related coding or transcribable sequence or gene (including transgenic) that is operably connected to the promoter, such as constitutive, developmental, tissue-specific, cell cycle-specific, inducible, etc.

[0084] In some embodiments, the promoter is operably linked to the 5' of the leader sequence. As used herein, the term "leader" refers to a DNA molecule separated from the non-translated 5' region (5'UTR) of a gene, generally defined as a nucleotide segment between the transcription start site (TSS) and the start site of a protein coding sequence. Alternatively, the leader region can be a DNA element produced or manipulated by artificial synthesis. The leader region can be used as a 5' regulatory element for regulating the expression of an operably linked transcribable DNA molecule. The leader molecule can be used together with a heterologous promoter or its natural promoter.

[0085] As used herein, "operably linked" refers to a functional connection between two or more elements. For example, an operable connection between a target polynucleotide and a regulatory sequence (e.g., a promoter) is a functional connection that allows the target polynucleotide to be expressed. Operably linked elements can be continuous or non-continuous.

[0086] Promoters that are expressed in a specific tissue of an organism but not in other tissues are referred to as "tissue-specific" promoters. Promoters that drive enhanced expression in certain tissues of an organism relative to other tissues of the organism are referred to as "tissue-preferred" promoters. Thus, "tissue-preferred" promoters cause relatively high or preferential expression in specific tissues of a plant, but expression levels are lower in other tissues of the plant. In another aspect, the promoters provided herein are tissue-specific promoters. In one other aspect, the promoters provided herein are tissue-preferred promoters. In one aspect, tissue-preferred promoters comprise tissue-specific promoters.

[0087] A promoter that is expressed in the meiotic cells of an organism but not expressed in non-meiotic cells is referred to as a "meiotic cell specific" or "meiosis specific" promoter. A promoter that promotes enhanced expression in the meiotic cells of an organism relative to other cells of the organism is referred to as a "meiotic cell preference" or "meiosis preference" promoter. Therefore, a "meiotic cell preference" or "meiosis preference" promoter causes relatively high or preferential expression in plant cells undergoing meiosis, but has a low expression level in other cells of the plant. In another aspect, the promoter provided herein is a meiosis specific promoter. In one other aspect, the promoter provided herein is a meiosis preference promoter. In one aspect, meiosis preference promoters include meiosis specific promoters. Promoters expressed in a cell cycle dependent manner are referred to as "cell cycle specific" promoters. In another aspect, the promoter provided herein is a cell cycle specific promoter. In one other aspect, the promoter provided herein is a cell cycle preference promoter. In one aspect, cell cycle preference promoters include cell cycle specific promoters.

[0088] Determination of promoter activity can be performed using any standard method in the art. For example, without limitation, a promoter of interest can be used to drive expression of a fluorophore or other reporter molecule, and the concentration of the expressed molecule can be used to determine promoter activity in different cell or tissue types.

[0089] Several embodiments described herein relate to the expression of DNA modifying enzymes, such as guide nucleases, preferably in egg, meiotic and / or embryonic cells of plants. Non-limiting examples of expression elements used in the compositions and methods described herein are provided in Table 1.

[0090] Table 1: Plant egg, embryo and meiotic cell promoters

[0091]

[0092]

[0093] In one embodiment, a fragment of a promoter sequence disclosed in Table 1 is provided. The promoter fragment may comprise egg, embryo and / or meiosis expression activity as described above, and may be used alone or in combination with other promoters and promoter fragments, such as for constructing a chimeric promoter, or in combination with other expression elements and expression element fragments. In some embodiments, a promoter fragment comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900 or at least about 1000 consecutive nucleotides or longer of a DNA molecule having a promoter activity disclosed herein is provided. In some embodiments, a promoter fragment is provided comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, at least about 1000, at least about 1050, at least about 1100 or at least about 1150 consecutive nucleotides or longer of a DNA sequence having promoter activity disclosed herein, wherein the DNA sequence comprises a TATA box and is identical to SEQ ID NO: 1. NO.1-6, 21-45, 65-69 or 75-88 have at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity or at least about 100% identity. Methods for producing this type of fragment from the starting promoter molecule are well known in the art.

[0094] In one aspect, the meiotic cell-preferred or meiotic-preferred promoter comprises a DMC1 promoter. In one aspect, the meiotic cell-preferred or meiotic-preferred promoter comprises an Mps1 promoter. In one aspect, the meiotic cell-preferred or meiotic-preferred promoter comprises an Adf1 promoter. In one aspect, the meiotic cell-preferred or meiotic-preferred promoter comprises a promoter selected from the group consisting of a DMC1 promoter, an Mps1 promoter, and an Adf1 promoter.

[0095] In one aspect, the expression elements described in Table 1 are operably connected to nucleic acids encoding DNA modification enzymes (such as guide nucleases). In one aspect, the expression elements described in Table 1 are operably connected to nucleic acids encoding Cas9 nucleases. In one aspect, the expression elements described in Table 1 are operably connected to nucleic acids encoding Cas12a nucleases. In one aspect, the expression elements described in Table 1 are operably connected to nucleic acids encoding CasX nucleases. In one aspect, the expression elements described in Table 1 are operably connected to nucleic acids encoding guide nucleic acids. In one aspect, the expression elements described in Table 1 are operably connected to nucleic acids encoding guide RNAs. In one aspect, the expression elements described in Table 1 are operably connected to nucleic acids encoding single guide RNAs. In some embodiments, the flank of guide RNA acid is a self-cleaving ribozyme. In one aspect, the expression elements described in Table 1 are operably connected to nucleic acids encoding recombinases (e.g., Cre recombinases). In one aspect, the expression elements described in Table 1 are operably connected to nucleic acids encoding TALEs.

[0096] In one aspect, the DMC1 promoter is operably connected to the nucleic acid encoding the guide nuclease. In one aspect, the DMC1 promoter is operably connected to the nucleic acid encoding the Cas9 nuclease. In one aspect, the DMC1 promoter is operably connected to the nucleic acid encoding the Cas12a nuclease. In one aspect, the DMC1 promoter is operably connected to the nucleic acid encoding the CasX nuclease. In one aspect, the DMC1 promoter is operably connected to the nucleic acid encoding the guide nucleic acid. In one aspect, the DMC1 promoter is operably connected to the nucleic acid encoding the guide RNA. In one aspect, the DMC1 promoter is operably connected to the nucleic acid encoding a single guide RNA. In some embodiments, the flank of the guide RNA acid is a self-cleaving ribozyme. In one aspect, the DMC1 promoter is operably connected to the nucleic acid encoding the recombinase (e.g., Cre recombinase). In one aspect, the DMC1 promoter is operably connected to the nucleic acid encoding the TALE.

[0097] In one aspect, the Mps1 promoter is operably connected to a nucleic acid encoding a guide nuclease. In one aspect, the Mps1 promoter is operably connected to a nucleic acid encoding a Cas9 nuclease. In one aspect, the Mps1 promoter is operably connected to a nucleic acid encoding a Cas12a nuclease. In one aspect, the Mps1 promoter is operably connected to a nucleic acid encoding a CasX nuclease. In one aspect, the Mps1 promoter is operably connected to a nucleic acid encoding a guide nucleic acid. In one aspect, the Mps1 promoter is operably connected to a nucleic acid encoding a guide RNA. In one aspect, the Mps1 promoter is operably connected to a nucleic acid encoding a single guide RNA. In some embodiments, the flank of the guide RNA acid is a self-cleaving ribozyme. In one aspect, the Mps1 promoter is operably connected to a nucleic acid encoding a recombinase (e.g., Cre recombinase). In one aspect, the Mps1 promoter is operably connected to a nucleic acid encoding a TALE.

[0098] In one aspect, the Adf1 promoter is operably linked to a nucleic acid encoding a guide nuclease. In one aspect, the Adf1 promoter is operably linked to a nucleic acid encoding a Cas9 nuclease. In one aspect, the Adf1 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In one aspect, the Adf1 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In one aspect, the Adf1 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In one aspect, the Adf1 promoter is operably linked to a nucleic acid encoding a guide RNA. In one aspect, the Adf1 promoter is operably linked to a nucleic acid encoding a single guide RNA. In some embodiments, the flank of the guide RNA acid is a self-cleaving ribozyme. In one aspect, the Adf1 promoter is operably linked to a nucleic acid encoding a recombinase (e.g., Cre recombinase). In one aspect, the Adf1 promoter is operably linked to a nucleic acid encoding a TALE.

[0099] In one aspect, the DMC1 promoter comprises a nucleic acid sequence having at least 80% identity with SEQ ID NO:4 or a functional fragment thereof. In one aspect, the DMC1 promoter comprises a nucleic acid sequence having at least 85% identity with SEQ ID NO:4 or a functional fragment thereof. In one aspect, the DMC1 promoter comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO:4 or a functional fragment thereof. In one aspect, the DMC1 promoter comprises a nucleic acid sequence having at least 95% identity with SEQ ID NO:4 or a functional fragment thereof. In one aspect, the DMC1 promoter comprises a nucleic acid sequence having at least 96% identity with SEQ ID NO:4 or a functional fragment thereof. In one aspect, the DMC1 promoter comprises a nucleic acid sequence having at least 97% identity with SEQ ID NO:4 or a functional fragment thereof. In one aspect, the DMC1 promoter comprises a nucleic acid sequence having at least 98% identity with SEQ ID NO:4 or a functional fragment thereof. In one aspect, the DMC1 promoter comprises a nucleic acid sequence having at least 99% identity with SEQ ID NO:4 or a functional fragment thereof. In one aspect, the DMC1 promoter comprises a nucleic acid sequence that is 100% identical to SEQ ID NO: 4 or a functional fragment thereof.

[0100] In one aspect, the Mps1 promoter comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO:5 or a functional fragment thereof. In one aspect, the Mps1 promoter comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO:5 or a functional fragment thereof. In one aspect, the Mps1 promoter comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO:5 or a functional fragment thereof. In one aspect, the Mps1 promoter comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO:5 or a functional fragment thereof. In one aspect, the Mps1 promoter comprises a nucleic acid sequence that is at least 96% identical to SEQ ID NO:5 or a functional fragment thereof. In one aspect, the Mps1 promoter comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO:5 or a functional fragment thereof. In one aspect, the Mps1 promoter comprises a nucleic acid sequence that is at least 98% identical to SEQ ID NO:5 or a functional fragment thereof. In one aspect, the Mps1 promoter comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO:5 or a functional fragment thereof. In one aspect, the Mps1 promoter comprises a nucleic acid sequence that is 100% identical to SEQ ID NO: 5 or a functional fragment thereof.

[0101] In one aspect, the Adf1 promoter comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO:6 or a functional fragment thereof. In one aspect, the Adf1 promoter comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO:6 or a functional fragment thereof. In one aspect, the Adf1 promoter comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO:6 or a functional fragment thereof. In one aspect, the Adf1 promoter comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO:6 or a functional fragment thereof. In one aspect, the Adf1 promoter comprises a nucleic acid sequence that is at least 96% identical to SEQ ID NO:6 or a functional fragment thereof. In one aspect, the Adf1 promoter comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO:6 or a functional fragment thereof. In one aspect, the Adf1 promoter comprises a nucleic acid sequence that is at least 98% identical to SEQ ID NO:6 or a functional fragment thereof. In one aspect, the Adf1 promoter comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO:6 or a functional fragment thereof. In one aspect, the Adf1 promoter comprises a nucleic acid sequence that is 100% identical to SEQ ID NO: 6 or a functional fragment thereof.

[0102] In one aspect, the meiocyte-preferred promoter comprises a nucleic acid sequence having at least 80% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6, 83-85, or functional fragments thereof. In one aspect, the meiocyte-preferred promoter comprises a nucleic acid sequence having at least 85% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6, 83-85, or functional fragments thereof. In one aspect, the meiocyte-preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6, 83-85, or functional fragments thereof. In one aspect, the meiocyte-preferred promoter comprises a nucleic acid sequence having at least 95% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6, 83-85, or functional fragments thereof. In one aspect, the meiocyte-preferred promoter comprises a nucleic acid sequence having at least 96% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6, 83-85, or functional fragments thereof. In one aspect, the meiocyte-preferred promoter comprises a nucleic acid sequence having at least 97% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6, 83-85, or a functional fragment thereof. In one aspect, the meiocyte-preferred promoter comprises a nucleic acid sequence having at least 98% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6, 83-85, or a functional fragment thereof. In one aspect, the meiocyte-preferred promoter comprises a nucleic acid sequence having at least 99% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6, 83-85, or a functional fragment thereof. In one aspect, the meiocyte-preferred promoter comprises a nucleic acid sequence having 100% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6, 83-85, or a functional fragment thereof.

[0103] As used herein, an "egg cell preferred promoter" refers to a promoter that exhibits higher or more preferential expression in egg cells compared to other cell or tissue types of a plant. An egg cell preferred promoter may show expression in nearby cells, such as, but not limited to, synergids, antipodal cells, central cells, integument cells, stigma cells, and style cells. An egg cell preferred promoter may also be expressed in other ovarian cells (e.g., ovules). An egg cell preferred promoter may also show expression in other plant tissues, such as, but not limited to, pollen cells, root cells, embryonic cells, stem cells, meristem cells, flower cells, and leaf cells, as long as the egg cell preferred promoter shows higher or more preferential expression in egg cells.

[0104] As used herein, "egg cell-specific promoter" refers to a promoter that is expressed only in egg cells. In one aspect, the egg cell-preferred promoter includes an egg cell-specific promoter.

[0105] As used herein, an "ovule tissue-preferred promoter" refers to a promoter that exhibits higher or more preferential expression in at least one or all ovule tissues compared to other cell or tissue types of a plant. In seed plants, the ovule is a structure that produces and contains the reproductive cells of the female plant. As used herein, the ovule is initially composed of unreduced tissue, the haploid tissue that produces the female gametophyte. The female gametophyte further develops into a "mature egg capsule" composed of four unique cell types: an egg cell, a central cell, two synergids, and three or more antipodal cells. As used herein, an ovule-preferred promoter can be expressed in an ovule before or after pollination. An ovule-preferred promoter can also show expression in other ovarian cells.

[0106] As used herein, "ovule tissue-specific promoter" refers to a promoter that is expressed only in ovules. In one aspect, the ovule tissue-preferred promoter includes an ovule tissue-specific promoter.

[0107] As used herein, "embryonic tissue-preferred promoter" refers to a promoter that exhibits higher or more preferential expression in embryonic tissue compared to other cell or tissue types of a plant. Embryonic tissue-preferred promoters can express in nearby cells, such as but not limited to endosperm cells, cotyledon cells, and seed coat cells. Embryonic tissue-preferred promoters can also express in other plant tissues, such as but not limited to pollen cells, root cells, egg cells, stem cells, meristem cells, flower cells, and leaf cells, as long as the embryonic tissue-preferred promoter exhibits higher or more preferential expression in embryonic tissue.

[0108] As used herein, "embryonic tissue-specific promoter" refers to a promoter that is expressed only in embryonic tissue. In one aspect, the embryonic tissue-preferred promoter includes an embryonic tissue-specific promoter.

[0109] As used herein, "zygotic cell-preferred promoter" refers to a promoter that exhibits higher or more preferential expression in the zygote compared to other cell or tissue types of the plant. After the haploid pollen cell is fertilized with the egg cell, a diploid zygote is formed, thereby producing an embryo. The zygotic cell-preferred promoter can also express in other plant cells, such as but not limited to pollen cells, egg cells, stem cells, meristem cells, endosperm cells, cotyledon cells, flower cells, leaf cells, and embryonic tissues, as long as the zygotic tissue-preferred promoter exhibits higher or more preferential expression in the zygote.

[0110] As used herein, "zygotic cell-specific promoter" refers to a promoter that is expressed only in the zygote. In one aspect, the zygotic cell-preferred promoter includes a zygotic cell-specific promoter.

[0111] It will be appreciated that the same promoter can be egg cell preferred, fertilized egg cell preferred, and embryonic tissue preferred, since the fertilized egg will develop into a zygote following pollination, thereby giving rise to embryonic tissue.

[0112] In one aspect, the embryonic tissue-preferred or embryonic tissue-specific promoter comprises the DSUL1 promoter. In one aspect, the egg cell-preferred or embryonic tissue-preferred promoter comprises the EA1 promoter. In one aspect, the egg cell-preferred or embryonic tissue-preferred promoter comprises the ES4 promoter. In one aspect, the egg cell-preferred or embryonic tissue-preferred promoter comprises the EAL1 promoter. In one aspect, the egg cell-preferred or embryonic tissue-preferred promoter comprises a promoter selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter.

