Increased use of meiosis and germline promoters in gene editing and site-directed integration events.

Abstract

This disclosure relates to compositions and methods related to the expression of inducible nucleases and guide nucleic acids in egg cells and embryonic tissues in plants. [Solution] This disclosure provides methods and compositions for increasing genome editing and site-directed integration events using inducible endonucleases and meiotic cell-preferential, egg cell-preferential, or embryonic tissue-preferential promoters.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 076,705, filed September 10, 2020, which is hereby incorporated by reference in its entirety.

[0002] This disclosure relates to compositions and methods related to the expression of inducible nucleases and guide nucleic acids in egg cells and embryo tissues in plants.

[0003] Incorporation of the Sequence Listing The sequence listing, which is 175,219 bytes (measured in MS - Windows®) and is contained in the file named "P34738WO00_SL.txt" created on September 9, 2021, is electronically submitted herewith and is hereby incorporated by reference in its entirety.

Background Art

[0004] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) nucleases (e.g., Cas12a, CasX, Cas9) are proteins that are guided by guide RNAs to target nucleic acid molecules, and the nuclease can cleave one or both strands of the target nucleic acid molecule.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

[0006] [Non-Patent Document 1] “The American Heritage Science Dictionary” (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York) [Non-Patent Document 2] "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York) [Non-Patent Document 3] "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York) [Non-Patent Document 4] Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition (2012) [Non-Patent Document 5] Current Protocols in Molecular Biology (FM Ausubel et al. (eds.) (1987)) [Non-Patent Document 6] Plant Breeding Methodology (NF Jensen, Wiley-Interscience (1988)) [Non-Patent Document 7] Methods in Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor (eds.) (1995)) [Non-Patent Document 8] Harlow and Lane (eds.) (1988) Antibodies, A Laboratory Manual [Non-Patent Document 9] Animal Cell Culture (RI Freshney (ed.) (1987)) [Non-Patent Document 10] Recombinant Protein Purification: Principles And Methods, 18-1142-75, GE Healthcare Life Sciences [Non-Patent Document 11] CN Stewart, A. Touraev, V. Citovsky, T. Tzfira (eds.) (2011) Plant Transformation Technologies (Wiley-Blackwell) [Non-Patent Document 12] RH Smith (2013) Plant Tissue Culture: Techniques and Experiments (Academic Press, Inc.) [Non-Patent Document 13] PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler (eds.), Cold Spring Harbor Laboratory Press, 1995 [Non-Patent Document 14] Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) [Non-licensed Document 15] Chenna R.ら, "Multiple sequence alignment with the Clustal series of programs", Nucleic Acids Research 31: 3497~3500 pages (2003) [Non-licensed Document 16] Thompson JD, "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 pages (1994) [Non-licensed Document 17] Larkin MAら, "Clustal W and Clustal X version 2.0", Bioinformatics 23: pages 2947~48 (2007) [Non-licensed Document 18] Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool", J. Mol. Biol. 215:403~410 pages (1990) [Non-licensed Document 19] Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001) [Non-licensed Document 20] Zhang and Madden, Genome Res., 1997, 7, pages 649~656 [Non-licensed Document 21] Smith and Waterman, Adv. Appl. Math., 1981, 2, pages 482~489 [Non-licensed Document 22] Schramm and Hernandez, 2002, Genes & Development, 16: 2593~2620 pages [Non-licensed Document 23] Lawton, Plant Molecular Biology (1987) 9: pages 315~324 [Non-licensed Document 24] Odell, Nature (1985) 313: pages 810~812 [Non-licensed Document 25] Yang and Russell, Proceedings of the National Academy of Sciences, USA (1990) 87: 4144~4148 pages [Non-licensed Document 26] Chandler, Plant Cell (1989) 1: 1175~1183 [Non-licensed Document 27] Depicker, Journal of Molecular and Applied Genetics (1982) 1: pages 561~573 [Non-licensed Document 28] Fraley, Proc. Natl. Acad. Sci. USA, 80: 4803~4807 pages (1983) [Non-licensed Document 29] www[dot]kazusa[dot]or[dot]jp[forwards slash]codon [Non-licensed Document 30] Nakamura, 2000, Nucl. Acids Res. 28:292 pages [Non-licensed Document 31] Campbell and Gowri, 1990, Plant Physiol., 92: pages 1~11 [Non-licensed Document 32] Murray et al., 1989, Nucleic Acids Res., 17:477-498. [Non-Patent Document 33] Compendium of Transgenic Crop Plants (2009) Blackwell Publishing [Non-Patent Document 34] www(dot)herbicide(dot)adjuvants(dot)com [Non-Patent Document 35] Kam et al. (2004) Am. Chem. Soc, 126 (22): pp. 6850-6851 [Non-Patent Document 36] Liu et al. (2009) Nano Lett, 9(3): pp. 1007-1010 [Non-Patent Document 37] Khodakovskaya et al. (2009) ACS Nano, 3(10):3221-3227 [Non-Patent Document 38] Gilles LM et al., Curr Biol. 2017 Oct 23;27(20):R1095~R1097 pp. [Non-Patent Document 39] Ravi and Chan. 2010. Nature. 464:615~6190 pages [Non-Patent Document 40] Wang et al., 2018, J. of Integrative Plant Biol, 60:8, pp. 626-631. [Overview of the Initiative] [Means for solving the problem]

[0007] In one embodiment, the disclosure provides a plant comprising (a) a first nucleic acid sequence encoding an inducible nuclease operably linked to a heterologous egg cell-preferential or embryonic tissue-preferential 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 can complex with the inducible nuclease and hybridize to a target sequence in the plant genome, the complex inducing a modification of the target sequence. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the modification is alternating cleavage within a double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgene. In some embodiments, the inducible 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 that is at least 90% identical to Sequence 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 includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the egg cell-preferential promoter is selected from the group consisting of the EA1 promoter and the ES4 promoter. In some embodiments, the egg cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82. In some embodiments, the embryonic tissue-preferential promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter. In some embodiments, the embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the PolIII promoter.In some embodiments, the second promoter is selected from the group consisting of tissue-preferential promoters, tissue-specific promoters, inductive promoters, and constitutive promoters. In some embodiments, the second promoter is a meiotic cell-preferential, egg cell-preferential, or embryonic tissue-preferential 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 the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid includes at least one guide RNA. In some embodiments, a 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 incorporated into the plant genome. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably incorporated into the genome of a haploid-inducible line. Several embodiments relate to seeds produced by the plant. In some embodiments, the seeds contain at least one mutation in the gene of interest containing the target sequence, compared to seeds derived from a control plant of the same variety lacking the first or second nucleic acid sequence.