[0113] In one aspect, the DSUL1 promoter is operably connected to the nucleic acid encoding the guide nuclease. In one aspect, the DSUL1 promoter is operably connected to the nucleic acid encoding the Cas9 nuclease. In one aspect, the DSUL1 promoter is operably connected to the nucleic acid encoding the Cas12a nuclease. In one aspect, the DSUL1 promoter is operably connected to the nucleic acid encoding the CasX nuclease. In one aspect, the DSUL1 promoter is operably connected to the nucleic acid encoding the guide nucleic acid. In one aspect, the DSUL1 promoter is operably connected to the nucleic acid encoding the guide RNA. In one aspect, the DSUL1 promoter is operably connected to the nucleic acid encoding a single guide RNA. In some embodiments, the flank of the guide RNA acid is a self-cleaving ribozyme. In one aspect, the DSUL1 promoter is operably connected to the nucleic acid encoding the recombinase (e.g., Cre recombinase). In one aspect, the DSUL1 promoter is operably connected to the nucleic acid encoding the TALE.

[0114] In one aspect, the EA1 promoter is operably connected to a nucleic acid encoding a guide nuclease. In one aspect, the EA1 promoter is operably connected to a nucleic acid encoding a Cas9 nuclease. In one aspect, the EA1 promoter is operably connected to a nucleic acid encoding a Cas12a nuclease. In one aspect, the EA1 promoter is operably connected to a nucleic acid encoding a CasX nuclease. In one aspect, the EA1 promoter is operably connected to a nucleic acid encoding a guide nucleic acid. In one aspect, the EA1 promoter is operably connected to a nucleic acid encoding a guide RNA. In one aspect, the EA1 promoter is operably connected to a nucleic acid encoding a single guide RNA. In some embodiments, the flank of the guide RNA acid is a self-cleaving ribozyme. In one aspect, the EA1 promoter is operably connected to a nucleic acid encoding a recombinase (e.g., Cre recombinase). In one aspect, the EA1 promoter is operably connected to a nucleic acid encoding a TALE.

[0115] In one aspect, the ES4 promoter is operably connected to the nucleic acid encoding the guide nuclease. In one aspect, the ES4 promoter is operably connected to the nucleic acid encoding the Cas9 nuclease. In one aspect, the ES4 promoter is operably connected to the nucleic acid encoding the Cas12a nuclease. In one aspect, the ES4 promoter is operably connected to the nucleic acid encoding the CasX nuclease. In one aspect, the ES4 promoter is operably connected to the nucleic acid encoding the guide nucleic acid. In one aspect, the ES4 promoter is operably connected to the nucleic acid encoding the guide RNA. In one aspect, the ES4 promoter is operably connected to the nucleic acid encoding a single guide RNA. In some embodiments, the flank of the guide RNA acid is a self-cleaving ribozyme. In one aspect, the ES4 promoter is operably connected to the nucleic acid encoding the recombinase (e.g., Cre recombinase). In one aspect, the ES4 promoter is operably connected to the nucleic acid encoding the TALE.

[0116] In one aspect, the EAL1 promoter is operably connected to a nucleic acid encoding a guide nuclease. In one aspect, the EAL1 promoter is operably connected to a nucleic acid encoding a Cas9 nuclease. In one aspect, the EAL1 promoter is operably connected to a nucleic acid encoding a Cas12a nuclease. In one aspect, the EAL1 promoter is operably connected to a nucleic acid encoding a CasX nuclease. In one aspect, the EAL1 promoter is operably connected to a nucleic acid encoding a guide nucleic acid. In one aspect, the EAL1 promoter is operably connected to a nucleic acid encoding a guide RNA. In one aspect, the EAL1 promoter is operably connected to a nucleic acid encoding a single guide RNA. In some embodiments, the flank of the guide RNA acid is a self-cleaving ribozyme. In one aspect, the EAL1 promoter is operably connected to a nucleic acid encoding a recombinase (e.g., Cre recombinase). In one aspect, the EAL1 promoter is operably connected to a nucleic acid encoding a TALE.

[0117] In one aspect, the DSUL1 promoter comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 1 or a functional fragment thereof. In one aspect, the DSUL1 promoter comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 1 or a functional fragment thereof. In one aspect, the DSUL1 promoter comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 1 or a functional fragment thereof. In one aspect, the DSUL1 promoter comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 1 or a functional fragment thereof. In one aspect, the DSUL1 promoter comprises a nucleic acid sequence that is at least 96% identical to SEQ ID NO: 1 or a functional fragment thereof. In one aspect, the DSUL1 promoter comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 1 or a functional fragment thereof. In one aspect, the DSUL1 promoter comprises a nucleic acid sequence that is at least 98% identical to SEQ ID NO: 1 or a functional fragment thereof. In one aspect, the DSUL1 promoter comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 1 or a functional fragment thereof. In one aspect, the DSUL1 promoter comprises a nucleic acid sequence that is 100% identical to SEQ ID NO: 1 or a functional fragment thereof.

[0118] In one aspect, the EA1 promoter comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO:2 or a functional fragment thereof. In one aspect, the EA1 promoter comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO:2 or a functional fragment thereof. In one aspect, the EA1 promoter comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO:2 or a functional fragment thereof. In one aspect, the EA1 promoter comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO:2 or a functional fragment thereof. In one aspect, the EA1 promoter comprises a nucleic acid sequence that is at least 96% identical to SEQ ID NO:2 or a functional fragment thereof. In one aspect, the EA1 promoter comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO:2 or a functional fragment thereof. In one aspect, the EA1 promoter comprises a nucleic acid sequence that is at least 98% identical to SEQ ID NO:2 or a functional fragment thereof. In one aspect, the EA1 promoter comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO:2 or a functional fragment thereof. In one aspect, the EA1 promoter comprises a nucleic acid sequence that is 100% identical to SEQ ID NO: 2 or a functional fragment thereof.

[0119] In one aspect, the ES4 promoter comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:3 or a functional fragment thereof. In one aspect, the ES4 promoter comprises a nucleic acid sequence having at least 85% identity to SEQ ID NO:3 or a functional fragment thereof. In one aspect, the ES4 promoter comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:3 or a functional fragment thereof. In one aspect, the ES4 promoter comprises a nucleic acid sequence having at least 95% identity to SEQ ID NO:3 or a functional fragment thereof. In one aspect, the ES4 promoter comprises a nucleic acid sequence having at least 96% identity to SEQ ID NO:3 or a functional fragment thereof. In one aspect, the ES4 promoter comprises a nucleic acid sequence having at least 97% identity to SEQ ID NO:3 or a functional fragment thereof. In one aspect, the ES4 promoter comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:3 or a functional fragment thereof. In one aspect, the ES4 promoter comprises a nucleic acid sequence having at least 99% identity to SEQ ID NO:3 or a functional fragment thereof. In one aspect, the ES4 promoter comprises a nucleic acid sequence that is 100% identical to SEQ ID NO: 3 or a functional fragment thereof.

[0120] In one aspect, the egg cell-preferred promoter, ovule tissue-preferred promoter, zygotic cell-preferred promoter, or embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 80% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-3, 21-45, 65-69, 75-82, 86-88, or a functional fragment thereof. In one aspect, the egg cell-preferred promoter or embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 85% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-3, 21-45, 65-69, 75-82, 86-88, or a functional fragment thereof. In one aspect, the egg cell-preferred promoter or embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 90% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-3, 21-45, 65-69, 75-82, 86-88, or a functional fragment thereof. In one aspect, the egg cell-preferred promoter or embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 95% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-3, 21-45, 65-69, 75-82, 86-88 or a functional fragment thereof. In one aspect, the egg cell-preferred promoter or embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 96% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-3, 21-45, 65-69, 75-82, 86-88 or a functional fragment thereof. In one aspect, the egg cell-preferred promoter or embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 97% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-3, 21-45, 65-69, 75-82, 86-88 or a functional fragment thereof. In one aspect, the egg cell-preferred promoter or embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 98% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 21-45, 65-69, 75-82, 86-88, or a functional fragment thereof. In one aspect, the egg cell-preferred promoter or embryonic tissue-preferred promoter comprises a nucleic acid sequence having at least 99% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 21-45, 65-69, 75-82, 86-88, or a functional fragment thereof. In one aspect, the egg cell-preferred promoter or embryonic tissue-preferred promoter comprises a nucleic acid sequence having 100% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 21-45, 65-69, 75-82, 86-88, or a functional fragment thereof.

[0121] It will be appreciated in the art that fragments of promoter sequences can be used to drive transcription of operably linked nucleic acid molecules. For example, but not limited to, if a 1000 bp promoter is truncated to 500 bp, and the 500 bp fragment is able to drive transcription, then the 500 bp fragment is referred to as a "functional fragment".

[0122] In one aspect, the egg cell preference promoter is operably connected to a nucleic acid sequence encoding a DNA modification enzyme (such as a guide nuclease). In one aspect, the egg cell preference promoter is operably connected to a nucleic acid sequence encoding a Cas9 nuclease. In one aspect, the egg cell preference promoter is operably connected to a nucleic acid sequence encoding a Cas12a nuclease. In one aspect, the egg cell preference promoter is operably connected to a nucleic acid sequence encoding a CasX nuclease. In one aspect, the egg cell preference promoter is operably connected to a nucleic acid sequence encoding a guide nucleic acid. In one aspect, the egg cell preference promoter is operably connected to a nucleic acid sequence encoding a guide RNA. In one aspect, the egg cell preference promoter is operably connected to a nucleic acid sequence encoding a single guide RNA. a nucleic acid sequence encoding asingle-guide RNA. In some embodiments, the flank of the guide RNA acid is a self-cleaving ribozyme. In one aspect, the egg cell preference promoter is operably connected to a nucleic acid sequence encoding a recombinase (e.g., Cre recombinase). In one aspect, the egg cell preference promoter is operably connected to a nucleic acid sequence encoding a TALE.

[0123] In one aspect, the egg cell specific promoter is operably connected to the nucleic acid sequence encoding DNA modification enzyme (such as guide nuclease). In one aspect, the egg cell specific promoter is operably connected to the nucleic acid sequence encoding Cas9 nuclease. In one aspect, the egg cell specific promoter is operably connected to the nucleic acid sequence encoding Cas12a nuclease. In one aspect, the egg cell specific promoter is operably connected to the nucleic acid sequence encoding CasX nuclease. In one aspect, the egg cell specific promoter is operably connected to the nucleic acid sequence encoding guide nucleic acid. In one aspect, the egg cell specific promoter is operably connected to the nucleic acid sequence encoding guide RNA. In one aspect, the egg cell specific promoter is operably connected to the nucleic acid sequence encoding single guide RNA. In some embodiments, the flank of guide RNA acid is self-cleaving ribozyme. In one aspect, the egg cell specific promoter is operably connected to the nucleic acid sequence encoding recombinase (for example, Cre recombinase). In one aspect, the egg cell specific promoter is operably connected to the nucleic acid sequence encoding TALE.

[0124] In one aspect, the embryonic tissue preference promoter is operably connected to the nucleic acid sequence encoding DNA modification enzymes (such as guide nucleases). In one aspect, the embryonic tissue preference promoter is operably connected to the nucleic acid sequence encoding Cas9 nuclease. In one aspect, the embryonic tissue preference promoter is operably connected to the nucleic acid sequence encoding Cas12a nuclease. In one aspect, the embryonic tissue preference promoter is operably connected to the nucleic acid sequence encoding CasX nuclease. In one aspect, the embryonic tissue preference promoter is operably connected to the nucleic acid sequence encoding guide nucleic acid. In one aspect, the embryonic tissue preference promoter is operably connected to the nucleic acid sequence encoding guide RNA. In one aspect, the embryonic tissue preference promoter is operably connected to the nucleic acid sequence encoding a single guide RNA. In some embodiments, the flank of the guide RNA acid is a self-cleaving ribozyme. In one aspect, the embryonic tissue preference promoter is operably connected to the nucleic acid sequence encoding recombinase (e.g., Cre recombinase). In one aspect, the embryonic tissue preference promoter is operably connected to the nucleic acid sequence encoding TALE.

[0125] In one aspect, the embryonic tissue-specific promoter is operably connected to the nucleic acid sequence encoding DNA modification enzymes (such as guide nucleases). In one aspect, the embryonic tissue-specific promoter is operably connected to the nucleic acid sequence encoding Cas9 nuclease. In one aspect, the embryonic tissue-specific promoter is operably connected to the nucleic acid sequence encoding Cas12a nuclease. In one aspect, the embryonic tissue-specific promoter is operably connected to the nucleic acid sequence encoding CasX nuclease. In one aspect, the embryonic tissue-specific promoter is operably connected to the nucleic acid sequence encoding guide nucleic acid. In one aspect, the embryonic tissue-specific promoter is operably connected to the nucleic acid sequence encoding guide RNA. In one aspect, the embryonic tissue-specific promoter is operably connected to the nucleic acid sequence encoding single guide RNA. In some embodiments, the flank of guide RNA acid is self-cleaving ribozyme. In one aspect, the embryonic tissue-specific promoter is operably connected to the nucleic acid sequence encoding recombinase (for example, Cre recombinase). In one aspect, the embryonic tissue-specific promoter is operably connected to the nucleic acid sequence encoding TALE.

[0126] Several embodiments described herein relate to methods and compositions for providing preferential or specific expression of a DNA modification enzyme, such as a guide nuclease (e.g., a CRISPR / Cas system) in eggs, embryos, and / or meiotic plant tissues from a constitutive promoter. In some embodiments, a transcribable polynucleotide encoding a DNA modification enzyme, such as a guide nuclease (e.g., a CRISPR / Cas system) is operably linked to a constitutive promoter by excising an intervening polynucleotide sequence preferentially or specifically in eggs, embryos, and / or meiotic plant tissues. In some embodiments, the intervening sequence is excised by a recombinase that is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In some embodiments, the intervening sequence is excised by Cre-mediated excision of an intervening Cre expression cassette, wherein Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues.

[0127] Promoters that drive expression in all or most tissues of a plant are referred to as "constitutive" promoters. Promoters that drive expression during certain developmental periods or stages are referred to as "developmental" promoters. "Inducible" promoters are promoters that initiate transcription in response to environmental stimuli (such as heat, cold, drought, light or other stimuli, such as injury or chemical application). Promoters can also be classified according to their source, such as heterologous, homologous, chimeric, synthetic, etc.

[0128] As used herein, the term "heterologous" refers to a promoter having a promoter sequence of a different origin and / or not naturally present in the plant species to be transformed relative to its associated transcribable DNA sequence, coding sequence or gene (or transgene). The term "heterologous" may more broadly refer to a combination of two or more DNA molecules or sequences, such as a promoter and an associated transcribable DNA sequence, coding sequence or gene, when such a combination is artificial and does not normally occur in nature.

[0129] In one aspect, the promoter provided herein is a constitutive promoter. In another aspect, the promoter provided herein is an inducible promoter. In one aspect, the promoter provided herein is selected from the following: a constitutive promoter, a tissue-specific promoter, a tissue-preferred promoter, and an inducible promoter.

[0130] RNA polymerase III (Pol III) promoter can be used to drive the expression of non-protein coding RNA molecules, such as guide RNA. In one aspect, the promoter provided herein is a Pol III promoter. In another aspect, the Pol III promoter provided herein is operably connected to a nucleic acid molecule encoding a non-protein coding RNA. In yet another aspect, the Pol III promoter provided herein is operably connected to a nucleic acid molecule encoding a guide nucleic acid. In yet another aspect, the Pol III promoter provided herein is operably connected to a nucleic acid molecule encoding a single guide RNA. In one other aspect, the Pol III promoter provided herein is operably connected to a nucleic acid molecule encoding CRISPR RNA (crRNA). In another aspect, the Pol III promoter provided herein is operably connected to a nucleic acid molecule encoding tracrRNA (tracrRNA). In some embodiments, a nucleic acid molecule encoding a non-protein coding RNA (e.g., gRNA, a single guide RNA, crRNA, tracrRNA, etc.) is operably connected to a Pol III promoter by preferentially or particularly excising an intervening polynucleotide sequence in an egg, embryo, and / or meiotic plant tissue. In some embodiments, the intervening sequence is excised by a recombinase that is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In some embodiments, the intervening sequence is excised by Cre-mediated excision of an intervening Cre expression cassette, wherein Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues.

[0131] Non-limiting examples of Pol III promoters include U6 promoter, H1 promoter, 5S promoter, adenovirus 2 (Ad2) VAI promoter, tRNA promoter and 7SK promoter. See, e.g., Schramm and Hernandez, 2002, Genes & Development, 16: 2593-2620, the entire contents of which are incorporated herein by reference. In one aspect, the Pol III promoter provided herein is selected from the following U6 promoter, H1 promoter, 5S promoter, adenovirus 2 (Ad2) VAI promoter, tRNA promoter and 7SK promoter. In another aspect, the guide RNA provided herein is operably linked to a promoter selected from the following: U6 promoter, H1 promoter, 5S promoter, adenovirus 2 (Ad2) VAI promoter, tRNA promoter and 7SK promoter. In another aspect, the single guide RNA provided herein is operably linked to a promoter selected from the group consisting of U6 promoter, H1 promoter, 5S promoter, adenovirus 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another aspect, the CRISPR RNA provided herein is operably linked to a promoter selected from the group consisting of U6 promoter, H1 promoter, 5S promoter, adenovirus 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another aspect, the tracer RNA provided herein is operably linked to a promoter selected from the group consisting of U6 promoter, H1 promoter, 5S promoter, adenovirus 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter.