[0008] In one embodiment, the disclosure provides a plant comprising (a) a first nucleic acid sequence encoding an inducible nuclease operably ligated to a heterologous meiotic cell-preferential promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably ligated to a heterologous second promoter, wherein the at least one guide nucleic acid can complex with the inducible nuclease and hybridize to a target sequence in the plant genome, the complex inducing a modification of the target sequence. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the modification is an alternating cleavage within a double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgene. In some embodiments, the inducible 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 that is at least 90% identical to Sequence 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 includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the meiotic cell-preferential promoter is selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter. In some embodiments, the meiotic cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the second promoter is the PolIII promoter. In some embodiments, the second promoter is selected from the group consisting of the tissue-preferential promoter, the tissue-specific promoter, the inductive promoter, and the constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferential, egg cell-preferential, or embryonic tissue-preferential 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 the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid includes at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably ligated to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably incorporated into the plant genome. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably incorporated into the genome of the haploid-inducible line.

[0009] In one embodiment, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease operably linked to a heterologous oocyte-preferential 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 can complex with the inducible nuclease and hybridize to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one oocyte of the plant, and the ribonucleoprotein generates at least one modification in the target sequence in the at least one oocyte. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the modification is an alternating cleavage within a double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgene. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical 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 includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the egg cell-preferential promoter is selected from the group consisting of the EA1 promoter and the ES4 promoter. In some embodiments, the egg cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the PolIII promoter.In some embodiments, the second promoter is selected from the group consisting of tissue-preferential promoters, tissue-specific promoters, inductive promoters, and constitutive promoters. In some embodiments, the second promoter is a meiotic cell-preferential, egg cell-preferential, or embryonic tissue-preferential 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 the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid includes at least one guide RNA. In some embodiments, a 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 incorporated into the plant genome. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably incorporated into the genome of a haploid-inducible line.

[0010] In one embodiment, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease operably linked to a heterologous meiotic cell-preferential 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 can complex with the inducible nuclease and hybridize to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one meiotic cell of the plant, and the ribonucleoprotein generates at least one modification in the target sequence in the at least one meiotic cell. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the modification is an alternating cleavage within a double-stranded DNA molecule of the genome. In some embodiments, the modification is the insertion of a transgene. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical 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 includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the meiotic cell-preferential promoter is selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter. In some embodiments, the meiotic cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the second promoter is the PolIII promoter.In some embodiments, the second promoter is selected from the group consisting of tissue-preferential promoters, tissue-specific promoters, inductive promoters, and constitutive promoters. In some embodiments, the second promoter is a meiotic cell-preferential, egg cell-preferential, or embryonic tissue-preferential 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 the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid includes at least one guide RNA. In some embodiments, a second nucleic acid sequence encoding at least one guide nucleic acid is operably ligated 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 plant genome. In some embodiments, meiotic cells are derived from haploid-inducible lines.

[0011] In one embodiment, the present disclosure provides a method for editing the genome of a plant cell, comprising the steps of (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease operably linked to a heterologous embryo-preferential promoter, and 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 can complex with the inducible nuclease and hybridize to a target sequence in the genome; and (b) obtaining at least one embryo from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one embryo, and the ribonucleoprotein generates at least one modification in a target sequence in at least one embryo. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the modification is an alternating 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 inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical 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 includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the embryonic tissue-preferential promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter. In some embodiments, the embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the PolIII promoter.In some embodiments, the second promoter is selected from the group consisting of tissue-preferential promoters, tissue-specific promoters, inductive promoters, and constitutive promoters. In some embodiments, the second promoter is a meiotic cell-preferential, egg cell-preferential, or embryonic tissue-preferential 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 the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid includes at least one guide RNA. In some embodiments, a 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 incorporated into the genome of a first plant. In some embodiments, the first plant is a hybrid derivative.

[0012] In one embodiment, the present disclosure provides a method for editing the genome of a plant cell, comprising the steps of (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease operably linked to a heterologous meiotic cell-preferential promoter, and 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 can complex with the inducible nuclease and hybridize to a target sequence in the genome; and (b) obtaining at least one embryo from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one meiotic cell, and the ribonucleoprotein generates at least one modification in the target sequence in at least one meiotic cell. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the modification is an alternating cleavage within a double-stranded DNA molecule of the genome. In some embodiments, the modification is the insertion of a transgene. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical 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 includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the meiotic cell-preferential promoter is selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter. In some embodiments, the meiotic cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the second promoter is the PolIII promoter.In some embodiments, the second promoter is selected from the group consisting of tissue-preferential promoters, tissue-specific promoters, inductive promoters, and constitutive promoters. In some embodiments, the second promoter is a meiotic cell-preferential, egg cell-preferential, or embryonic tissue-preferential 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 the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid includes at least one guide RNA. In some embodiments, a 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 incorporated into the plant genome. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably incorporated into the genome of a haploid-inducible line.

[0013] In one embodiment, the present disclosure provides a method for editing a plant genome, comprising the steps of (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease operably linked to a heterologous embryo-preferential 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 can complex with the inducible nuclease and hybridize to a target sequence in the genome; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing at least one plant to produce at least one embryo, wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one embryo derived from step (c), and the ribonucleoprotein generates at least one modification in the target sequence in at least one embryo. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the modification is an alternating break within the double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgene. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical 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 includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the embryonic tissue-preferential promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter. In some embodiments, the embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 PolIII promoter.In some embodiments, the second promoter is selected from the group consisting of tissue-preferential promoters, tissue-specific promoters, inductive promoters, and constitutive promoters. In some embodiments, the second promoter is a meiotic cell-preferential, egg cell-preferential, or embryonic tissue-preferential 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 the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid includes at least one guide RNA. In some embodiments, a 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 incorporated into the genome of a first plant. In some embodiments, the first plant is a hybrid derivative.