[0132] In one aspect, the promoter provided herein is the dahlia mosaic virus (DaMV) promoter. In another aspect, the promoter provided herein is the α U6 promoter. In another aspect, the promoter provided herein is the actin promoter. In one aspect, the promoter provided herein is the cauliflower mosaic virus (CaMV) 35S promoter. In one aspect, the promoter provided herein is the ubiquitin promoter.

[0133] In one aspect, the constitutive promoter is selected from the group consisting of a CaMV 35S promoter, an actin promoter, and an ubiquitin promoter.

[0134] Examples of promoters described for use herein include, but are not limited to, U.S. Pat. No. 6,437,217 (maize RS81 promoter), U.S. Pat. No. 5,641,876 (rice actin promoter), U.S. Pat. No. 6,426,446 (maize RS324 promoter), U.S. Pat. No. 6,429,362 (maize PR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter), U.S. Pat. No. 6,177,611 (constitutive maize promoter), U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and 5,530,196 (35S promoter), U.S. Pat. Nos. 6,433,2 52 (maize L3 oleoprotein promoter), U.S. Patent No. 6,429,357 (rice actin 2 promoter and rice actin 2 intron), U.S. Patent No. 5,837,848 (root-specific promoter), U.S. Patent No. 6,294,714 (light-inducible promoter), U.S. Patent No. 6,140,078 (salt-inducible promoter), U.S. Patent No. 6,252,138 (pathogen-inducible promoter), U.S. Patent No. 6,175,060 (phosphate deficiency-inducible promoter), U.S. Patent No. 6,635,806 (γ-coixin promoter) and U.S. Patent Application Serial No. 09 / 757,089 (maize chloroplast aldolase promoter). Other promoters that can be used are the nopaline synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (which is carried on a tumor-inducing plasmid in Agrobacterium tumefaciens), cauliflower mosaic virus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Molecular Biology (1987) 9:315-324), the CaMV 35S promoter (Odell et al., Nature (1985) 313:810-812), the fig mosaic virus 35S promoter (U.S. Pat. Nos. 6,051,753; 5,378,619), the sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA (1990) 87:4144-4148), the R gene complex promoter (Chandler et al., Plant Molecular Biology (1991) 88:4157-4159), the sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA (1992) 86:4161-4163), the sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA (1993) 85:4162-4164), the sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA (1994) 86:4163-4165), the sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA (1994) 86:4161-4163), the sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA (1994 Cell (1989) 1: 1175-1183) and the chlorophyll a / b binding protein gene promoter PC1SV (U.S. Pat. No. 5,850,019) and AGRtu.nos (GenBank Accession No. V00087; Depicker et al., Journal of Molecular and Applied Genetics (1982) 1: 561-573; Bevan et al., 1983) promoter.

[0135] Promoter hybrids can also be used and constructed to enhance transcriptional activity (see U.S. Patent number 5,106,739), or in combination with desired transcriptional activity, inducibility and tissue specificity or developmental specificity. Promoters that work in plants include but are not limited to inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated and spatiotemporally regulated promoters. Other promoters of tissue enhancement, tissue specificity or developmental regulation are also well known in the art and are envisioned to have practicality in the practice of the present disclosure.

[0136] In one aspect, the intervening Cre expression cassette is excised by Cre mediation, and a constitutive promoter is operably connected to a nucleic acid sequence encoding a guide nuclease, wherein Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In one aspect, the intervening Cre expression cassette is excised by Cre mediation, and a constitutive promoter is operably connected to a nucleic acid sequence encoding a Cas9 nuclease, wherein Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In one aspect, the intervening Cre expression cassette is excised by Cre mediation, and a constitutive promoter is operably connected to a nucleic acid sequence encoding a Cas12a nuclease, wherein Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In one aspect, the intervening Cre expression cassette is excised by Cre mediation, and a constitutive promoter is operably connected to a nucleic acid sequence encoding a CasX nuclease, wherein Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In one aspect, the intervening Cre expression cassette is excised by Cre mediation, and a constitutive promoter is operably linked to a nucleic acid sequence encoding a guide nucleic acid, wherein Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In one aspect, the intervening Cre expression cassette is excised by Cre mediation, and a constitutive promoter is operably linked to a nucleic acid sequence encoding a guide RNA, wherein Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In one aspect, the intervening Cre expression cassette is excised by Cre mediation, and a constitutive promoter is operably linked to a nucleic acid sequence encoding a single guide RNA, wherein Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues.

[0137] In one aspect, an inducible promoter is operably connected to a nucleic acid sequence encoding a guide nuclease. In one aspect, an inducible promoter is operably connected to a nucleic acid sequence encoding a Cas9 nuclease. In one aspect, an inducible promoter is operably connected to a nucleic acid sequence encoding a Cas12a nuclease. In one aspect, an inducible promoter is operably connected to a nucleic acid sequence encoding a CasX nuclease. In one aspect, an inducible promoter is operably connected to a nucleic acid sequence encoding a guide nuclease. In one aspect, an inducible promoter is operably connected to a nucleic acid sequence encoding a guide RNA. In one aspect, an inducible promoter is operably connected to a nucleic acid sequence encoding a single guide RNA. In some embodiments, the flank of the guide RNA acid is a self-cleaving ribozyme. In one aspect, an inducible promoter is operably connected to a nucleic acid sequence encoding a recombinase (e.g., Cre recombinase). In one aspect, an inducible promoter is operably connected to a nucleic acid sequence encoding a TALE.

[0138] In one aspect, the developmental promoter is operably connected to the nucleic acid sequence encoding the guide nuclease. In one aspect, the developmental promoter is operably connected to the nucleic acid sequence encoding the Cas9 nuclease. In one aspect, the developmental promoter is operably connected to the nucleic acid sequence encoding the Cas12a nuclease. In one aspect, the developmental promoter is operably connected to the nucleic acid sequence encoding the CasX nuclease. In one aspect, the developmental promoter is operably connected to the nucleic acid sequence encoding the guide nucleic acid. In one aspect, the developmental promoter is operably connected to the nucleic acid sequence encoding the guide RNA. In one aspect, the developmental promoter is operably connected to the nucleic acid sequence encoding a single guide RNA. In some embodiments, the flank of the guide RNA acid is a self-cleaving ribozyme. In one aspect, the developmental promoter is operably connected to the nucleic acid sequence encoding a recombinase (e.g., Cre recombinase). In one aspect, the developmental promoter is operably connected to the nucleic acid sequence encoding TALE.

[0139] As used herein, the term "leader sequence" refers to a nucleotide fragment between the transcription start site (TSS) of a gene and the start site of a protein coding sequence. It is separated from the untranslated 5' region of the genomic copy of the gene. The leader sequence can be used as a 5' regulatory element to regulate the expression of an operably linked transcribable polynucleotide molecule. The leader sequence molecule can be used with a heterologous promoter or with its native promoter.

[0140] As used herein, the term "3' transcription terminator" or "3'UTR" refers to a DNA sequence that is used to produce the 3' untranslated region (3'UTT) of an mRNA molecule during transcription. The 3' untranslated region of an mRNA molecule can be produced by specific cleavage and 3' polyadenylation (i.e., polyA tail). The 3'UTR can be operably linked to a transcribable polynucleotide molecule and located downstream thereof, and can include a polynucleotide that provides a polyadenylation signal and other regulatory signals that can affect transcription, mRNA processing, or gene expression. It is believed that the PolyA tail plays a role in mRNA stabilization and translation initiation. Examples of 3' transcriptional terminal molecules in the art are nopaline synthase 3' region (see, Fraley, et al., Proc. Natl. Acad. Sci. USA, 80: 4803-4807 (1983)); wheat hsp17 3' region; pea ribulose small subunit 3' region; cotton E6 3' region (U.S. Pat. No. / 6,096,950); the 3' region disclosed in WO0011200A2; and coixin 3'UTR (U.S. Pat. No. 6,635,806). 3'UTRs are generally beneficial for recombinant expression of specific genes. 3'UTRs can be used as 3' regulatory elements to regulate the expression of operably linked transcribable polynucleotide molecules. 3'UTRs can be used as 3' regulatory elements to regulate the tissue / cell-preferred expression of operably linked transcribable polynucleotide molecules. 3'UTRs can be used with heterologous promoters or their native promoters. Non-limiting examples of 3'UTRs for use in the practice of the various embodiments described herein include SEQ ID NOs 46-64, 70-74, 89-102.

[0141] DNA modifying enzymes

[0142] Several embodiments relate to compositions and methods for preferentially or specifically expressing one or more components of a genome editing system in eggs, embryos, and / or meiotic plant tissues. Several embodiments relate to gene regulatory elements as described in Table 1, which are operably linked to heterologous transcribable DNA molecules encoding one or more components of a genome editing system. The genome editing system can be used to introduce one or more insertions, deletions, substitutions, base modifications, translocations, or inversions into the genome of a host cell. In some embodiments, the gene regulatory elements as described in Table 1 are operably linked to heterologous transcribable DNA molecules encoding sequence-specific DNA modification enzymes, such as CRISPR-Cas effector proteins, zinc finger proteins, or transcriptional activation (TAL) proteins. In some embodiments, the sequence-specific DNA modification enzyme may be a fusion protein. In some embodiments, the sequence-specific DNA modification enzyme may be a guide nuclease.

[0143] A guide nuclease is a nuclease that forms a complex (e.g., a ribonucleoprotein) with a guide nucleic acid molecule (e.g., a guide RNA) and then guides the complex to a target site within a target sequence. A non-limiting example of a guide nuclease is a CRISPR nuclease.

[0144] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) nucleases (e.g., Cas9, CasX, Cas12a (also known as Cpf1), CasY) are proteins found in bacteria that are directed by a guide RNA ("gRNA") to a target nucleic acid molecule, where the endonuclease can then cleave one or both strands of the target nucleic acid molecule. Although the origin of CRISPR nucleases is bacterial, many CRISPR nucleic acids have been shown to function in eukaryotic cells.

[0145] Although not limited by any particular scientific theory, CRISPR nuclease forms a complex with a guide RNA (gRNA), which hybridizes with a complementary target site, thereby guiding the CRISPR nuclease to the target site. In the Class II CRISPR-Cas system, the CRISPR array (including the spacer sequence) is transcribed upon encountering recognized invading DNA and processed into small interfering CRISPR RNA (crRNA). The crRNA contains a repetitive sequence and a spacer sequence that is complementary to a specific original spacer sequence in the invading pathogen. The spacer sequence can be designed to be complementary to the target sequence in the eukaryotic genome.

[0146] In some embodiments, a gene regulatory element as described herein is operably linked to a heterologous transcribable DNA molecule encoding a CRISPR-Cas effector protein. In some embodiments, the CRISPR-Cas effector protein is selected from a type I CRISPR-Cas system, a type II CRISPR-Cas system, a type III CRISPR-Cas system, a type IV CRISPR-Cas system, a type V CRISPR-Cas system, or a type VI CRISPR-Cas system. Examples of CRISPR-Cas effector proteins include, but are not limited to, Cas9, C2c1, C2c3, C2c4, C2c5, C2c8, C2c9, C2c10, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', C as3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Cs x16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), Csf5, Cas14a, Cas14b and Cas14c effector proteins. In some embodiments, the gene regulatory elements as described herein are operably linked to CRISPR-Cas effector proteins, and the effector proteins contain mutations in their nuclease active sites (e.g., RuvC, HNH and / or NUC domains). The CRISPR-Cas effector protein mutates in its nuclease active site and therefore no longer has nuclease activity, and is generally referred to as "dead", such as dCas. In some embodiments, a CRISPR-Cas effector protein domain or polypeptide having a mutation in its nuclease active site may have impaired activity or reduced activity compared to the same CRISPR-Cas effector protein without mutation. In some embodiments, the gene regulatory elements described herein are operably linked to a CRISPR-Cas effector protein, and the effector protein has a mutation in its nuclease active site.

[0147] CRISPR effector proteins bind to their respective crRNAs in their active form. CasX, similar to the class II endonuclease Cas9, requires another noncoding RNA component, called trans-activating crRNA (tracrRNA), to be functionally active. The nucleic acid molecules provided herein can combine crRNA and tracrRNA into one nucleic acid molecule, referred to herein as a "single guide RNA" (sgRNA). Cas12a does not require tracrRNA to be guided to the target site; crRNA alone is sufficient for Cas12a. The gRNA guides the active CRISPR nuclease complex to the target site, where the CRISPR nuclease can cleave the target site.

[0148] When the CRISPR effector protein and the guide RNA form a complex, the entire system is referred to as a "ribonucleoprotein." The ribonucleoproteins provided herein also include other nucleic acids or proteins.

[0149] In one aspect, the CRISPR effector protein and the guide nucleic acid form ribonucleoproteins in the egg cell. In another aspect, the CRISPR effector protein and the guide nucleic acid form ribonucleoproteins in embryonic tissues. In one aspect, the Cas9 nuclease and the guide nucleic acid form ribonucleoproteins in the egg cell. In another aspect, the Cas9 nuclease and the guide nucleic acid form ribonucleoproteins in the embryonic tissue. In one aspect, the Cas12a nuclease and the guide nucleic acid form ribonucleoproteins in the egg cell. In another aspect, the Cas12a nuclease and the guide nucleic acid form ribonucleoproteins in the embryonic tissue. In one aspect, the CasX nuclease and the guide nucleic acid form ribonucleoproteins in the egg cell. In another aspect, the CasX nuclease and the guide nucleic acid form ribonucleoproteins in the embryonic tissue. In one aspect, the CRISPR effector protein and the guide RNA form ribonucleoproteins in the egg cell. In another aspect, the CRISPR effector protein and the guide RNA form ribonucleoproteins in the embryonic tissue. In one aspect, the Cas9 nuclease and the guide RNA form ribonucleoproteins in the egg cell. In another aspect, the Cas9 nuclease and the guide RNA form ribonucleoproteins in embryonic tissues. In one aspect, the Cas12a nuclease and the guide RNA form ribonucleoproteins in egg cells. In another aspect, the Cas12a nuclease and the guide RNA form ribonucleoproteins in embryonic tissues. In one aspect, the CasX nuclease and the guide RNA form ribonucleoproteins in egg cells. In another aspect, the CasX nuclease and the guide RNA form ribonucleoproteins in embryonic tissues. In one aspect, the guide nuclease and the single guide RNA form ribonucleoproteins in egg cells. In another aspect, the guide nuclease and the single guide RNA form ribonucleoproteins in embryonic tissues. In another aspect, the CasX nuclease and the single guide RNA form ribonucleoproteins in embryonic tissues. In another aspect, the Cas9 nuclease and the single guide RNA form ribonucleoproteins in embryonic tissues.

[0150] In one aspect, the ribonucleoprotein generates at least one double-strand break in a target site in an egg cell. In one aspect, the ribonucleoprotein generates at least one double-strand break in a target site in an embryonic tissue. In one aspect, the ribonucleoprotein generates at least one single-strand break in a target site in an egg cell. In one aspect, the ribonucleoprotein generates at least one single-strand break in a target site in an embryonic tissue.

[0151] A prerequisite for CRISPR ribonucleoprotein to cut the target site is the presence of a conserved protospacer adjacent motif (PAM) near the target site. Depending on the CRISPR nuclease, cutting can occur within a certain number of nucleotides at the PAM site (e.g., between 18-23 nucleotides for Cas12a). PAM sites are only used for type I and type II CRISPR-associated proteins, and different CRISPR endonucleases recognize different PAM sites. Without limitation, Cas12a is at least able to recognize the following PAM sites: TTTN and YTN; and CasX is at least able to recognize the following PAM sites: TTCN, TTCA, and TTC (wherein T is thymine; C is cytosine; A is adenine; Y is thymine or cytosine and N is thymine, cytosine, guanine, or adenine).

[0152] Cas12a is an RNA-guided nuclease of the Class II, Type V CRISPR / Cas system. Cas12a nuclease produces staggered cuts when cleaving double-stranded DNA molecules. Staggered cuts of double-stranded DNA produce single-stranded DNA overhangs of at least one nucleotide. This is in contrast to blunt-end cuts (such as those produced by Cas9), which do not produce single-stranded DNA overhangs when cleaving double-stranded DNA.

[0153] In one aspect, the Cas12a nuclease provided herein is a Lachnospiraceae bacterial Cas12a (LbCas12a) nuclease. In another aspect, the Cas12a nuclease provided herein is a Francisella Cas12a (FnCas12a) nuclease. In one aspect, the Cas12a nuclease is selected from the following: LbCas12a and FnCas12a.

[0154] In one aspect, the Cas12a nuclease or the nucleic acid encoding the Cas12a nuclease is derived from a genus selected from the group consisting of Streptococcus, Campylobacter, Nitrocellus, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Lepidococcus, Lactobacillus, Eubacterium, Corynebacterium, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Fusobacterium, Lachnospira, Clostridiaridium, Leptotrichia, Francisella , Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroides, Vulnificus, Leptospira, Desulfovibrio, Desulfovibrio, Toyosporium, Tuberculosis, Bacillus, Bacillus, Bacillus brevis, Methylobacterium, Acidaminococcus, Peregrinibacteria, Butyrivibrio, Parcubacteria, Smithella, Candidatus, Moraxella, and Leptospira.

[0155] In one aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence having at least 80% identity with a polynucleotide selected from the group consisting of: SEQ ID NO: 7. In another aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence having at least 85% identity with a polynucleotide selected from the group consisting of: SEQ ID NO: 7. In another aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence having at least 90% identity with a polynucleotide selected from the group consisting of: SEQ ID NO: 7. In another aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence having at least 95% identity with a polynucleotide selected from the group consisting of: SEQ ID NO: 7. In another aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence having at least 96% identity with a polynucleotide selected from the group consisting of: SEQ ID NO: 7. In another aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence having at least 97% identity with a polynucleotide selected from the group consisting of: SEQ ID NO: 7. In another aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence having at least 98% identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 7. In another aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence having at least 99% identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 7. In another aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence having 100% identity to a polynucleotide selected from the group consisting of: SEQ ID NO: 7.

[0156] CasX is a class II CRISPR-Cas nuclease that has been identified in the bacterial phyla Proteobacteria and Planctomyces. Similar to Cas12a, CasX nucleases produce staggered cuts when cleaving double-stranded DNA molecules. However, unlike Cas12a, CasX nucleases require crRNA and tracrRNA or a single guide RNA to target and cleave the target nucleic acid.

[0157] In one aspect, the CasX nuclease provided herein is a CasX nuclease from Proteobacteria. In another aspect, the CasX nuclease provided herein is a CasX nuclease from Planctomycetes. Without limitation, other suitable CasX nucleases are those shown in WO 2019 / 084148, the entire contents of which are incorporated herein by reference.

[0158] In one aspect, the guide nuclease capable of generating staggered cuts in a double-stranded DNA molecule is selected from the group consisting of Cas12a and CasX. In one aspect, the guide nuclease is selected from the group consisting of Cas12a and CasX.

[0159] In one aspect, the guide nuclease is an RNA guide nuclease. In another aspect, the guide nuclease is a CRISPR nuclease. In another aspect, the guide nuclease is a Cas12a nuclease. In another aspect, the guide nuclease is a CasX nuclease.

[0160] As used herein, "nuclear localization signal" (NLS) refers to an amino acid sequence that "tags" a protein to be imported into the nucleus. In one aspect, the nucleic acid molecules provided herein encode a nuclear localization signal. In another aspect, the nucleic acid molecules provided herein encode two or more nuclear localization signals.

[0161] In one aspect, the CRISPR effector protein provided herein comprises a nuclear localization signal. In one aspect, the Cas9 effector protein provided herein comprises a nuclear localization signal. In one aspect, the nuclear localization signal is located at the N-terminus of the Cas12a nuclease. In one other aspect, the nuclear localization signal is located at the C-terminus of the Cas9 effector protein. In yet another aspect, the nuclear localization signal is located at the N-terminus and C-terminus of the Cas9 effector protein.

[0162] In one aspect, the Cas12a effector protein provided herein comprises a nuclear localization signal. In one aspect, the nuclear localization signal is located at the N-terminus of the Cas12a effector protein. In one other aspect, the nuclear localization signal is located at the C-terminus of the Cas12a effector protein. In yet another aspect, the nuclear localization signal is located at the N-terminus and C-terminus of the Cas12a effector protein.

[0163] In one aspect, the CasX effector protein provided herein comprises a nuclear localization signal. In one aspect, the nuclear localization signal is located at the N-terminus of the CasX effector protein. In one other aspect, the nuclear localization signal is located at the C-terminus of the CasX effector protein. In yet another aspect, the nuclear localization signal is located at the N-terminus and C-terminus of the CasX effector protein.

[0164] In one aspect, the ribonucleoprotein comprises at least one nuclear localization signal. In another aspect, the ribonucleoprotein comprises at least two nuclear localization signals. In one aspect, the nuclear localization signal provided herein is encoded by SEQ ID NO: 8 or 9.

[0165] Various species show special biases for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is generally related to the translation efficiency of messenger RNA (mRNA), which in turn is believed to depend on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The advantage of selected tRNAs in cells generally reflects the most commonly used codons in peptide synthesis. Therefore, based on codon optimization, genes can be customized for optimal gene expression in a given organism. Codon usage tables are easily available, for example, in the "Codon Usage Database" available from www[dot]kazusa[dot]or[dot]jp[forwards slash]codon, and these tables can be adjusted in a variety of ways. See Nakamura et al., 2000, Nucl.Acids Res.28:292. Computer algorithms for codon optimization of specific sequences for expression in specific plant cells are also available, such as Gene Forge (Aptagen; Jacobus, PA).

[0166] As used herein, "codon optimization" refers to the process of modifying a nucleic acid sequence to enhance expression in a desired plant cell by replacing at least one codon (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the sequence with codons that are more frequently or most frequently used in the genes of the plant cell while maintaining the original amino acid sequence (e.g., introducing silent mutations).

[0167] In one aspect, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all codons) in the sequence encoding the guide nuclease correspond to the most frequently used codons for a particular amino acid. In another aspect, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all codons) in the sequence encoding the Cas9 effector protein, Cas12a effector protein, or CasX effector protein correspond to the most frequently used codons for a particular amino acid. For codon usage in plants, see Campbell and Gowri, 1990, Plant Physiol., 92: 1-11; and Murray et al., 1989, Nucleic Acids Res., 17: 477-98, the entire contents of each of which are incorporated herein by reference.

[0168] In one aspect, the nucleic acid molecule encodes a guide nuclease optimized for plant codons. In one aspect, the nucleic acid molecule encodes a Cas9 effector protein optimized for plant codons. In one aspect, the nucleic acid molecule encodes a Cas12a effector protein optimized for plant codons. In one aspect, the nucleic acid molecule encodes a CasX effector protein optimized for plant codons.

[0169] In another aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for plant cells. In another aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for monocot species. In another aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for dicot species. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for gymnosperm species. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for angiosperm species. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for corn cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for soybean cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for rice cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for wheat cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for cotton cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for sorghum cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for alfalfa cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for sugarcane cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for Arabidopsis cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for tomato cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for cucumber cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for potato cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for onion cells.

[0170] In another aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for plant cell codons. In another aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for monocot species codons. In another aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for dicot species codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for gymnosperm species codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for angiosperm species codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for corn cell codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for soybean cell codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for rice cell codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for wheat cell codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for cotton cell codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for sorghum cell codons. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases optimized for alfalfa cell codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for sugarcane cell codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for Arabidopsis cell codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for tomato cell codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for cucumber cell codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for potato cell codons. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins optimized for onion cell codons.

[0171] In another aspect, the nucleic acid molecules provided herein encode CasX effector proteins optimized for plant cell codons. In another aspect, the nucleic acid molecules provided herein encode CasX effector proteins optimized for monocot species codons. In another aspect, the nucleic acid molecules provided herein encode CasX effector proteins optimized for dicot species codons. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins optimized for gymnosperm species codons. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins optimized for angiosperm species codons. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins optimized for corn cell codons. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins optimized for soybean cell codons. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins optimized for rice cell codons. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins optimized for wheat cell codons. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins optimized for cotton cell codons. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins that are codon-optimized for sorghum cells. In one other aspect, the nucleic acid molecules provided herein encode guide nucleases that are codon-optimized for alfalfa cells. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins that are codon-optimized for sugarcane cells. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins that are codon-optimized for Arabidopsis cells. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins that are codon-optimized for tomato cells. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins that are codon-optimized for cucumber cells. In one other aspect, the nucleic acid molecules provided herein encode CasX effector proteins that are codon-optimized for potato cells. In one other aspect, the nucleic acid molecules provided herein encode Cas12a effector proteins that are codon-optimized for onion cells.

[0172] Guide nucleic acid

[0173] As used herein, "guide nucleic acid" refers to a nucleic acid that forms a ribonucleoprotein (e.g., a complex) with a CRISPR effector protein (e.g., but not limited to Cas9, Cas12a, CasX), and then guides the ribonucleoprotein to a specific sequence in a target nucleic acid molecule, wherein the guide nucleic acid and the target nucleic acid molecule share a complementary sequence. In one aspect, the ribonucleoprotein provided herein comprises at least one guide nucleic acid.

[0174] In one aspect, the guide nucleic acid comprises DNA. In another aspect, the guide nucleic acid comprises RNA. In one aspect, the guide nucleic acid comprises DNA, RNA, or a combination thereof. In one aspect, the guide nucleic acid is single-stranded. In another aspect, the guide nucleic acid is at least partially double-stranded.

[0175] When the guide nucleic acid comprises RNA, it can be referred to as a "guide RNA". In another aspect, the guide nucleic acid comprises DNA and RNA. In another aspect, the guide RNA is single-stranded. In another aspect, the guide RNA is double-stranded. In one other aspect, the guide RNA is partially double-stranded.

[0176] In one aspect, the guide nucleic acid comprises a guide RNA. In another aspect, the guide nucleic acid comprises at least one guide RNA. In another aspect, the guide nucleic acid comprises at least two guide RNAs. In another aspect, the guide nucleic acid comprises at least three guide RNAs. In another aspect, the guide nucleic acid comprises at least five guide RNAs. In another aspect, the guide nucleic acid comprises at least ten guide RNAs.

[0177] In another aspect, the guide nucleic acid comprises at least 10 nucleotides. In another aspect, the guide nucleic acid comprises at least 11 nucleotides. In another aspect, the guide nucleic acid comprises at least 12 nucleotides. In another aspect, the guide nucleic acid comprises at least 13 nucleotides. In another aspect, the guide nucleic acid comprises at least 14 nucleotides. In another aspect, the guide nucleic acid comprises at least 15 nucleotides. In another aspect, the guide nucleic acid comprises at least 16 nucleotides. In another aspect, the guide nucleic acid comprises at least 17 nucleotides. In another aspect, the guide nucleic acid comprises at least 18 nucleotides. In another aspect, the guide nucleic acid comprises at least 19 nucleotides. In another aspect, the guide nucleic acid comprises at least 20 nucleotides. In another aspect, the guide nucleic acid comprises at least 21 nucleotides. In another aspect, the guide nucleic acid comprises at least 22 nucleotides. In another aspect, the guide nucleic acid comprises at least 23 nucleotides. In another aspect, the guide nucleic acid comprises at least 24 nucleotides. In another aspect, the guide nucleic acid comprises at least 25 nucleotides. In another aspect, the guide nucleic acid comprises at least 26 nucleotides. In another aspect, the guide nucleic acid comprises at least 27 nucleotides. In another aspect, the guide nucleic acid comprises at least 28 nucleotides. In another aspect, the guide nucleic acid comprises at least 30 nucleotides. In another aspect, the guide nucleic acid comprises at least 35 nucleotides. In another aspect, the guide nucleic acid comprises at least 40 nucleotides. In another aspect, the guide nucleic acid comprises at least 45 nucleotides. In another aspect, the guide nucleic acid comprises at least 50 nucleotides.

[0178] In another aspect, the guide nucleic acid comprises between 10 nucleotides and 50 nucleotides. In another aspect, the guide nucleic acid comprises between 10 nucleotides and 40 nucleotides. In another aspect, the guide nucleic acid comprises between 10 nucleotides and 30 nucleotides. In another aspect, the guide nucleic acid comprises between 10 nucleotides and 20 nucleotides. In another aspect, the guide nucleic acid comprises between 16 nucleotides and 28 nucleotides. In another aspect, the guide nucleic acid comprises between 16 nucleotides and 25 nucleotides. In another aspect, the guide nucleic acid comprises between 16 nucleotides and 20 nucleotides.

[0179] In one aspect, the guide nucleic acid comprises at least 70% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 75% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 80% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 85% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 90% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 91% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 92% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 93% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 94% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 95% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 96% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 97% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 98% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises at least 99% sequence complementarity to the target site. In one aspect, the guide nucleic acid comprises 100% sequence complementarity to the target site. In another aspect, the guide nucleic acid comprises 70% to 100% sequence complementarity to the target site. In another aspect, the guide nucleic acid comprises 80% to 100% sequence complementarity to the target site. In another aspect, the guide nucleic acid comprises 90% to 100% sequence complementarity to the target site. In one aspect, the guide nucleic acid is capable of hybridizing with the target site.

[0180] As described above, some guide nucleases, such as CasX and Cas9, require another non-coding RNA component, called trans-activating crRNA (tracrRNA), to be functionally active. The guide nucleic acid molecules provided herein can combine crRNA and tracrRNA into one nucleic acid molecule, referred to herein as "single guide RNA" (sgRNA). The gRNA guides the active CasX complex to the target site in the target sequence, where CasX can cut the target site. In other embodiments, crRNA and tracrRNA are provided as separate nucleic acid molecules. In one aspect, the guide nucleic acid comprises crRNA. In another aspect, the guide nucleic acid comprises tracrRNA. In one other aspect, the guide nucleic acid comprises sgRNA.

[0181] Target site

[0182] As used herein, "target sequence" refers to a selected sequence or region in a DNA molecule that requires modification (eg, cleavage, deamination, site-directed integration). The target sequence comprises a target site.

[0183] As used herein, "target site" refers to the portion of a target sequence that is modified (e.g., cleaved) by a CRISPR effector protein. A target site includes significant complementarity with a guide nucleic acid or guide RNA compared to a non-target nucleic acid (e.g., non-target ssDNA) or non-target region.

[0184] In one aspect, the target site is 100% complementary to the guide nucleic acid. In another aspect, the target site is 99% complementary to the guide nucleic acid. In another aspect, the target site is 98% complementary to the guide nucleic acid. In another aspect, the target site is 97% complementary to the guide nucleic acid. In another aspect, the target site is 96% complementary to the guide nucleic acid. In another aspect, the target site is 95% complementary to the guide nucleic acid. In another aspect, the target site is 94% complementary to the guide nucleic acid. In another aspect, the target site is 93% complementary to the guide nucleic acid. In another aspect, the target site is 92% complementary to the guide nucleic acid. In another aspect, the target site is 91% complementary to the guide nucleic acid. In another aspect, the target site is 90% complementary to the guide nucleic acid. In another aspect, the target site is 85% complementary to the guide nucleic acid. In another aspect, the target site is 80% complementary to the guide nucleic acid.

[0185] In one aspect, the target site comprises at least one PAM site. In one aspect, the target site is adjacent to a nucleic acid sequence comprising at least one PAM site. In another aspect, the target site is within 5 nucleotides of at least one PAM site. In one other aspect, the target site is within 10 nucleotides of at least one PAM site. In another aspect, the target site is within 15 nucleotides of at least one PAM site. In another aspect, the target site is within 20 nucleotides of at least one PAM site. In another aspect, the target site is within 25 nucleotides of at least one PAM site. In another aspect, the target site is within 30 nucleotides of at least one PAM site.

[0186] In one aspect, the target site is located within the gene DNA. In another aspect, the target site is located within the gene. In another aspect, the target site is located within the target gene. In another aspect, the target site is located within the gene exon. In another aspect, the target site is located within the gene intron. In another aspect, the target site is located within the gene 5'-UTR. In another aspect, the target site is located within the gene 3'-UTR. In another aspect, the target site is located within the intergenic DNA.

[0187] In one aspect, the target DNA molecule is single-stranded. In another aspect, the target DNA molecule is double-stranded.

[0188] In one aspect, the target sequence comprises genomic DNA. In one aspect, the target sequence is located within the nuclear genome. In one aspect, the target sequence comprises chromosomal DNA. In one aspect, the target sequence comprises plasmid DNA. In one aspect, the target sequence is located within a plasmid. In one aspect, the target sequence comprises mitochondrial DNA. In one aspect, the target sequence is located within the mitochondrial genome. In one aspect, the target sequence comprises plastid DNA. In one aspect, the target sequence is located within the plastid genome. In one aspect, the target sequence comprises chloroplast DNA. In one aspect, the target sequence is located within the chloroplast genome. In one aspect, the target sequence is located within a genome selected from the group consisting of: a nuclear genome, a mitochondrial genome, and a plastid genome.