[0014] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease operably linked to a heterologous egg cell-preferential promoter; (ii) one or more guide nucleic acids operably linked to a heterologous second promoter, which can (A) form a complex with the inducible nuclease and hybridize to a target sequence in the plant genome; and (B) hybridize to a first and second site flanking the nucleic acid sequence encoding the gene of interest; The method provides a step of (iii) introducing a second nucleic acid sequence encoding one or more guide nucleic acids; (iii) introducing a third nucleic acid sequence encoding a gene of interest; and (b) regenerating at least one plant from the plant cells of step (a); wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one egg cell of the plant, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest is incorporated into the target site in at least one egg cell. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the modification is alternating breaks in the double-stranded DNA molecule of the genome. In some embodiments, the modification is an insertion of a transgene. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a) and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical to sequence number 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 includes a nucleic acid sequence selected from the group consisting of sequence numbers 8 and 9. In some embodiments, the egg cell-preferential promoter is selected from the group consisting of the EA1 promoter and the ES4 promoter. In some embodiments, the egg cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of sequence numbers 2-3, 21-38, 41-45, and 65-82.In some embodiments, the second promoter is the PolIII promoter. In some embodiments, the second promoter is selected from the group consisting of tissue-preferential promoters, tissue-specific promoters, inductive promoters, and constitutive promoters. In some embodiments, the second promoter is a meiotic cell-preferential, oocyte-preferential, or embryonic tissue-preferential promoter. In some embodiments, the second promoter is a meiotic cell-specific, oocyte-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the 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 second nucleic acid sequence encoding at least one guide nucleic acid is operably ligated 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 plant genome. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of a haploid-inducible line.

[0015] In one embodiment, the present disclosure provides a method for generating site-directed integration in a plant, comprising: (a) a plant cell containing (i) a first nucleic acid sequence encoding an inducible nuclease operably linked to a heterologous embryo-preferential promoter; (ii) one or more guide nucleic acids that can (A) form a complex with the inducible nuclease and hybridize to a target sequence in the plant genome; and (B) one or more guide nucleic acids operably linked to a heterologous second promoter that can hybridize to a first and second site flanking the nucleic acid sequence encoding the gene of interest; The present invention provides a method comprising: (iii) introducing a second nucleic acid sequence; (b) introducing a third nucleic acid sequence encoding a gene of interest; (c) regenerating at least one plant from the plant cells of step (a); and (d) fertilizing at least one plant derived from step (b) to produce at least one embryo; wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one embryo, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest is incorporated into the target site in at least one embryo. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the modification is alternating breaks in the double-stranded DNA molecule of the genome. In some embodiments, the modification is the insertion of a transgene. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical 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 includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the embryonic tissue-preferential promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter.In some embodiments, the embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the PolIII promoter. In some embodiments, the second promoter is selected from the group consisting of tissue-preferential promoters, tissue-specific promoters, inductive promoters, and constitutive promoters. In some embodiments, the second promoter is a meiotic cell-preferential, oocyte-preferential, or embryonic tissue-preferential promoter. In some embodiments, the second promoter is a meiotic cell-specific, oocyte-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the CaMV 35S promoter, the actin promoter, the Rab15 promoter, and the ubiquitin promoter. In some embodiments, the at least one guide nucleic acid includes at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably ligated to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably incorporated into the genome of a first plant. In some embodiments, the first plant is a hybrid derivative.

[0016] In one embodiment, the disclosure provides a recombinant DNA construct comprising (a) a first nucleic acid sequence encoding an inducible nuclease operably ligated to a heterologous oocyte-preferential promoter, a meiotic cell-preferential promoter, or an embryonic tissue-preferential promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably ligated to a heterologous second promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the plant genome. In some embodiments, the inducible 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 that is at least 90% identical 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 egg cell-preferential promoter is selected from the group consisting of the EA1 promoter and the ES4 promoter. In some embodiments, the egg cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82. In some embodiments, the embryonic tissue-preferential promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter. In some embodiments, the embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88. In some embodiments, the meiotic cell-preferential promoter is selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter. In some embodiments, the meiotic cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85.In some embodiments, the second promoter is the PolIII promoter. In some embodiments, the second promoter is selected from the group consisting of tissue-preferential promoters, tissue-specific promoters, inductive promoters, and constitutive promoters. In some embodiments, the second promoter is a meiotic cell-preferential, oocyte-preferential, or embryonic tissue-preferential promoter. In some embodiments, the second promoter is a meiotic cell-specific, oocyte-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the 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 second nucleic acid sequence encoding at least one guide nucleic acid is operably ligated to one or more self-cleaving ribozymes. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably incorporated into the plant genome. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both are stably incorporated into the genome of a haploid-inducible line. In some embodiments, a recombinant DNA construct is incorporated into the genome of a haploid-inducible line.

[0017] Some embodiments relate to a recombinant DNA construct comprising (a) a first nucleic acid sequence encoding a DNA-modifying enzyme operably ligated to one or more TALE binding sites and a minimal promoter; and (b) a second nucleic acid sequence encoding a TALE operably ligated to an egg-preferential promoter, a meiotic-preferential promoter, or an embryonic-preferential promoter, wherein the minimal promoter does not drive the expression of the DNA-modifying enzyme in the absence of a TALE bound 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 operably ligated to a third promoter. In some embodiments, the DNA-modifying enzyme is an inducible nuclease. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the first nucleic acid sequence encoding the DNA-modifying enzyme and the minimal promoter is operably ligated to one, two, three, four, five, six, seven, eight, nine, ten or more TALE binding sites. In some embodiments, the CRISPR effector protein is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical 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 includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the egg cell-preferential promoter is selected from the group consisting of the EA1 promoter and the ES4 promoter. In some embodiments, the egg cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82. In some embodiments, the embryonic tissue-preferential promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter.In some embodiments, the embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88. In some embodiments, the meiotic cell-preferential promoter is selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter. In some embodiments, the meiotic cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the third promoter is the PolIII promoter. In some embodiments, the third promoter is selected from the group consisting of the tissue-preferential promoter, the tissue-specific promoter, the inductive promoter, and the constitutive promoter. In some embodiments, the third promoter is a meiotic cell-preferential, oocyte-preferential, or embryonic tissue-preferential promoter. In some embodiments, the third promoter is a meiotic cell-specific, oocyte-specific, or embryonic tissue-specific promoter. In some embodiments, the third promoter is selected from the group consisting of the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the third promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88. In some embodiments, the third promoter is a constitutive promoter selected from the group consisting of the CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid includes at least one guide RNA. In some embodiments, the third nucleic acid sequence encoding at least one guide nucleic acid is operably ligated 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 incorporated into the plant genome. In some embodiments, the plant is derived from a haploid-inducible line.In some embodiments, guide nucleic acids are provided to the plant by bombardment. Some embodiments relate to a plant containing a recombinant DNA construct. Some embodiments relate to seeds produced by a plant containing a recombinant DNA construct. In some embodiments, the recombinant DNA construct is incorporated into the genome of a haploid-inducible line.

[0018] Some embodiments relate to a recombinant DNA construct comprising (a) a first nucleic acid sequence encoding at least one guide nucleic acid operably ligated to one or more TALE binding sites and a minimal promoter; and (b) a second nucleic acid sequence encoding a TALE operably ligated to an egg cell-preferential promoter, a meiotic cell-preferential promoter, or an embryonic tissue-preferential promoter, wherein the minimal promoter does not drive the expression of the guide nucleic acid in the absence of a TALE bound to one or more TALE binding sites. In some embodiments, the recombinant DNA construct further comprises a third nucleic acid sequence encoding an inducible nuclease operably ligated to a third promoter. In some embodiments, the inducible 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 ligated 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 group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the third nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical 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 includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the egg cell-preferential promoter is selected from the group consisting of the EA1 promoter and the ES4 promoter. In some embodiments, the egg cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82. In some embodiments, the embryonic tissue-preferential promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter.In some embodiments, the embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88. In some embodiments, the meiotic cell-preferential promoter is selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter. In some embodiments, the meiotic cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the third promoter is selected from the group consisting of the tissue-preferential promoter, the tissue-specific promoter, the inductive promoter, and the constitutive promoter. In some embodiments, the third promoter is a meiotic cell-preferential, oocyte-preferential, or embryonic tissue-preferential promoter. In some embodiments, the third promoter is a meiotic cell-specific, oocyte-specific, or embryonic tissue-specific promoter. In some embodiments, the third promoter is selected from the group consisting of the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the third promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88. In some embodiments, the third promoter is a constitutive promoter selected from the group consisting of the CaMV 35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. In some embodiments, at least one guide nucleic acid includes at least one guide RNA. In some embodiments, the first nucleic acid sequence encoding at least one guide nucleic acid is operably ligated 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 incorporated into the plant genome. In some embodiments, the plant is derived from a haploid-inducible line. Some embodiments relate to plants comprising recombinant DNA constructs.Some embodiments relate to seeds produced by plants containing recombinant DNA constructs. In some embodiments, the recombinant DNA constructs are incorporated into the genome of haploid-inducible lines.

[0019] Some embodiments relate to a recombinant DNA construct comprising (a) a first nucleic acid sequence encoding an inducible nuclease; (b) a second nucleic acid sequence encoding a first promoter; and (c) a third nucleic acid sequence encoding a DNA-modifying enzyme operably linked to an egg-preferential promoter, a meiotic cell-preferential promoter, or an embryonic tissue-preferential promoter, wherein the third nucleic acid is located between the first and second nucleic acids, and the third nucleic acid includes a first target site for the DNA-modifying enzyme at its 5' end and a second target site for the DNA-modifying enzyme at its 5' end. In some embodiments, the recombinant DNA construct further comprises a fourth nucleic acid sequence encoding one or more guide nucleic acids operably linked to the third promoter. In some embodiments, the DNA-modifying enzyme is a recombinase. In some embodiments, the first and second target sites are Lox sites. In some embodiments, the DNA-modifying enzyme is an endonuclease. In some embodiments, the DNA-modifying enzyme is a CRISPR effector protein. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical 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 includes 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-preferential, egg cell-preferential, or embryonic tissue-preferential promoter. In some embodiments, the first promoter is selected from the group consisting of the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter.In some embodiments, the first promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88. In some embodiments, the first promoter is a constitutive promoter selected from the group consisting of the CaMV 35S promoter, the actin promoter, the Rab15 promoter, and the ubiquitin promoter. In some embodiments, at least one guide nucleic acid includes at least one guide RNA. In some embodiments, the egg cell-preferential promoter is selected from the group consisting of the EA1 promoter and the ES4 promoter. In some embodiments, the egg cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82. In some embodiments, the embryonic tissue-preferential promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter. In some embodiments, the embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88. In some embodiments, the meiotic cell-preferential promoter is selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter. In some embodiments, the meiotic cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the third promoter is the PolIII promoter. Some embodiments relate to plants containing recombinant DNA constructs. Some embodiments relate to seeds produced by plants containing recombinant DNA constructs. In some embodiments, the plants are derived from haploid-inducible lines. In some embodiments, the recombinant DNA constructs are incorporated into the genome of the haploid-inducible line.

[0020] In some embodiments, high levels of DNA-modifying enzymes, such as inducible nucleases (e.g., CRISPR / Cas systems), in egg, embryo, and / or meiotic tissue-specific expression are achieved by providing plant cells with: 1) an expression construct comprising a promoter listed in Table 1, operably linked to a sequence encoding a CRISPR effector protein, such as dCas12a or dCas9 fused to a transcription activator; 2) an expression construct comprising one or more target sites operably linked to a minimal promoter and a sequence encoding a DNA-modifying enzyme; and 3) an expression construct encoding a guide RNA that hybridizes with one or more target sites; and generating plants therefrom. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten or more target sites are operably linked to the minimal promoter. In some embodiments, the plant cells are derived from haploid-inducible lines. In some embodiments, female plants expressing high levels of DNA-modifying enzymes in eggs, embryos, and / or meiotic tissue are generated. In some embodiments, female plants are crossbred to identify a population of R1 plants containing unique edits.