[0189] In one aspect, the target sequence comprises gene DNA. As used herein, "gene DNA" refers to DNA encoding one or more genes. In another aspect, the target sequence comprises intergenic DNA. Unlike gene DNA, "intergenic DNA" comprises non-coding DNA and lacks DNA encoding genes. In one aspect, intergenic DNA is located between two genes.

[0190] In one aspect, the target sequence encodes a gene. As used herein, "gene" refers to a polynucleotide capable of producing a functional unit (e.g., not limited to, for example, a protein or a non-coding RNA molecule). A gene can include a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, 5'-UTR, 3'-UTR, or any combination thereof." Gene sequence" can include a polynucleotide sequence encoding a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, 5'-UTR, 3'-UTR, or any combination thereof. In one aspect, a gene encodes a non-protein coding RNA molecule or a precursor thereof. In another aspect, a gene encodes a protein. In some embodiments, the target sequence is selected from the group consisting of a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, an exon, an intron, a 5'-UTR, a 3'-UTR, a protein coding sequence, a non-protein coding sequence, a miRNA, a pre-miRNA, and a miRNA binding site.

[0191] Non-limiting examples of non-protein coding RNA molecules include microRNA (miRNA), miRNA precursors (pre-miRNA), small interfering RNA (siRNA), small RNA (18 to 26 nucleotides in length) and identical coding precursors, heterochromatin siRNA (hc-siRNA), Piwi interacting RNA (piRNA), hairpin double-stranded RNA (hairpin dsRNA), trans-activating siRNA (ta-siRNA), naturally occurring antisense siRNA (nat-siRNA), CRISPR RNA (crRNA), tracer RNA (tracrRNA), guide RNA (gRNA) and single guide RNA (sgRNA). In one aspect, non-protein coding RNA molecules include miRNA. In one aspect, non-protein coding RNA molecules include siRNA. In one aspect, non-protein coding RNA molecules include ta-siRNA. In one aspect, non-protein coding RNA molecules are selected from the following: miRNA, siRNA and ta-siRNA.

[0192] As used herein, "target gene" refers to a polynucleotide sequence encoding a protein or non-protein coding RNA molecule that will be integrated into the target sequence, or, alternatively, an endogenous polynucleotide sequence encoding a protein or a non-protein coding RNA molecule edited by a ribonucleoprotein. In one aspect, the target gene encodes a protein. In another aspect, the target gene encodes a non-protein coding RNA molecule. In one aspect, the target gene is exogenous to the targeting DNA molecule. In one aspect, the target gene replaces the endogenous gene in the target DNA molecule.

[0193] mutation

[0194] In one aspect, the ribonucleoproteins or methods provided herein generate at least one mutation in a target sequence in an egg, embryo, and / or meiotic cell.

[0195] In one aspect, seeds produced by plants provided herein comprise at least one mutation in a gene of interest comprising a target site, compared to seeds of a control plant of the same line or variety lacking a first nucleic acid sequence encoding a guide nuclease operably linked to an egg cell-preferred promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a heterologous second promoter. In one aspect, seeds produced by plants provided herein comprise at least one mutation in a gene of interest comprising a target site, compared to seeds of a control plant of the same line or variety lacking a first nucleic acid sequence encoding a guide nuclease operably linked to an embryo tissue-preferred promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a heterologous second promoter.

[0196] In one aspect, seeds produced by plants provided herein comprise at least one mutation in a gene of interest comprising a target site, compared to seeds of a control plant of the same line or variety lacking a first nucleic acid sequence encoding a guide nuclease operably linked to a heterologous promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to an egg cell-preferred promoter. In one aspect, seeds produced by plants provided herein comprise at least one mutation in a gene of interest comprising a target site, compared to seeds of a control plant of the same line or variety lacking a first nucleic acid sequence encoding a guide nuclease operably linked to a heterologous promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to an embryonic tissue-preferred promoter.

[0197] As used herein, "mutation" refers to a non-natural change in a nucleic acid or amino acid sequence compared to a naturally occurring reference nucleic acid or amino acid sequence from the same organism. It should be appreciated that when identifying a mutation, the reference sequence should be from the same nucleic acid (e.g., gene, non-coding RNA) or amino acid (e.g., protein). When determining whether the difference between two sequences includes a mutation, it will be appreciated in the art that comparisons should not be made between homologous sequences from two different species or between homologous sequences from two different varieties of a single species. Instead, comparisons should be made between edited (e.g., mutant) sequences and endogenous, unedited (e.g., "wild type") sequences from the same organism.

[0198] Several types of mutations are well known in the art. In one aspect, mutations include insertions. "Insertion" refers to adding one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, mutations include deletions. "Deletion" refers to removing one or more nucleotides or amino acids from a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, mutations include substitutions. "Substitution" refers to replacing a given polynucleotide or amino acid sequence, respectively, with one or more nucleotides or amino acids, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, mutations include inversions. "Inversion" refers to the end-to-end reversal of a segment of a polynucleotide or amino acid sequence. In one aspect, mutations provided herein include mutations selected from the following: insertions, deletions, substitutions, and inversions.

[0199] In one aspect, the plant or seed comprises at least one mutation in the target gene, wherein the at least one mutation results in the deletion of one or more amino acids from the protein encoded by the target gene compared to the wild-type protein.

[0200] In one aspect, the plant or seed comprises at least one mutation in the target gene, wherein the at least one mutation results in the substitution of one or more amino acids in the protein encoded by the target gene compared to the wild-type protein.

[0201] In one aspect, the plant or seed comprises at least one mutation in a target gene, wherein the at least one variation results in the insertion of one or more amino acids into the protein encoded by the target gene compared to the wild-type protein.

[0202] When the mutated messenger RNA (mRNA) is translated into a protein or polypeptide, a mutation in the gene coding region (e.g., an exon mutation) can result in the production of a truncated protein or polypeptide. In one aspect, the present disclosure provides mutations that result in truncation of a protein or polypeptide. As used herein, a "truncated" protein or polypeptide comprises at least one less amino acid compared to an endogenous control protein or polypeptide. For example, if endogenous protein A comprises 100 amino acids, a truncated version of protein A may comprise between 1 and 99 amino acids.

[0203] Without being limited by any scientific theory, a method for causing protein or polypeptide truncation is to introduce an early termination codon in the mRNA transcript of an endogenous gene. In one aspect, the present disclosure provides a mutation that causes the production of an early termination codon in an endogenous gene mRNA transcript. As used herein, "termination codon" refers to a nucleotide triplet in an mRNA transcript that signals the termination of protein translation. "Early termination codon" refers to a termination codon located earlier (e.g., on the 5' side) than a normal termination codon position in an endogenous mRNA transcript. Without limitation, several termination codons are well known in the art, including "UAG", "UAA", "UGA", "TAG", "TAA" and "TGA".

[0204] In one aspect, the seed or plant comprises at least one mutation, wherein the at least one variation results in the introduction of a premature stop codon in the messenger RNA encoded by the gene of interest compared to the wild-type messenger RNA.

[0205] In one aspect, the mutations provided herein include null mutations. As used herein, "null mutations" refer to mutations that completely lose function of a protein encoded by a gene comprising a mutation, or mutations that completely lose function of a small RNA encoded by a genomic locus. Null mutations can result in a lack of mRNA transcription products, a lack of small RNA transcription products, a lack of protein function, or a combination thereof.

[0206] The mutations provided herein can be located in any part of an endogenous gene. In one aspect, the mutations provided herein can be located in an exon of an endogenous gene. In another aspect, the mutations provided herein can be located in an intron of an endogenous gene. In one other aspect, the mutations provided herein are located in the 5'-untranslated region of an endogenous gene. In yet another aspect, the mutations provided herein are located in the 3'-untranslated region of an endogenous gene. In yet another aspect, the mutations provided herein are located in the promoter of an endogenous gene.

[0207] In one aspect, mutation is located at the splice site within the gene. The mutation of the splice site can interfere with the splicing of exons during mRNA processing. If one or more nucleotides are inserted, deleted or replaced at the splice site, splicing may be interfered with. Interference in splicing can lead to introns in the mature mRNA sequence not being spliced, exons being missing, or both. Usually, although not always, a "GU" sequence is required at the 5' end of the intron, and an "AG" sequence is required at the 3' end of the intron, so as to perform correct splicing. If any one of these splice sites mutates, splicing interference will occur.

[0208] In one aspect, the seed or plant comprises at least one mutation, wherein the at least one mutation comprises deleting one or more splice sites from the target gene. In another aspect, the seed or plant comprises at least one mutation, wherein the at least one mutation is located within one or more splice sites of the target gene.

[0209] In one aspect, the mutation comprises site-directed integration. In one aspect, the site-directed integration comprises inserting all or part of a desired sequence into a target sequence.

[0210] As used herein, "site-directed integration" refers to all or part of a desired sequence (e.g., an exogenous gene, an edited endogenous gene) inserted or integrated into a desired site or locus in a plant genome (e.g., a target sequence). As used herein, a "desired sequence" refers to a DNA molecule comprising a nucleic acid sequence that will be integrated into the genome of a plant or plant cell. The desired sequence may comprise a transgene or a construct. In one aspect, the nucleic acid molecule comprising the desired sequence comprises one or two homology arms flanking the desired sequence to promote targeted insertion events by homologous recombination and / or homology-directed repair.

[0211] In one aspect, the methods provided herein comprise site-directed integration of a desired sequence into a target sequence.

[0212] Any site or locus within the plant genome can be selected for site-directed integration of a transgene or construct of the present disclosure. In one aspect, the target sequence is located within a B chromosome or an extra chromosome.

[0213] For site-directed integration, a double-strand break (DSB) or a gap can first be generated at the target sequence by a guide nuclease or ribonucleoprotein provided herein. In the presence of a desired sequence, the DSB or gap can be repaired by homologous recombination (HR) between the homology arms of the desired sequence and the target sequence or by non-homologous end joining (NHEJ), resulting in site-directed integration of all or part of the desired sequence into the target sequence to generate a target insertion event at the DSB or gap site.

[0214] In one aspect, site-directed integration comprises using the cell's endogenous NHEJ repair mechanism. In another aspect, site-directed integration comprises using the cell's endogenous HR repair mechanism.

[0215] In one aspect, repair of the double strand break produces at least one mutation in the target gene compared to a control plant of the same line or variety.

[0216] In one aspect, the mutation comprises integrating at least 5 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation comprises integrating at least 10 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation comprises integrating at least 15 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation comprises integrating at least 20 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation comprises integrating at least 25 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation comprises integrating at least 50 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation comprises integrating at least 100 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation comprises integrating at least 250 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation comprises integrating at least 500 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation comprises integrating at least 1000 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation comprises incorporation of at least 2000 contiguous nucleotides of the desired sequence into the target sequence.

[0217] In one aspect, the mutation includes integrating between 5 consecutive nucleotides and 3500 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 5 consecutive nucleotides and 2500 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 5 consecutive nucleotides and 1500 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 5 consecutive nucleotides and 750 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 5 consecutive nucleotides and 500 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 5 consecutive nucleotides and 250 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 5 consecutive nucleotides and 150 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 25 consecutive nucleotides and 2500 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 25 consecutive nucleotides and 1500 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 25 consecutive nucleotides and 750 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 50 consecutive nucleotides and 2500 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 50 consecutive nucleotides and 1500 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 50 consecutive nucleotides and 750 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 100 consecutive nucleotides and 2500 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 100 consecutive nucleotides and 1500 consecutive nucleotides of the desired sequence into the target sequence. In one aspect, the mutation includes integrating between 100 consecutive nucleotides and 750 consecutive nucleotides of the desired sequence into the target sequence.

[0218] In one aspect, the method provided herein includes detecting editing or mutation in the target sequence. The screening and selection of mutagenic or edited plants or plant cells can be by any method known to those of ordinary skill in the art. Examples of screening and selection methods include, but are not limited to, Southern analysis, PCR amplification for detecting polynucleotides, Northern blotting, RNase protection, primer extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, enzyme assays, protein gel electrophoresis, Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assays for detecting polypeptides and polynucleotides for the next generation sequencing technology (e.g., Illumina, PacBio, Ion Torrent, 454) of enzyme or ribozyme activity. Other techniques such as in situ hybridization, enzyme staining, and immunostaining can also be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods for performing all of the above techniques are well known in the art.

[0219] Recombinase

[0220] Several embodiments described herein relate to methods and compositions for preferentially or particularly inducing site-specific recombination in eggs, embryos, and / or meiotic plant tissues. Several embodiments described herein relate to methods and compositions for providing preferential or specific expression of recombinases in eggs, embryos, and / or meiotic plant tissues. Several embodiments described herein relate to methods and compositions for preferentially or specifically expressing DNA modification enzymes such as guide nucleases (e.g., CRISPR / Cas systems) in eggs, embryos, and / or meiotic plant tissues by inducing site-specific recombination to operably connect polynucleotides encoding DNA modification enzymes to constitutive promoters. In some embodiments, transcribable polynucleotides encoding DNA modification enzymes (e.g., guide nucleases (e.g., CRISPR / Cas systems) are operably connected to constitutive promoters by preferentially or specifically excising intervening polynucleotide sequences mediated by recombinases in eggs, embryos, and / or meiotic plant tissues.

[0221] When DNA strand exchange occurs between DNA segments with at least some sequence homology to each other, site-specific recombination occurs. Site-specific recombinases can recognize and bind to "recombination sites", which are short, specific DNA sequences cut by recombinases, allowing exchange of DNA strands, followed by strand repair. Typically, each recombinase protein is bound to a specific, unique recombination site. As used herein, "recombinase" refers to an enzyme that can catalyze a specific site recombination event in a DNA. Recombinases can excise DNA, insert DNA, invert DNA, translocate DNA and / or exchange DNA.

[0222] In one aspect, the present disclosure provides methods and compositions for specifically or preferentially providing recombinases in eggs, embryos, and / or meiotic plant tissues. In one aspect, the present disclosure provides nucleic acid sequences encoding recombinases operably connected to the promoters described in Table 1. In one aspect, the recombinant nucleic acid construct comprises a sequence encoding at least one recombinase operably connected to the promoters described in Table 1. In one aspect, a recombinant nucleic acid construct comprising a sequence encoding at least one recombinase operably connected to the promoters described in Table 1 is provided to a plant cell in combination with a recombinant nucleic acid construct comprising a polynucleotide encoding a DNA modification enzyme, an insertion sequence flanked by a recombination site, and a constitutive promoter, wherein excision of the insertion sequence operably connects the polynucleotide encoding the DNA modification enzyme to a constitutive promoter in an egg, an embryo, and / or meiotic tissue.

[0223] In one aspect, the recombinase is a tyrosine recombinase. In one aspect, the tyrosine recombinase is selected from the group consisting of: Cre recombinase and Flp recombinase.

[0224] In one aspect, the recombinase is a Cre recombinase. Cre-lox is a site-specific recombination system derived from bacteriophage P1. Cre-lox can be used to invert a nucleic acid sequence, delete a nucleic acid sequence, or translocate a nucleic acid sequence. In the system, the Cre recombinase recombines a pair of lox nucleic acid sequences. The lox site contains 34 nucleotides, of which the first and last 13 nucleotides (arms) are palindromic. During the recombination process, the Cre recombinase protein binds to two lox sites on different nucleic acids and cuts at the lox sites. The cut nucleic acids are spliced ​​together (reciprocal translocation) and the recombination is complete.

[0225] In one aspect, the recombinase is a flippase (Flp). The Flp-FRT site-specific recombination system is from the 2μ plasmid of the baker's yeast Saccharomyces cerevisiae, similar to the Cre-lox system. Flp can induce the recombination between the flippase recognition target (FRT) sites. The FRT site contains 34 nucleotides. Flp binds to the "arm" of the FRT site (one arm is in the reverse direction) and cuts the FRT site at both ends of the inserted nucleic acid sequence. After cutting, Flp recombines the nucleic acid sequence between the two FRT sites.

[0226] In one aspect, the recombination site is a lox site. In one aspect, the lox site is selected from the following: loxP site, lox 2272 site, loxN site, lox 511 site, lox5171 site, lox71 site, lox66 site, loxLTR site, M2 site, M3 site, M7 site and M11 site. In one aspect, the recombination site is a FRT site.

[0227] TALE

[0228] Several embodiments provided herein relate to preferential expression of DNA modification enzymes, such as guide nucleases (e.g., CRISPR / Cas systems) in eggs, embryos, and / or meiotic plant tissues using TALE activators. In several embodiments, high-level egg, embryo, and / or meiotic tissue-specific expression of DNA modification enzymes, such as guide nucleases (e.g., CRISPR / Cas systems), is provided to plant cells by providing 1) an expression construct comprising a promoter operably connected to a sequence encoding TALE as described in Table 1 and 2) an expression construct comprising one or more TALE binding sites (TB) operably connected to a minimal promoter and a sequence encoding a DNA modification enzyme, and producing plants therefrom. In some embodiments, an expression construct encoding one or more guide nucleic acids is also provided. In some embodiments, the egg, embryo, and / or meiotic tissue-specific expression level of a DNA modification enzyme can be adjusted by changing the number of TBs.