[0021] In some embodiments, high levels of egg, embryo, and / or meiotic tissue-specific expression of DNA-modifying enzymes such as inducible nucleases (e.g., CRISPR / Cas systems) are achieved by providing plant cells with an expression construct comprising 1) a promoter described in Table 1 operably linked to a sequence encoding a TALE, and 2) an expression construct comprising a minimal promoter and one or more TALE-binding sites (TBs) operably linked to a sequence encoding a DNA-modifying enzyme; and generating plants therefrom. In some embodiments, an expression construct encoding one or more guide nucleic acids is further provided. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten or more TBs are operably linked to the minimal promoter. In some embodiments, the plant cells are derived from haploid-inducible lines. In some embodiments, female plants expressing high levels of DNA-modifying enzymes in eggs, embryos, and / or meiotic tissue are generated. In some embodiments, the female plants are crossbred to identify a population of R1 plants containing unique editing.

[0022] Some embodiments of the method provide a way to produce two or more offspring plants having unique edits from a single transformed plant cell, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease operably linked to a heterologous meiotic preferential 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 can hybridize to a target sequence in the genome; and (b) regenerating a first plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one meiotic cell of the first plant, and the ribonucleoprotein generates at least one modification in the target sequence in the at least one meiotic cell; (c) pollinating the first plant of step (b); and (d) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique edits. In some embodiments, the modification is an alternating break within a double-stranded DNA molecule of the genome. In some embodiments, the target sequence includes gene DNA. In some embodiments, the target sequence includes intergene DNA. In some embodiments, the target sequence is within the gene of interest. In some embodiments, the gene of interest encodes a protein or non-protein-coding RNA. In some embodiments, the gene of interest encodes a non-protein-coding RNA selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, or its precursor. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical to Sequence 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, at least one nuclear localization signal includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the meiotic cell-preferential promoter is selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter. In some embodiments, the meiotic cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the second promoter is the PolIII promoter. In some embodiments, the second promoter is selected from the group consisting of the tissue-preferential promoter, the tissue-specific promoter, the inductive promoter, and the constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferential, oocyte-preferential, or embryonic tissue-preferential promoter. In some embodiments, the second promoter is a meiotic cell-specific, oocyte-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, the DMC1 promoter, the Mps1 promoter, the Adf1 promoter, and the EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the CaMV 35S promoter, the actin promoter, the Rab15 promoter, and the ubiquitin promoter. In some embodiments, the at least one guide nucleic acid includes at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably ligated to one or more self-cleaving ribozymes. In some embodiments, the first plant is self-pollinated. In some embodiments, the first plant is cross-pollinated. In some embodiments, the plant cells are derived from a haploid-inducible line. In some embodiments, the offspring plants are haploid. In some embodiments, the method further includes the step of screening haploid offspring for modification at a target site.In some embodiments, the method further includes the step of inducing genome duplication of a haploid plant.

[0023] Some embodiments of the method provide a way to produce two or more offspring plants having unique edits from a single transformed plant cell, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease operably linked to a first heterologous promoter; and a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous meiotic preferential promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; and (b) regenerating a first plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one meiotic cell of the first plant, and the ribonucleoprotein generates at least one modification in the target sequence in the at least one meiotic cell; (c) pollinating the first plant of step (b); and (d) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique edits. In some embodiments, the modification is an alternating break within a double-stranded DNA molecule of the genome. In some embodiments, the target sequence includes gene DNA. In some embodiments, the target sequence includes intergene DNA. In some embodiments, the target sequence is within the gene of interest. In some embodiments, the gene of interest encodes a protein or non-protein-coding RNA. In some embodiments, the gene of interest encodes a non-protein-coding RNA selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, or its precursor. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical to Sequence 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, at least one nuclear localization signal includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9. In some embodiments, the meiotic cell-preferential promoter is selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter. In some embodiments, the meiotic cell-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the first promoter is selected from the group consisting of the tissue-preferential promoter, the tissue-specific promoter, the inductive promoter, and the constitutive promoter. In some embodiments, the first promoter is a meiotic cell-preferential, oocyte-preferential, or embryonic tissue-preferential promoter. In some embodiments, the first promoter is a meiotic cell-specific, oocyte-specific, or embryonic tissue-specific promoter. In some embodiments, the first promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, the DMC1 promoter, the Mps1 promoter, the Adf1 promoter, and the EAL1 promoter. In some embodiments, the first promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88. In some embodiments, the first promoter is a constitutive promoter selected from the group consisting of the CaMV 35S promoter, the actin promoter, the Rab15 promoter, and the ubiquitin promoter. In some embodiments, the at least one guide nucleic acid includes at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably ligated 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 cells are derived from a haploid-inducible line. In some embodiments, the offspring plants are haploid. In some embodiments, the method further includes the step of screening haploid offspring for modification at a target site.In some embodiments, the method further includes the step of inducing genome duplication of a haploid plant.

[0024] A method for generating two or more offspring plants having unique edits from a single transformed plant cell, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease operably linked to a heterologous oocell-preferential 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 can hybridize to a target sequence in the genome; and (b) regenerating a first plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one oocell of the first plant, and the ribonucleoprotein generates at least one modification in the target sequence in at least one oocell; (c) pollinating the first plant of step (b); and (d) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique edits. In some embodiments, the modification is an alternating break within a double-stranded DNA molecule of the genome. In some embodiments, the target sequence includes gene DNA. In some embodiments, the target sequence includes intergene DNA. In some embodiments, the target sequence is within the gene of interest. In some embodiments, the gene of interest encodes a protein or non-protein-coding RNA. In some embodiments, the gene of interest encodes a non-protein-coding RNA selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, or its precursor. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical to Sequence 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, at least one nuclear localization signal includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9.In some embodiments, the oocyte-preferential promoter is selected from the group consisting of the EA1 promoter and the ES4 promoter. In some embodiments, the oocyte-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the PolIII promoter. In some embodiments, the second promoter is selected from the group consisting of the tissue-preferential promoter, tissue-specific promoter, inductive promoter, and constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferential, oocyte-preferential, or embryonic tissue-preferential promoter. In some embodiments, the second promoter is a meiotic cell-specific, oocyte-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the CaMV 35S promoter, the actin promoter, the Rab15 promoter, and the ubiquitin promoter. In some embodiments, the at least one guide nucleic acid includes at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably ligated to one or more self-cleaving ribozymes. In some embodiments, the first plant is self-pollinated. In some embodiments, the first plant is cross-pollinated. In some embodiments, the plant cells are derived from a haploid-inducing line. In some embodiments, the offspring plants are haploid. In some embodiments, the method further includes the step of screening haploid offspring for modifications at a target site. In some embodiments, the method further includes the step of inducing genome duplication of a haploid plant.