[0229] As used herein, "TALE protein" refers to a transcription activator-like effector (TALE) protein or its homolog. TALE proteins were originally identified as virulence factors from plant pathogenic bacteria of the genus Xanthomonas or Ralstonia. These proteins are secreted by plant pathogens to alter the transcription of host genes in plant cells. TALE proteins bind to DNA in the nucleus through the domain of DNA binding repeats, where they act as transcription activators, thereby promoting virulence. TALE moves to the nucleus, where it recognizes and binds to specific DNA sequences in the regulatory region of specific genes in the host genome. TALE has a central DNA binding domain consisting of 13-28 repeating monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, except for the highly variable amino acid residues at positions 12 and 13. The two variable amino acids are referred to as recombinant variable diresidues (RVD). The amino acid pairs NI, NG, HD and NN of RVD preferentially recognize adenine, thymine, cytosine and guanine / adenine, respectively, and the regulation of RVD can recognize continuous DNA bases. This simple relationship between amino acid sequence and DNA recognition allows the design of specific DNA binding domains by selecting combinations of repeat segments containing appropriate RVDs. As used herein, a "TALE binding site" (TBS) refers to a specific DNA sequence recognized and bound by a "TALE DNA binding domain" of a TALE protein.

[0230] plant

[0231] Any plant or plant cell can be used with the methods and compositions provided herein. In one aspect, the plant is selected from the following: corn plants, rice plants, sorghum plants, wheat plants, alfalfa plants, barley plants, millet plants, rye plants, sugarcane plants, cotton plants, soybean plants, rape plants, tomato plants, onion plants, cucumber plants, Arabidopsis plants and potato plants. In one aspect, the plant is an angiosperm. In one aspect, the plant is a gymnosperm. In one aspect, the plant is a monocot. In one aspect, the plant is a dicot. In one aspect, the plant is a plant selected from the following families: Allium family, Anacardiaceae, Apiaceae, Palm family, Asteraceae, Cruciferae, Sorbus family, Cucurbitaceae, Ericaceae, Leguminosae, Juglandaceae, Malvaceae, Mandragaceae, Moraceae, Musaceae, Orchidaceae, Fabaceae, Pinaceae, Poaceae, Rosaceae, Rutaceae, Rubiaceae and Solanaceae.

[0232] In one aspect, the plant cell is selected from the following: corn cells, rice cells, sorghum cells, wheat cells, alfalfa cells, barley cells, millet cells, rye cells, sugarcane cells, cotton cells, soybean cells, rapeseed cells, tomato cells, onion cells, cucumber cells, Arabidopsis cells and potato cells. In one aspect, the plant cell is an angiosperm cell. In one aspect, the plant cell is a gymnosperm cell. In one aspect, the plant cell is a monocotyledonous plant cell. In one aspect, the plant cell is a dicotyledonous plant cell. In one aspect, the plant cell is a plant cell selected from the following families: Allium family, Anacardiaceae, Apiaceae, Palm family, Asteraceae, Cruciferae, Sorbus family, Cucurbitaceae, Ericaceae, Leguminosae, Juglandaceae, Malvaceae, Mangiferae, Moraceae, Musaceae, Orchidaceae, Fabaceae, Pinaceae, Poaceae, Rosaceae, Rutaceae, Rubiaceae and Solanaceae.

[0233] As used herein, "variety" refers to a group of plants within a species (e.g., but not limited to corn) that have certain genetic traits that distinguish them from other possible varieties within the species. Varieties can be inbred or hybrids, but commercial plants are usually hybrids to take advantage of hybrid vigor. Individuals in a hybrid variety are homogeneous and almost genetically identical, with most loci in a heterozygous state.

[0234] As used herein, the term "inbred line" refers to a line bred for genetic homogeneity. In one aspect, the seeds provided herein are inbred seeds. In one aspect, the plants provided herein are inbred plants.

[0235] As used herein, the term "hybrid" refers to the offspring of mating between at least two genetically different parents. Without limitation, examples of mating schemes include single cross, improved single cross, double improved single cross, tri-cross, improved tri-cross and double cross, wherein at least one parent in the improved cross is the offspring of hybridization between sister lines. In one aspect, the seed provided herein is a hybrid seed. In one aspect, the plant provided herein is a hybrid plant.

[0236] Conversion

[0237] The methods can involve transient transformation or stable integration of any nucleic acid molecule into any plant or plant cell provided herein.

[0238] As used herein, "stable integration" or "stably integrated" refers to the transfer of DNA into the genomic DNA of a target cell or plant, so that the target cell or plant passes the transferred DNA to the next generation of transformed organisms. Stable transformation requires the integration of the transferred DNA into the reproductive cells of the transformed organism. As used herein, "transient transformation" or "transiently transformed" refers to the transfer of DNA into a cell without transferring it to the next generation of transformed organisms. In transient transformation, the transformed DNA is generally not integrated into the genomic DNA of the transformed cell. In one aspect, a method for stably transforming a plant cell or plant with one or more nucleic acid molecules provided herein. In another aspect, a method for transiently transforming a plant cell or plant with one or more nucleic acid molecules provided herein.

[0239] In one aspect, nucleic acid molecules encoding guide nucleases are stably integrated into plant genomes. In one aspect, nucleic acid molecules encoding Cas12a nucleases are stably integrated into plant genomes. In one aspect, nucleic acid molecules encoding CasX nucleases are stably integrated into plant genomes. In one aspect, nucleic acid molecules encoding guide nucleic acids are stably integrated into plant genomes. In one aspect, nucleic acid molecules encoding guide RNAs are stably integrated into plant genomes. In one aspect, nucleic acid molecules encoding single guide RNAs are stably integrated into plant genomes.

[0240] Many methods for transforming cells with recombinant nucleic acid molecules or constructs are known in the art and can be used according to the methods of the present application. According to the methods of the present invention, any suitable method or technique for cell transformation known in the art can be used. Effective methods for plant transformation include bacterial-mediated transformation, such as Agrobacterium-mediated or Rhizobium-mediated transformation and microprojectile bombardment-mediated transformation. A variety of methods are known in the art for transforming explants with transformation vectors by bacterial-mediated transformation or microprojectile bombardment and then culturing these explants to regenerate or develop transgenic plants.

[0241] In one aspect, a method comprises providing a cell with a nucleic acid molecule transformed by Agrobacterium-mediated transformation. In one aspect, a method comprises providing a cell with a nucleic acid molecule transformed by polyethylene glycol-mediated transformation. In one aspect, a method comprises providing a cell with a nucleic acid molecule transformed by a gene gun. In one aspect, a method comprises providing a cell with a nucleic acid molecule by liposome-mediated transfection. In one aspect, a method comprises providing a cell with a nucleic acid molecule transduced by a virus. In one aspect, a method comprises providing a cell with a nucleic acid molecule by using one or more delivery particles. In one aspect, a method comprises providing a cell with a nucleic acid molecule by microinjection. In one aspect, a method comprises providing a cell with a nucleic acid molecule by electroporation.

[0242] In one aspect, the nucleic acid molecule is provided to the cell by a method selected from the group consisting of Agrobacterium-mediated transformation, polyethylene glycol-mediated transformation, biolistic transformation, liposome-mediated transfection, viral transduction, use of one or more delivery particles, microinjection, and electroporation.

[0243] Other methods for transformation, such as vacuum infiltration, pressure, ultrasound, and silicon carbide fiber agitation, are also known in the art and are contemplated for use with any of the methods provided herein.

[0244] Methods for transforming cells are well known to those of ordinary skill in the art. For example, specific instructions for transforming plant cells by microbombardment with particles coated with recombinant DNA (e.g., gene gun transformation) are described in U.S. Patent Nos. 5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,153,812, and Agrobacterium-mediated transformation is described in U.S. Patent Nos. 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871; 5,463,174; and 5,188,958, the entire contents of which are incorporated herein by reference. Other methods for transforming plants can be found in, for example, Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Any suitable method known to those skilled in the art can be used to transform plant cells with any nucleic acid molecule provided herein.

[0245] Lipofection is described in, for example, U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355, and lipofection reagents are sold commercially (e.g., Transfectam TM and Lipofectin TMCationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include Felgner, WO 91 / 17424; WO 91 / 16024. Delivery to cells (eg, in vitro or ex vivo administration) or target tissues (eg, in vivo administration) is also possible.

[0246] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expressing one or more elements of nucleic acid molecules as used in WO 2014 / 093622. In one aspect, a method of providing nucleic acid molecules or proteins to cells includes delivery by delivery particles. In one aspect, a method of providing nucleic acid molecules to plant cells or plants includes delivery by delivery vesicles. In one aspect, the delivery vesicles are selected from the following: exosomes and liposomes. In one aspect, a method of providing nucleic acid molecules to plant cells or plants includes delivery by viral vectors. In one aspect, the viral vectors are selected from the following: adenoviral vectors, lentiviral vectors and adeno-associated viral vectors. In another aspect, a method of providing nucleic acid molecules to plant cells or plants includes delivery by nanoparticles. In one aspect, a method of providing nucleic acid molecules to plant cells or plants includes microinjection. In one aspect, a method of providing nucleic acid molecules to plant cells or plants includes polycations. In one aspect, a method of providing nucleic acid molecules to plant cells or plants includes cationic oligopeptides.

[0247] In one aspect, the delivery particles are selected from the following: exosomes, adenoviral vectors, lentiviral vectors, adeno-associated viral vectors, nanoparticles, polycations, and cationic oligopeptides. In one aspect, the methods provided herein include the use of one or more delivery particles. In another aspect, the methods provided herein include the use of two or more delivery particles. In another aspect, the methods provided herein include the use of three or more delivery particles.

[0248] Suitable agents for promoting transfer of nucleic acids into plant cells include agents that increase the external permeability of the plant or increase the permeability of plant cells to oligonucleotides or polynucleotides. Such agents for promoting transfer of compositions into plant cells include chemical agents or physical agents or combinations thereof. Chemical agents for conditioning include (a) surfactants, (b) organic solvents, aqueous solutions or aqueous mixtures of organic solvents, (c) oxidants, (e) acids, (f) bases, (g) oils, (h) enzymes or combinations thereof.

[0249] Organic solvents that can be used to condition plants to be permeated with polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, propylene glycol, other solvents that are miscible with water or will dissolve nucleotide phosphates in non-aqueous systems (e.g., for synthesis reactions). Naturally derived or synthetic oils with or without surfactants or emulsifiers can be used, for example, oils of plant origin, crop oils (such as those listed in the 9th edition of the Compendium of Herbicide Adjuvants, which can be publicly available online from www (dot) herbicide (dot) adjuvants (dot) com), for example, paraffin oils, polyol fatty acid esters, or oils with short chain molecules modified with amides or polyamines such as polyethylene imine or N-pyrrolidine.

[0250] Examples of useful surfactants include sodium or lithium salts of fatty acids (such as tallow or tallow amine or phospholipids) and silicone surfactants. Other useful surfactants include silicone surfactants, including nonionic silicone surfactants, for example, trisiloxane ethoxylate surfactants or silicone polyether copolymers, such as copolymers of polyoxyalkylene-modified heptamethyltrisiloxane and allyloxypropylene glycol methyl ether (which can be used as L-77 purchased).

[0251] Useful physical agents may include (a) abrasives such as corundum, corundum, sand, calcite, pumice, garnet, etc., (b) nanoparticles such as carbon nanotubes or (c) physical forces. Carbon nanotubes are disclosed by Kam et al., (2004) Am. Chem. Soc, 126 (22): 6850-6851, Liu et al., (2009) Nano Lett, 9 (3): 1007-1010, and Khodakovskaya et al., (2009) ACS Nano, 3 (10): 3221-3227. Physical force agents may include heating, cooling, applying positive pressure or ultrasonic treatment. Embodiments of the method may optionally include an incubation step, a neutralization step (e.g., neutralizing an acid, base or oxidant, or inactivating an enzyme), a rinse step or a combination thereof. The method of the present invention may also include the use of other agents that have an enhanced effect due to the silencing of certain genes. For example, when a polynucleotide is designed to modulate a gene that confers herbicide resistance, subsequent applications of the herbicide may have a significant effect on the herbicide's efficacy.

[0252] Agents used for laboratory conditioning of plant cells for polynucleotide permeability include, for example, chemical agents, enzymatic treatment, application of heat or cooling, positive or negative pressure treatment, or sonication. Agents used to condition plants in the field include chemicals such as surfactants and salts.

[0253] In one aspect, the cell that transforms or transfects is a plant cell. The receptor plant cell or explant target for transformation includes but is not limited to seed cell, fruit cell, leaf cell, cotyledon cell, hypocotyl cell, meristem cell, embryo cell, endosperm cell, root cell, bud cell, stem cell, pod cell, flower cell, inflorescence cell, main stem cell, peduncle cell, style cell, stigma cell, receptacle cell, petal cell, sepal cell, pollen cell, anther cell, filamentous cell, ovary cell, ovule cell, pericarp cell, phloem cell, bud cell or vascular tissue cell. In another aspect, the disclosure provides plant chloroplast. In one other aspect, the disclosure provides epidermal cell, guard cell, hair cell, root hair cell, storage root cell or tuber cell. In another aspect, the disclosure provides protoplast. In another aspect, the disclosure provides plant callus cell. Any cell that can be regenerated from fertile plants is considered to be useful receptor cell in the practice of the disclosure. Callus can be started from various tissue sources, including but not limited to immature embryos or partial embryos, seedling apical meristems, microspores, etc. Those cells that can be proliferated into callus can be used as the recipient cells of transformation. For example, in U.S. Patent Nos. 6,194,636 and 6,232,526 and U.S. Patent Application Publication 2004 / 0216189, the implementation transformation method and materials (for example, various culture media and receptor target cells, the transformation of immature embryos and the regeneration of fertile transgenic plants subsequently) for making transgenic plants of the present disclosure are disclosed, all of which are incorporated herein by reference. The explant, cell or tissue of transformation can undergo additional culture steps, such as callus induction, selection, regeneration, etc. well known in the art. The transformed cell, tissue or explant containing the recombinant DNA insert can be grown, developed or regenerated into a transgenic plant according to methods well known in the art in culture, plugs or soil. In one aspect, the present disclosure provides plant cells that are not reproductive materials and do not mediate the natural reproduction of plants. In another aspect, the present disclosure also provides plant cells as reproductive materials and mediate the natural reproduction of plants. In another aspect, the present disclosure provides plant cells that cannot sustain themselves through photosynthesis. In another aspect, the present disclosure provides somatic plant cells. In contrast to germline cells, somatic cells do not mediate plant reproduction. In one aspect, the present disclosure provides non-reproductive plant cells.

[0254] Uses of Haploid / Haploid Inducing Lines

[0255] Several embodiments relate to the purposes of the method and composition as described herein in combination with haploid induction technology.As used herein, "haploid" cells or nuclei include one set of unpaired chromosomes (x).In contrast, "diploid" cells or nuclei include two complete sets of chromosomes (2x) and can carry out homologous pairing.As used herein, "haploid plant" describes a sporophyte comprising a plurality of cells, which include a haploid nuclear genome.The haploid plant provided herein can be a maternal haploid plant, which means that it has lost the paternal nuclear genome while retaining the maternal nuclear genome.Or, the haploid plant provided herein can be a paternal haploid plant, which means that it has lost the maternal nuclear genome while retaining the paternal nuclear genome.Usually, maternal mitochondria and plastid (e.g., chloroplast) genomes are retained in maternal and paternal haploid plants.

[0256] In some embodiments, a "doubled haploid (DH)" method is used to rapidly produce homozygous plants. The offspring of DH plants are genetically homogeneous materials, allowing breeders to evaluate their target traits on genetically fixed materials in the early stages of the breeding cycle, thereby improving breeding efficiency (see Gilles LM et al., Curr Biol. 2017 Oct 23; 27 (20): R1095-R1097). DH technology relies on two main steps: (1) a haploid induction system to produce haploid embryos or plants, and (2) a chromosome doubling step to restore the diploidy of these plants.

[0257] "Haploid induction (HI)" is a phenomenon in some plants characterized by parental induced chromosome loss during embryo development. As used herein, a "haploid induced (HI) plant" is a plant that can induce haploidy in offspring plants by eliminating a set of chromosomes. Maternal haploid induction is triggered by the pollinator (male plant) father. Paternal haploid induction is triggered by the mother.

[0258] Haploid induction lines are commonly used in plant breeding, especially corn. There are many known haploid induction corn lines, including but not limited to: stock 6, MHI (Moldova haploid induction line), indeterminate gametophyte (ig 1) mutation, KEMS, RWK, ZEM, ZMS, KMS. By using the centromere histone 3 (CENH3) variant of through engineering approaches, haploid induction lines have also been produced in Arabidopsis, mustard and corn (see Ravi and Chan.2010.Nature.464:615-6190). In some aspects, haploid induction lines as described herein include maize Stock 6 lines, corn plants with ig-1 locus mutation, MHI induction lines, KEMS induction lines, RWK induction lines, ZEM induction lines, ZMS induction lines and KMS induction lines. In some aspects, the haploid inducible lines comprise a modified MATRILINEAL / NOT LIKE DAD / ZmPROPOLIPLASE-A1 (MATL / NLD / ZmPLA1) gene. In some aspects, the haploid inducible lines described herein comprise a modified CEN H3 variant.

[0259] The benefits that haploid induction systems provide to crop breeding programs are diverse, as DH technology can be used in combination with several different molecular techniques to overcome various limitations in crop improvement. One example is the use of haploid induction systems to expand the application of genome editing technology in crops. Genome editing components (e.g., guide nucleases) can be introduced into haploid induction lines, which are then crossed with non-induced corn lines. The haploid offspring are then screened for nuclease-induced mutations, followed by induction of genome doubling to produce diploid, non-editing component, genome-edited varieties. The method has been described in detail in US20190169596 (application US16 / 275200), and is incorporated herein by reference in its entirety.

[0260] The present disclosure will now be described with reference to the following examples. It should be appreciated that these examples are not intended to limit the scope of the claims to the present disclosure; rather, they are intended to be examples of certain embodiments. Any variations in the exemplary methods that occur to those skilled in the art are intended to fall within the scope of the present disclosure.

[0261] Example

[0262] Example 1: Expression of Cas12a in meiotic oocytes or embryonic tissues to produce germinal mutations or targeted integration of template DNA.

[0263] Several Agrobacterium T-DNA vectors were generated to preferentially express Cas12a in maize egg cells and / or maize embryo cells (meiotic cells) undergoing meiosis. See Table 2.

[0264] Table 2: Expression cassettes designed for preferential or sole expression of Cas12a in maize egg cells and / or maize embryo cells and / or cells undergoing meiosis.

[0265]

[0266] The plant codon-optimized LbCas12a sequences (SEQ ID NO:7) in these expression cassettes are flanked by NLS sequences (SEQ ID:8 and SEQ ID:9) at the 5' and 3' ends and are operably connected to the transcription termination sequence from the rice lipid transfer protein (LTP) gene (described as SEQ ID NO:8 in US20200080096). Each vector also contains an expression cassette encoding a Cas12a-gRNA targeting a unique corn genomic site (ZmTS1) under the control of the Pol IIIZmU6 promoter (SEQ ID NO:10); an expression cassette flanked by a ZmTS1 target site, wherein the cassette comprises a constitutive promoter operably connected to the target gene (GOI) and an expression cassette for a selective marker for conferring resistance to the herbicide glyphosate. The above-mentioned vectors were used to transform corn 01DKD2 variety embryos by Agrobacterium-mediated transformation, and R0 plants were regenerated from the transformed corn cells. DNA was extracted from leaf samples of 59-153 R0 seedlings generated from each construct. The genomic target sites were sequenced and analyzed for the presence of targeted mutations. Taqman-based assays were also performed to identify the copy number of constructs carrying Cas12a. For all reproductive promoters except ZmDSUL, no mutations were detected at the ZmTS1 site in the R0 generation. ZmDSULbCas12a (construct 1) showed a target site mutation rate of about 32% in R0 seedlings (see Table 3). It is expected that the ZmTS1 target site in the newly transformed (or R0) plants lacks mutations, in which LbCas12a expression is limited to reproductive tissues.

[0267] 20 R0 lines from each transformation construct were grown to maturity, and at least one ear from each transformed corn plant was self-pollinated. 10 R1 lines were selected, and each line germinated up to 16 seedlings, screened for mutations in ZmTS1, and calculated mutation rate (cutting rate). Taqman assay was also performed to determine the presence and copy number of the LbCas12a expression cassette. The total target site mutation rate in all lines produced from constructs 2-5 was in the range of about 1.1% to 14%. The average mutation rate is shown in Table 3. 4 strains expressing ZmDSUL::LbCas12a (construct 1) were tested, and no mutation was observed. The co-expression of Cas12a and its cognitive gRNA in cells undergoing meiosis is expected to produce double-strand breaks in the ZmTS1 target site, and then incomplete DNA repair will produce unique mutations in egg cells and embryos produced by pollination.

[0268] Table 3: Average mutation rates observed at ZmTS1 target sites in R0 and R1 plants

[0269]

[0270] The mutation rate in the R1 plant produced by a single R0 line can vary, as shown in Tables 4-8. For example, in the highest cutting promoter ZmES4, some lines with a single copy of the Cpf1 box showed a 0% mutation rate, while another line showed about 11% mutation rate. The highest mutation rate observed was 46%, which was derived from the R0 line with two copies of ZmES4:LbCas12a. In ZmES4:Cas12a plants, among all the lines tested, the R0 lines with 2 copies of Cas12a had a higher target mutation rate than the 1 copy R0 lines (see Table 7).

[0271] Table 4: Mutation rate in R1 seedlings of maize plants transformed with pZmDMC1::LbCas12a

[0272]

[0273] Table 5: Mutation rate in R1 seedlings of maize plants transformed with pZmMPS4::LbCas12a

[0274]

[0275] Table 6: Mutation rate in R1 seedlings of maize plants transformed with pZmAdf1::LbCas12a

[0276]

[0277] Table 7: Mutation rate in R1 seedlings of maize plants transformed with pZmES4::LbCas12a

[0278]

[0279] Table 8: Mutation rate in R1 seedlings of maize plants transformed with pZmEA1::LbCas12a

[0280]

[0281] Table 9: Mutation rate in R1 seedlings of maize plants transformed with pZmEDSUL1::LbCas12a

[0282]

[0283] To increase confidence in the targeted mutation rate, three R0 lines were selected from ZmES4:LbCas12a transformants. For the selected lines, 152, 112, and 86 R1 seedlings were screened. The mutation rates were 74.2%, 49.4%, and 8.3%, respectively. The lines with mutation rates of 74.2% and 49.4% were observed to be R1 seeds from R0 lines with 2 copies of ZmES4:Cas12a.

[0284] Unique targeted mutations in R1 plants: Two R0 lines (ZM_S222321326 and ZM_S22221323) of ZmES4::LbCas12a, which are expected to selectively express LbCas12 in egg cells and synergids, were analyzed to evaluate the types of mutations produced. Five mutant plants were obtained from the ZM_S222321326 line. Sequencing results of the ZmTS1-gRNA target site showed that all five plants had unique mutations at the target site. 49 mutant seedlings were analyzed from the ZM_S222321323R0 line with two copies of ZmES4:LbCas12a. Among the 49 mutant seedlings, 56 target site mutations were identified. This indicates that some plants contain heterologous mutations. There are 23 unique mutations in the 49 plants.

[0285] Table 10: Types and numbers of unique mutations observed in R1 plants of the ZmES4::LbCas12a R0ZM_S222321326 event.

[0286]

[0287] Table 11: Types and numbers of unique mutations observed in R1 plants of the ZmES4::LbCas12a R0ZM_S22321323 event.

[0288]

[0289]

[0290] Site-directed integration testing of template DNA in plants expressing Cas12a

[0291] In addition to the Cas12a and gRNA expression cassettes, each vector also contains an expression cassette for a target gene (GOI) flanked by the ZmTS1-gRNA target sequence. Cas12a expression in reproductive tissues is expected to produce double-strand breaks on both sides of the GOI box, releasing it from the T-DNA. This released DNA can serve as a donor for targeted insertion into the genomic ZmTS1 target site. When the CRISPR-Cpf1 complex cuts the target site within the genome, the non-homologous end joining (NHEJ) DNA repair pathway can insert the donor GOI box into the genomic target site. This form of SDI is also called trans-fragment targeting (TFT). To test SDI by TFT, a flanking PCR assay similar to that described in WO2019084148 was used to identify putative targeted insertions. Primers were designed for PCR amplification of the expected insertion flanking sequences. Four separate PCR reactions were performed: left flank PCR and right flank PCR for locating potential inserts in the sense direction, and left flank PCR, and right flank PCR for locating inserts in the antisense direction. In the initial screening of the R0 line, two plants showed flanking positive PCR. Both were identified in the ZmES4:LbCas12a line and both produced only one flanking PCR product. While one flanking positive plant was consistent with the determined mutation rate (11.11%), the other flanking negative plant was consistent with the plant with a mutation rate of 0%.

[0292] In summary, these data show that reproductive editing can be achieved when LbCas12a is expressed under the control of the promoters DMC1, Mps1, or Adf1, promoter-preferred or expressed alone in cells undergoing meiosis. Reproductive editing can also be achieved when LbCas12a is expressed by egg or embryo-expressing promoters such as ZmES4 and ZmEA1. In addition, the data show that a single R0 plant can produce many R1 progeny, each with a unique target site edit. This suggests that these promoters can be used to drive the expression of nucleases, thereby increasing the frequency of unique edits produced by each transformed plant.

[0293] Germline mutations can be passed on to the F1 generation

[0294] Two R1 individuals of ZmES4::LbCas12a R0 event ZM_S222321323 were grown to maturity and cross-pollinated with wild-type 01DKD2 tester. R1 plant-1 contains a 7bp deletion at 15 nucleotides of the target site (see mutation 3 in Table 11), and R1 plant-2 contains a 9bp deletion at 16 nucleotides of the target site. 32 seedlings of each F1 system were planted, and the inheritance of ZmTS1 target site mutations was screened. 11 hybridizations were germinated from 32 F1 seedlings of plant-1, of which 6 inherited R1 event-specific mutations. 5 hybridizations were germinated from 32 seedlings of plant-2, of which 3 inherited R1 event-specific mutations.

[0295] Example 2: Expression of Cas12a in cells undergoing meiosis to generate germinal mutations

[0296] Several constructs were generated to preferentially express Cas12a in maize cells undergoing meiosis, maize egg cells, and / or maize embryos. See Table 12.

[0297] Table 12: Expression cassettes designed to express Cas12a preferentially or solely in cells undergoing meiosis, corn egg cells, and / or corn embryos

[0298]

[0299] The plant codon-optimized LbCas12a sequence (SEQ ID NO:7) in the expression cassette described in Table 12 is flanked by NLS sequences (SEQ ID:8 and SEQ ID:9) at the 5' and 3' ends. Under the control of the Pol III ZmU6 promoter (SEQ ID NO:10), each construct described in Table 12 is introduced into corn cells simultaneously with a construct encoding a gRNA complementary to the target site ("gRNA construct"). Alternatively, the construct described in Table 12 can be transformed into cells containing gRNA constructs biologically or by Agrobacterium T-DNA vectors. The resulting transformed corn cells include one of constructs 7-13 and a gRNA construct. Corn plants are regenerated and grown to maturity by the transformed corn cells. At least one ear of each transformed corn plant is pollinated. The seeds produced by pollination are screened for target site mutations, and the number and type of mutations produced using constructs 8-13 are compared with the transformed corn plants produced using construct 7. Co-expression of Cas12a from constructs 8-13 and its cognitive guide RNA from the gRNA constructs is expected to result in double-strand breaks in the genomic DNA at the target site, with subsequent DNA repair generating one or more unique mutations.

[0300] Example 3: Expression of gRNA in eggs or embryonic tissues with constitutively expressed Cas12a to generate germinal mutations

[0301] In meiotic cells, corn egg cells and / or corn embryos, under the control of the Pol II promoter, several constructs are produced to preferentially express guide RNAs (gRNAs) complementary to the target site. See Table 3. After transcription, the Pol II product is rapidly modified with a 5' cap and a poly-A tail and exported from the nucleus. These modifications and altered positioning may prevent the effective use of gRNA. In order to optimize the availability and performance of gRNA, self-cleaving ribozymes are introduced into the gRNA box design. It is reported that self-cleaving ribozymes help to cut / process gRNA transcripts from transcripts expressed by Pol II to produce accurate guide molecules (see Wang et al., 2018, J. of Integrative Plant Biol, 60: 8, 626-631).

[0302] Table 13: Design of constructs to express gRNAs preferentially or solely in maize egg cells and / or maize embryos

[0303] Construct Promoter::gRNA Promoter SEQ ID NO. 14(Control) ZmUbqM1::ribozyme-gRNA-ribozyme 11 15 ZmDSUL1::ribozyme-gRNA-ribozyme 1 16 ZmEA1::ribozyme-gRNA-ribozyme 2 17 ZmES4::ribozyme-gRNA-ribozyme 3 18 ZmDMC1::ribozyme-gRNA-ribozyme 4 19 ZmMps1::ribozyme-gRNA-ribozyme 5 20 ZmAdf1::ribozyme-gRNA-ribozyme 6

[0304] The construct described in Table 13 is stably introduced into corn cells using transformation methods routinely used in the art. In addition, a construct ("Cas12a construct") comprising a plant codon-optimized nucleic acid sequence encoding a Cas12a protein (SEQ ID NO: 1) is co-introduced with each construct provided in Table 13, wherein the Cas12a protein (SEQ ID NO: 1) is flanked by NLS sequences at 5' and 3' ends (SEQ ID: 2 and SEQ ID: 3) and is under the control of the ubiquitous ZmUbqM1 promoter (SEQ ID NO: 11). The resulting transformed corn cells comprise one of constructs 14-20 and a Cas12a construct. Corn plants are regenerated and grown to maturity by the transformed corn cells. At least one ear of each transformed corn plant is pollinated. The seeds produced by pollination are screened for target site mutations, and the number and type of mutations produced using constructs 15-20 are compared with the transformed corn plants produced using construct 14. Selective expression of gRNA is expected to produce one or more unique mutations in each meiotic cell, egg cell, or each embryo produced by pollination.

[0305] Example 4: Expression of Cas12a and gRNA as a single transcript in meiotic cells, oocytes or embryonic tissues to generate germinal mutations

[0306] Several constructs were generated to preferentially express LbCas12a and a guide RNA (gRNA) complementary to the target site as a single transcript in cells undergoing meiosis, corn egg cells, and / or corn embryos. See Table 14.

[0307] Table 14: Constructs designed for expression of LbCas12a and gRNA in a single transcript flanked by self-cleaving ribozymes in meiotic cells, maize egg cells, and / or maize embryos.

[0308] Construct Promoter::gRNA Promoter SEQ ID NO. 21(Control) ZmUbqM1::LbCas12a-ribozyme-gRNA-ribozyme 11 22 ZmDSUL1::LbCas12a-ribozyme-gRNA-ribozyme 1 23 ZmEA1::LbCas12a-ribozyme-gRNA-ribozyme 2 24 ZmES4::LbCas12a-ribozyme-gRNA-ribozyme 3 25 ZmDMC1::LbCas12a-ribozyme-gRNA-ribozyme 4 26 ZmMps1::LbCas12a-ribozyme-gRNA-ribozyme 5 27 ZmAdf1::LbCas12a-ribozyme-gRNA-ribozyme 6

[0309] Each construct described in Table 14 is stably introduced into corn cells using the biotransformation method or Agrobacterium transformation method conventionally used in the art. The resulting transformed corn cells include one of constructs 21-27. Corn plants are regenerated and grown to maturity by the transformed corn cells. At least one ear of each transformed corn plant is pollinated. LbCas12a and gRNA are transcribed as part of a single transcript in cells expressed by the promoter. Subsequently, ribozyme-mediated cutting occurs, releasing the gRNA segment. The LbCas12a protein transcribed from the transcript forms a ribonucleoprotein (RNP) with gRNA. RNP will produce double-strand breaks at the target site, and subsequent repair will produce one or more unique mutations in each meiotic cell, egg cell or each embryo produced by pollination. The seeds produced by pollination are screened for target site mutations, and the number and type of mutations produced using constructs 22-27 are compared with the transformed corn plants produced using construct 21.

[0310] Example 5: Generation of mutations by hybridization

[0311] Generate a transgenic corn plant (see Example 2, Table 2) comprising one of constructs 8-13 and grow to the flowering stage. Also generate another transgenic corn plant comprising the gRNA construct of Example 2 and grow to the flowering stage. The corn plant comprising one of constructs 8-13 is hybridized with the corn plant comprising the gRNA construct to generate an offspring corn plant comprising Cas12a, and the gRNA is expressed in the resulting embryo.

[0312] Alternatively, a transgenic corn plant (see Example 3, Table 13) comprising one of constructs 15-20 is generated and grown to the flowering stage.Another transgenic corn plant comprising the Cas12a construct of Example 3 is also generated and grown to the flowering stage. A corn plant comprising one of constructs 15-20 is hybridized with a corn plant comprising a Cas12a construct to generate an offspring corn plant comprising Cas12b, and gRNA is expressed in the resulting embryo.