[0025] A method for generating two or more offspring plants having unique edits from a single transformed plant cell, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease operably linked to a first heterologous promoter; and a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous oocyte-preferential promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; and (b) regenerating a first plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one oocyte of the first plant, and the ribonucleoprotein generates at least one modification in the target sequence in at least one oocyte; (c) pollinating the first plant of step (b); and (d) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique edits. In some embodiments, the modification is an alternating break within a double-stranded DNA molecule of the genome. In some embodiments, the target sequence includes gene DNA. In some embodiments, the target sequence includes intergene DNA. In some embodiments, the target sequence is within the gene of interest. In some embodiments, the gene of interest encodes a protein or non-protein-coding RNA. In some embodiments, the gene of interest encodes a non-protein-coding RNA selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, or its precursor. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical to Sequence 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, at least one nuclear localization signal includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9.In some embodiments, the oocyte-preferential promoter is selected from the group consisting of the EA1 promoter and the ES4 promoter. In some embodiments, the oocyte-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82. In some embodiments, the first promoter is selected from the group consisting of the tissue-preferential promoter, the tissue-specific promoter, the inductive promoter, and the constitutive promoter. In some embodiments, the first promoter is a meiotic cell-preferential, oocyte-preferential, or embryonic tissue-preferential promoter. In some embodiments, the first promoter is a meiotic cell-specific, oocyte-specific, or embryonic tissue-specific promoter. In some embodiments, the first promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, the DMC1 promoter, the Mps1 promoter, the Adf1 promoter, and the EAL1 promoter. In some embodiments, the first promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88. In some embodiments, the first promoter is a constitutive promoter selected from the group consisting of the CaMV 35S promoter, the actin promoter, the Rab15 promoter, and the ubiquitin promoter. In some embodiments, at least one guide nucleic acid includes at least one guide RNA. In some embodiments, a second nucleic acid sequence encoding at least one guide nucleic acid is operably ligated to one or more self-cleaving ribozymes. In some embodiments, the first plant is self-pollinated. In some embodiments, the first plant is cross-pollinated. In some embodiments, the plant cells are derived from a haploid-inducing line. In some embodiments, the offspring plants are haploid. In some embodiments, the method further includes the step of screening haploid offspring for modifications at a target site. In some embodiments, the method further includes the step of inducing genome duplication of a haploid plant.

[0026] A method for producing two or more offspring plants having unique edits from a single transformed plant cell, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease operably linked to a heterologous embryonic-preferential 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 can hybridize to a target sequence in the genome; (b) regenerating a first plant from the plant cell of step (a); (c) pollinating the first plant of step (b) such that the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the embryonic cell, the ribonucleoprotein generating at least one modification in the target sequence in the embryonic cell; and (d) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique edits. In some embodiments, the modification is an alternating break within a double-stranded DNA molecule of the genome. In some embodiments, the target sequence includes gene DNA. In some embodiments, the target sequence includes intergenetic DNA. In some embodiments, the target sequence is located within the gene of interest. In some embodiments, the gene of interest encodes a protein or non-protein-coding RNA. In some embodiments, the gene of interest encodes non-protein-coding RNA selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, or its precursors. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical 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 includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9.In some embodiments, the embryonic tissue-preferential promoter is selected from the group consisting of the DSUL1 promoter, EA1 promoter, ES4 promoter, and EAL1 promoter. In some embodiments, the embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the PolIII promoter. In some embodiments, the second promoter is selected from the group consisting of the tissue-preferential promoter, tissue-specific promoter, inductive promoter, and constitutive promoter. In some embodiments, the second promoter is a meiotic cell-preferential, oocyte-preferential, or embryonic tissue-preferential promoter. In some embodiments, the second promoter is a meiotic cell-specific, oocyte-specific, or embryonic tissue-specific promoter. In some embodiments, the second promoter is selected from the group consisting of the DSUL1 promoter, EA1 promoter, ES4 promoter, DMC1 promoter, Mps1 promoter, Adf1 promoter, and EAL1 promoter. In some embodiments, the second promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence 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 the CaMV 35S promoter, the actin promoter, the Rab15 promoter, and the ubiquitin promoter. In some embodiments, the at least one guide nucleic acid includes at least one guide RNA. In some embodiments, the second nucleic acid sequence encoding at least one guide nucleic acid is operably ligated to one or more self-cleaving ribozymes. In some embodiments, the first plant is self-pollinated. In some embodiments, the first plant is cross-pollinated. In some embodiments, the plant cells are derived from a haploid-inducing line. In some embodiments, the offspring plants are haploid. In some embodiments, the method further includes the step of screening haploid offspring for modifications at a target site. In some embodiments, the method further includes the step of inducing genome duplication of a haploid plant.