[0313] Co-expression of Cas12a and gRNA generates a double-strand break within the target site, thereby generating a unique mutation in each cell expressing both components of the CRISPR system. The resulting embryos or plants generated from the resulting embryos are screened to identify mutations at the target site.

[0314] Example 6: Cas12a is expressed in oocytes or embryonic tissues of F1 intercrosses during meiosis to produce different germinal mutations

[0315] Plant R1 seeds containing a T-DNA vector that preferentially expresses Cas12a in maize meiotic tissue, eggs, and / or maize embryo cells as described in Example 1. See Table 15 for a list of constructs.

[0316] Table 15: Expression cassettes that preferentially or solely express Cas12a in maize zygotes or embryos

[0317]

[0318] 30 R1 individuals from each transformation construct were grown on behalf of 4-10 independent events of each construct, and attempts were made to intercross with all individuals. F1 seeds were recovered from 6-29 hybridizations in each direction of each construct. Up to 72 seedlings were sprouted from each F1 spike, the sudden change of the ZmTS1 target site was screened, and the mutation rate (cutting rate) was calculated. Referring to Table 16.

[0319] Table 16: Total events and samples screened in F1 intercross progeny

[0320]

[0321] Taqman assay was performed to determine the copy number of the LbCas12a expression cassette. The total target site mutation rate of all F1s was 0-14% and showed directional expression ( Figure 1 and Table 16).

[0322] The F1 mutation rate was compared to the mutation rate from the event parent (R1) to determine how many new edits were generated (present in the F1 but not in the R1 parent). These data are summarized in Figure 1 The ZmES4:LbCas12a construct showed the highest cleavage rate in female plants (on average 12% of samples contained new edits). The ZmDMC1:LbCas12a construct showed the highest cleavage rate in male plants (on average 14% of samples contained new edits).

[0323] ZmES4:LbCas12a not only produced a high proportion of F1 plants with new edits when carried by the maternal parent, but also generated a large number of unique edits in the F1 plants ( Figure 1, Table 17). When carried by the maternal parent, ZmES4:LbCas12a detected 39 different mutation types in F1 plants. The other promoters tested produced up to 4 unique editing types in 1 progeny (see Figure 1 ).

[0324] The highest abundance of new edits was observed in ZmES4:LbCas12a F1 plants. Sequencing of the target site showed that 40%-100% of the sequencing reads contained mutant alleles, indicating that these edits were performed early in embryonic / zygotic development and were therefore fixed in the plant (Table 17). Edits fixed in plants will appear in the reproductive system of the plant and are therefore inherited in its offspring.

[0325] ZmES4 maintains high embryo / zygotic expression from generation to generation, and many independent events show the functional activity of LbCas12a. Almost every editing plant in each event contains a unique edit (Table 17). In addition, except for one ZmES4 event, all tested events produced new edits, with a mutation rate of up to 20% for each event (Table 17).

[0326] Table 17: Summary of ZmES4::LbCas12a editing in F1 by events

[0327]

[0328] *Total samples with edits include samples with both de novo and heritable edits.

[0329] ** New edits are defined as edits in the F1 that were not detected in the R1 parent.

[0330] $ We defined a heritable edit as an edit that was detected by sequencing with at least 40% of the total sequencing reads from a sample mapping to the edit.

[0331] & Distinct edits refer to the number of unique edits identified.

[0332] Example 7: Expression of Cas12a in corn egg cells, meiotic cells or embryos to achieve editing of the target genome from a haploid induction line.

[0333] Vectors are produced to preferentially express Cas12a in zygotes, embryos, eggs and / or meiotic cells of haploid induction (HI) lines, which produce haploids when hybridized with another line. Non-limiting examples of promoters and regulatory sequences for driving expression embryos, eggs and / or meiotic cells are provided in Table 1. An expression cassette is provided, wherein the plant codon-optimized LbCas12a sequence is flanked by NLS sequences at the 5' and 3' ends, and is optionally connected to the promoter described in Table 1. Each vector may also include an expression cassette encoding one or more Cas12a-gRNAs targeting unique maize genomic target sites; and optionally, an expression cassette encoding a selective marker that confers resistance to the herbicide glyphosate. The vector can be directly converted into a haploid induction line, or an event containing the vector can be produced in different germplasms, which is then hybridized into a haploid induction line. The "editing induction line" produced will include a haploid induction trait and an expression cassette encoding Cas12a, and optionally, a gRNA pointing to the target site. In some embodiments, it is believed that the gRNA can be delivered separately from the expression cassette expressing Cas12a to the haploid inducible line or WT germplasm.

[0334] Editing induced by DNA modifying enzymes such as guide nucleases expressed from the genome of a haploid inducer (e.g., CRISPR / Cas systems) is limited by the efficiency of editing, which may occur shortly after fertilization but before the genome contributed by the haploid inducer is eliminated. In systems where haploid induction is maternally driven, robust expression of genome editing components in the maternal genome before or shortly after fertilization is a desirable feature. Therefore, it is highly desirable to identify and use promoters that are expressed at high levels in maternal tissues (e.g., in egg and embryonic tissues), such as the ZmES4 promoter (see Example 6).

[0335] The induced line (edited induced line) carrying the event containing the above-mentioned expression construct is hybridized as a female plant with the wild-type germplasm of the male plant. After the pollen from the male wild-type parent contacts the female haploid induced parent, there will be a period of time (before fertilization / during fertilization and / or shortly after fertilization) during which the editing mechanism (e.g., CRISPR / Cas system) expressed from the induced parent will contact the genome inherited from the male parent and induce modification. In some embodiments, genome editing occurs when the offspring of the induced hybrid are in the zygote stage of their life cycle. In some embodiments, genome editing takes longer and may span several rounds of mitosis in the offspring plant tissues.

[0336] After editing the wild-type genome, the induction line genome and the expression cassette (e.g., Cas12a and gRNA) encoding the editing mechanism will be lost from the cell in a certain frequency of offspring by a genome elimination mechanism, thereby producing a haploid offspring plant of corresponding frequency, which includes the editor in the male plant genome, but there is no expression cassette encoding the editing mechanism. Haploid offspring can be identified using any method known in the art, including genomic DNA sequencing, molecular marker gene typing and plant or seed marker phenotyping. Haploid offspring can be edited and screened at the target site by any method known in the art (including sequencing of the target site). The haploid edited is selected and then multiplied using any method known in the art (such as cold treatment). Double haploid is a homozygote of the editing allele, which can be used for breeding pipelines.

[0337] A single editing-inducible line can be used to create multiple unique edits in multiple germplasms, thereby alleviating germplasm-dependent transformation limitations. In addition, inducible lines that express editing machinery (e.g., Cas12a and gRNA) in eggs, embryos, and / or meiotic tissues can generate a wide range of unique edits, eliminating the need to create multiple transgenic events to identify specific edits or to generate a range of edited alleles.

[0338] Example 8: TALE activators can promote the expression of LbCas12a in corn protoplasts

[0339] This example describes the use of TALEs to induce robust and specific expression of transcribable polynucleotides in eggs, embryos and / or meiotic plant cells.

[0340] Transcription activator-like effectors (TALEs) are transcription factors containing a C-terminal activation domain that activate / increase the expression of an operably linked transcribable polynucleotide once the TALE binds to a TALE binding site at or near the promoter. To test whether TALEs can promote the expression of Cas12a, several expression cassettes were designed that used a minimal 35S (-46) promoter operably linked to a maize DNAK intron and placed upstream of the Cas12a polynucleotide sequence (see Figure 2). The minimum 35S (-46) promoter has a TATA box, but lacks a transcription factor binding site for inducible gene expression. Three expression constructs were generated, each comprising 35S (-46): Lb. Cas12a box, with one to six TALE protein binding sites (TB) (SEQ ID NO 15), which are located upstream of the minimum 35S (-46) promoter (see Table 7). As a control, the polynucleotide sequence encoding LbCas12a is operably connected to a constitutive ubiquitin promoter. Finally, a separate expression construct comprising a polynucleotide sequence encoding a TALE protein (SEQ ID NO 16) was also produced, and the TALE protein had a C-terminal activation domain (SEQ ID NO 17) operably connected to a full 35S promoter.

[0341] Table 18: Expression cassettes designed for preferential expression of Cas12a in the presence of TALE activating proteins or alone. TB = Tale binding site

[0342]

[0343] The constructs described in Table 18 were transfected into corn leaf protoplasts with and without TALE expression constructs. After 18-24 hours, RNA was isolated and the expression of Cas12a and TALE was quantified using TaqMan assays (see Figure 3 ).

[0344] The data show that in the absence of TALE expression, 1XTB:Cas12a, 3XTB:Cas12a, and 6XTB:Cas12a constructs are less expressed than the Ubq:Cas12a control (see Figure 3 ). However, in the case of TALE expression, high expression of Cas12a was observed, and expression increased with the number of TALE binding sites. The 3XTB:Cas12a and 6XTB:Cas12a constructs showed higher Cas12a expression than the Ubq:Cas12a control group (see Figure 3 ). The data indicate that TALE proteins can not only induce high expression of genes operably linked to TALE binding sites, but also that the expression can be regulated depending on the number of TALE binding sites contained.

[0345] Example 9: TALE-induced meiocyte / embryo / oocyte-preferred expression of LbCas12a

[0346] A potential disadvantage of tissue / cell-preferred promoters is that they are often not stably expressed. This example describes a construct that has been generated that overcomes this limitation and induces robust expression of transcribable polynucleotides such as Cas12a in a tissue / cell-preferred manner.

[0347] Several constructs were generated for expressing TALE-induced Cas12a in eggs, embryos and / or meiotic cells with preference. The generated construct includes a plant codon-optimized LbCas12a coding sequence, which is flanked by 5' and 3' end NLS sequences and is operably connected to an OsLTP transcription termination sequence and a minimum 35S (-46) promoter with 1, 3 or 6 TALE binding sites. An expression construct comprising a TALE coding sequence (SEQ ID NO 16) was also generated, the sequence being operably connected to a promoter expressed in eggs, embryos and / or meiotic cells with preference or alone. Non-limiting examples of promoters and regulatory sequences for driving preference expressed in eggs, embryos and / or meiotic cells are provided in Table 1. An expression cassette comprising a TALE coding sequence operably connected to a constitutive ubiquitin promoter was generated as a control. Under the control of plant Pol III, corn 01DKD2 cultivated embryos were transformed with a vector comprising an expression cassette as described above and an expression cassette encoding a Cas12a-gRNA complementary to a unique corn genomic target site (ZmTS1) and an expression cassette encoding a Cas12a-gRNA complementary to a unique corn genomic target site (ZmTS1) under the control of a plant Pol III promoter and an expression cassette for a selective marker for imparting herbicide glyphosate resistance by Agrobacterium-mediated transformation, and R0 plants were produced from transformed corn cells. Several R0 lines from each transformation construct were grown to maturity, and at least one ear from each transformed corn plant was pollinated. Several R1 lines were selected, germinated and screened for LbCas12a-induced editing in the target site, and the editing rate was calculated. It is expected that when plants comprising the above-mentioned Cas12a, gRNA and TALE expression vectors reach the reproductive stage, TALEs that are preferentially expressed in meiotic ce...

Claims

1. A method for editing a plant genome, comprising: (a) Introducing into plant cells: (i) a first nucleic acid sequence encoding a CRISPR effector protein operably linked to a heterologous first promoter comprising a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 26 or a functional fragment thereof; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein said CRISPR effector protein and at least one guide nucleic acid form a ribonucleoprotein within at least one egg cell or zygote of said plant, and wherein said ribonucleoprotein produces at least one modification within said target sequence in said at least one egg cell or zygote.

2. The method of claim 1, wherein the CRISPR effector protein is selected from the group consisting of Cas9, Cas12a, Cas12b and CasX.

3. The method of claim 1, further comprising hybridizing the plants to produce progeny plants.

4. The method of claim 3, wherein the plant is a haploid inducer line and the progeny plant is haploid.

5. The method of claim 4, further comprising treating cells of the progeny plant with colchicine to produce a doubled haploid plant.

6. A method for editing a plant genome, comprising: (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding a CRISPR effector protein operably linked to a heterologous promoter comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 26 or a functional fragment thereof; and wherein the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) obtaining at least one embryo from the hybridization of step (a), wherein said CRISPR effector protein and said at least one guide nucleic acid form a ribonucleoprotein within at least one egg cell or zygote, and wherein said ribonucleoprotein produces at least one modification within said target sequence in said at least one egg cell or zygote.

7. The method of claim 6, wherein the CRISPR effector protein is selected from the group consisting of Cas9, Cas12a, Cas12b and CasX.

8. The method of claim 6, wherein the heterologous second promoter is a Pol III promoter.

9. A plant produced by the method of claim 1, wherein the plant comprises a modified target sequence.

10. A recombinant DNA construct comprising (a) a first nucleic acid sequence encoding a CRISPR effector protein, which is operably linked to one or more TALE binding sites and a minimal promoter; and (b) a second nucleic acid sequence encoding a TALE, which is operably linked to a promoter comprising a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 26 or a functional fragment thereof, wherein in the absence of TALE bound to the one or more TALE binding sites, the minimal promoter does not drive expression of the DNA modification enzyme.

11. The recombinant DNA construct of claim 10, further comprising a third nucleic acid sequence encoding a guide nucleic acid operably linked to a third promoter.

12. The recombinant DNA construct of claim 10, wherein the one or more TALE binding sites consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TALE binding sites.

13. The recombinant DNA construct of claim 10, wherein the CRISPR effector protein is selected from the group consisting of Cas9, Cas12a, Cas12b and CasX.

14. The recombinant DNA construct of claim 10, wherein the minimal promoter is a 35S (-46) promoter.

15. A plant comprising in its genome the recombinant DNA construct according to claim 10.

16. A recombinant DNA construct comprising (a) a first nucleic acid sequence encoding a CRISPR effector protein; (b) a second nucleic acid sequence encoding a first promoter; and (c) a third nucleic acid sequence encoding a DNA modifying enzyme, which is operably linked to a heterologous second promoter comprising a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 26 or a functional fragment thereof, wherein the third nucleic acid is located between the first nucleic acid and the second nucleic acid, and wherein the third nucleic acid comprises a first target site for the DNA modifying enzyme at the 5' end and a second target site for the DNA modifying enzyme at the 3' end.

17. The recombinant DNA construct of claim 16, further comprising a fourth nucleic acid encoding a guide nucleic acid operably linked to a third promoter.

18. The recombinant DNA construct of claim 16, wherein the CRISPR effector protein is selected from the group consisting of Cas9, Cas12a, Cas12b and CasX.

19. The recombinant DNA construct of claim 16, wherein the first promoter is selected from the group consisting of an OCS promoter, a CaMV 19S promoter, a CaMV 35S promoter, an actin promoter, and an ubiquitin promoter.

20. The recombinant DNA construct of claim 16, wherein the DNA modifying enzyme is Cre recombinase.

21. The recombinant DNA construct of claim 20, wherein the first target site and the second target site are lox sites.

22. The recombinant DNA construct of claim 16, wherein the DNA modifying enzyme is a CRISPR effector protein.

23. The recombinant DNA construct of claim 22, wherein the first target site and the second target site are target sites for a guide nucleic acid.

24. A plant comprising in its genome the recombinant DNA construct of claim 16.

25. A method of producing two or more progeny plants from a single transformed plant cell using unique editing, the method comprising: (a) introducing into the plant cell: (i) a first nucleic acid sequence encoding a CRISPR effector protein operably linked to a heterologous first promoter comprising a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 26 or a functional fragment thereof; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) regenerating a first plant from the plant cell of step (a), wherein the CRISPR effector protein and at least one guide nucleic acid form a ribonucleoprotein in at least one egg cell or zygote of the first plant, and wherein the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one egg cell or zygote; (c) pollinating the first plant of step (b); (d) germinating the two or more seeds produced from step (c) to produce two or more sub-generation plants having unique edits.

26. The method of claim 25, wherein the CRISPR effector protein is selected from the group consisting of: Cas9, Cas12a, Cas12b, and CasX.

27. The method of claim 25, wherein the heterologous second promoter is a PolIII promoter.

28. The method of claim 25, wherein the first plant is a haploid inducer line.

29. The method of claim 25, wherein the first plant is a female plant.

30. A recombinant DNA construct comprising a nucleic acid sequence encoding a DNA modification enzyme, which is operably linked to a heterologous promoter comprising a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 26 or a functional fragment thereof.

31. The recombinant DNA construct of claim 30, wherein the DNA modifying enzyme is Cre recombinase.

32. A plant comprising in its genome the recombinant DNA construct according to claim 30.

33. A plant comprising in its genome the recombinant DNA construct according to claim 31.

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