[0027] A method for generating two or more offspring plants having unique edits from a single transformed plant cell, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease operably linked to a first heterologous promoter; and a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous embryonic cell-preferential promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; (b) regenerating a first plant from the plant cell of step (a); (c) pollinating the first plant of step (b) such that the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the embryonic cell, the ribonucleoprotein generating at least one modification in the target sequence in the embryonic cell; and (d) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique edits. In some embodiments, the modification is an alternating break in a double-stranded DNA molecule of the genome. In some embodiments, the target sequence includes gene DNA. In some embodiments, the target sequence includes intergenetic DNA. In some embodiments, the target sequence is located within the gene of interest. In some embodiments, the gene of interest encodes a protein or non-protein-coding RNA. In some embodiments, the gene of interest encodes non-protein-coding RNA selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, or its precursors. In some embodiments, the inducible nuclease is a CRISPR effector protein. In some embodiments, the inducible nuclease is selected from the group consisting of Cas9, Cas12a (e.g., LbCas12a, FnCas12a), and CasX. In some embodiments, the first nucleic acid sequence includes a nucleic acid sequence that is at least 90% identical 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 includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9.In some embodiments, the embryonic tissue-preferential promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter. In some embodiments, the embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88. In some embodiments, the first promoter is selected from the group consisting of the tissue-preferential promoter, the tissue-specific promoter, the inductive promoter, and the constitutive promoter. In some embodiments, the first promoter is a meiotic cell-preferential, oocyte-preferential, or embryonic tissue-preferential promoter. In some embodiments, the first promoter is a meiotic cell-specific, oocyte-specific, or embryonic tissue-specific promoter. In some embodiments, the first promoter is selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, the DMC1 promoter, the Mps1 promoter, the Adf1 promoter, and the EAL1 promoter. In some embodiments, the first promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, 21-45, and 65-88. In some embodiments, the first promoter is a constitutive promoter selected from the group consisting of the CaMV 35S promoter, the actin promoter, the Rab15 promoter, and the ubiquitin promoter. In some embodiments, at least one guide nucleic acid includes at least one guide RNA. In some embodiments, a second nucleic acid sequence encoding at least one guide nucleic acid is operably ligated to one or more self-cleaving ribozymes. In some embodiments, the first plant is self-pollinated. In some embodiments, the first plant is cross-pollinated. In some embodiments, the plant cells are derived from a haploid-inducing line. In some embodiments, the offspring plants are haploid. In some embodiments, the method further includes the step of screening haploid offspring for modifications at a target site. In some embodiments, the method further includes the step of inducing genome duplication of a haploid plant. [Brief explanation of the drawing]

[0028] [Figure 1] Editing in forward and reverse F1 plants using a reproductive promoter. Transgenic R1 lines were used as female or male to generate forward and reverse F1s. Black bars represent the percentage of events that exhibit active LbCas12a when provided by the female or male parent, as indicated by the new edits present in the F1 plants. Gray bars represent the percentage of F1 individuals containing new edits. Light gray bars represent the number of unique edits found in the F1 generation. [Figure 2] A vector designed for TALE-inducible expression of LbCas12a is shown. 35S(-46) is the 35S minimal promoter. TB indicates the TALE binding site. [Figure 3] RNA expression of Cas12a and TALE in maize leaf protoplasts. [Figure 4] A T-DNA vector designed for TALE-induced meiotic cell / embryo / egg cell preferential expression of LbCas12a in plant tissue is illustrated. LB indicates the left boundary. RB indicates the right boundary. P indicates the promoter. 35S(-46) is the 35S minimal promoter. TB is the TALE binding site. [Figure 5] A T-DNA vector designed for preferential expression of Cas12a in meiotic cells / embryos / eggs, driven by a strong constitutive promoter, is illustrated. P indicates the promoter. Cre refers to Cre recombinase. Arrowheads indicate directionality. [Modes for carrying out the invention]

[0029] Unless otherwise defined, all technical and scientific terms used have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Where a term is provided in the singular form, the inventors also intend aspects of this disclosure described by that term in the plural form. Where there are differences in terms and definitions used in references incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their common meanings in the industry in which they are used, as exemplified by various industry-specific dictionaries, such as "The American Heritage® Science Dictionary" (Editors of the American Heritage Dictionaries, 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 limit themselves to mechanisms or modes of operation. The references are provided for illustrative purposes only.

[0030] Unless otherwise noted, the implementation of this disclosure includes, but is not limited to, prior art in biochemistry, chemistry, molecular biology, microbiology, cell biology, plant biology, genomics, biotechnology, and genetics, which are within the scope of the art in this field. For example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition (2012); Current Protocols In Molecular Biology (FM Ausubel et al. (eds.) (1987)); Plant Breeding Methodology (NF Jensen, Wiley-Interscience (1988)); Methods In Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor (eds.) (1995)); Harlow and Lane (eds.) (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; CN Stewart, A. Touraev, V. Citovsky, T. Tzfira (eds.) (2011) Plant Transformation Technologies See Wiley-Blackwell and RH Smith (2013) Plant Tissue Culture: Techniques and Experiments (Academic Press, Inc.).

[0031] For example, all references cited herein, including all patents, published patent applications, and non-patent publications, are incorporated herein by reference in their entirety.

[0032] When a set of options is presented, all possible combinations of the members constituting that set of options are specifically envisioned. For example, if an item is selected from a group consisting of A, B, C, and D, the inventors specifically envision each option individually (e.g., A only, B only, etc.), as well as combinations such as A, B, and D; A and C; B and C, etc.

[0033] As used herein, singular terms and singular forms of “a,” “an,” and “the” refer to multiple subjects, for example, unless otherwise explicitly indicated by the text.

[0034] Any composition, nucleic acid molecule, polypeptide, cell, plant, etc., provided herein is specifically intended for use in conjunction with any method provided herein.

[0035] Some embodiments described herein relate to compositions and methods for preferentially expressing DNA-modifying enzymes, such as inducible nucleases, in plant eggs, meiotic cells, and / or embryonic cells. Some embodiments provide compositions and methods for preferentially expressing components of a CRISPR / Cas editing system in plant eggs, meiotic cells, and / or embryonic cells. Some embodiments relate to compositions and methods for producing offspring having unique edits from a parent comprising an expression cassette that preferentially provides a DNA-modifying enzyme, such as an inducible nuclease, in eggs, meiotic cells, and / or embryonic cells. Some embodiments provide a female parent plant that preferentially expresses a DNA-modifying enzyme, such as an inducible nuclease, in eggs, meiotic cells, and / or embryonic cells. Some embodiments provide a male parent plant that preferentially expresses a DNA-modifying enzyme, such as an inducible nuclease, in eggs, meiotic cells, and / or embryonic cells. In some embodiments, a population of seeds is provided in which two or more seeds contain unique edits, and which are preferentially produced in eggs, meiosis, and / or embryonic cells from a parent expressing a DNA-modifying enzyme, such as an inducible nuclease. Non-limiting examples of expression elements useful in the compositions and methods described herein are provided in Table 1.

[0036] As used herein, “egg cell” refers to a haploid egg cell produced by the female gametophyte of a plant. Upon fertilization by a haploid pollen cell, a diploid zygote is formed, giving rise to an embryo. As used herein, “embryonic tissue” refers to the precursor tissues for leaf, stem, and root tissues, as well as diploid tissues containing one or more cotyledons. The embryonic tissue is eventually incorporated into the seed. Once the embryo begins to germinate, a seedling or plant body is produced. As used herein, meiosis refers to the process of cell division in sexually reproducing organisms that produce gametes. Meiosis consists of two cell divisions that ultimately produce four haploid cells. Meiotic cells refer to cells undergoing meiosis.

[0037] In one aspect, the disclosure provides a recombinant DNA construct comprising (b) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous egg cell-preferential 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 can hybridize to a target sequence in the plant genome. In one aspect, the disclosure provides a recombinant DNA construct comprising (b) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous embryo tissue-preferential 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 can hybridize to a target sequence in the plant genome. In one embodiment, the disclosure provides a recombinant DNA construct comprising: (b) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous meiotic preferential 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 can hybridize to a target sequence in the genome of a plant cell.

[0038] In one embodiment, the disclosure provides a recombinant DNA construct comprising (b) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating 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-preferential promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the plant genome. In one embodiment, the disclosure provides a recombinant DNA construct comprising: (b) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating 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 meiotic preferential promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome of a plant cell.

[0039] In one embodiment, the disclosure provides a plant comprising a recombinant DNA construct comprising (b) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous egg cell-preferential 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 can hybridize to a target sequence in the plant genome. In one embodiment, the disclosure provides a plant comprising a recombinant DNA construct comprising a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous embryo tissue-preferential 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 can hybridize to a target sequence in the plant genome. In one embodiment, the present disclosure provides a plant comprising a recombinant DNA construct comprising (b) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous meiotic preferential 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 can hybridize to a target sequence in the plant genome.

[0040] In one embodiment, the disclosure provides a plant comprising a recombinant DNA construct comprising (b) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating 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-preferential promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the plant genome. In one embodiment, the disclosure provides a plant comprising a recombinant DNA construct comprising a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating 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 embryo tissue-preferential promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the plant genome. In one embodiment, the disclosure provides a plant comprising a recombinant DNA construct comprising (b) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating 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 meiotic preferential promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the plant genome.

[0041] In some embodiments, the disclosure provides seeds of any plant provided herein.

[0042] Nucleic acids and amino acids The use of the terms “polynucleotide” or “nucleic acid molecule” is not intended to limit this disclosure to polynucleotides containing deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also envisioned. Those skilled in the art will recognize that polynucleotides and nucleic acid molecules may include deoxyribonucleotides, ribonucleotides, or combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides of this disclosure also encompass, but are not limited to, all forms of sequences, including single-stranded, double-stranded, hairpin, stem-loop structures, etc. In some embodiments, the nucleic acid molecule provided herein is a DNA molecule. In other embodiments, the nucleic acid molecule provided herein is an RNA molecule. In some embodiments, the nucleic acid molecule provided herein is single-stranded. In other embodiments, the nucleic acid molecule provided herein is double-stranded.

[0043] As used herein, the term “recombinant” refers to nucleic acid (DNA or RNA) molecules, proteins, constructs, vectors, etc., which include combinations of polynucleotides or protein sequences that do not naturally exist adjacent to or very close to each other without human intervention, and / or polynucleotide molecules, proteins, constructs, etc., which include at least two polynucleotides or protein sequences that are heterogeneous with respect to each other, which are artificial, not normally found in nature, and / or exist in circumstances in which they are not normally found in nature.

[0044] In one embodiment, the methods and compositions provided herein include a vector. As used herein, the term “vector” refers to a DNA molecule used as a vehicle for transporting exogenous genetic material into a cell.

[0045] In one embodiment, one or more polynucleotide sequences derived from the vector are stably integrated into the plant genome. In another embodiment, one or more polynucleotide sequences derived from the vector are stably integrated into the genome of a plant cell.

[0046] In one embodiment, the first nucleic acid sequence and the second nucleic acid sequence are provided in a single vector. In another embodiment, the first nucleic acid sequence is provided in the first vector, and the second nucleic acid sequence is provided in the second vector.

[0047] As used herein, the term “polypeptide” refers to a chain of at least two covalently linked amino acids. Polypeptides may be encoded by polynucleotides provided herein. An example of a polypeptide is a protein. Proteins provided herein may be encoded by nucleic acid molecules provided herein.

[0048] Nucleic acids can be isolated using techniques commonplace in this art. For example, nucleic acids can be isolated using any method, including, but not limited to, recombinant nucleic acid techniques and / or polymerase chain reaction (PCR). Common PCR techniques are described, for example, in *PCR Primer: A Laboratory Manual*, edited by Dieffenbach & Dveksler, Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate nucleic acids. Isolated nucleic acids can also be chemically synthesized 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 an expression vector. Furthermore, purified polypeptides can be obtained by chemical synthesis. The purity of polypeptides can be measured using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0049] Nucleic acids can be detected using hybridization, though not exclusively. Hybridization between nucleic acids is discussed in detail by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0050] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. The antibodies provided herein may be polyclonal or monoclonal antibodies. Antibodies having specific binding affinity to the polypeptides provided herein can be produced using methods known in the art. The antibodies provided herein can be attached to a solid support such as a microtiter plate using methods known in the art.

[0051] As used herein in reference to two or more nucleotide or protein sequences, the term “percent identity” or “percent identical” is calculated by (i) comparing two optimally aligned sequences (nucleotides or proteins) on a comparison window; (ii) determining the number of positions in both sequences where identical nucleic acid bases (for nucleotide sequences) or amino acid residues (for proteins) exist, thereby obtaining the number of matching positions; (iii) dividing the number of matching positions by the total number of positions in the comparison window; and then (iv) multiplying this quotient by 100% to obtain the percentage identity. When “percent identity” is calculated with respect to a reference sequence without specifying a particular comparison window, the percentage identity is determined by dividing the number of matching positions on the alignment region by the total length of the reference sequence. Therefore, for the purposes of this application, when two sequences (query and target) are optimally aligned (allowing for gaps in their alignment), the "percent identity" of the query sequence is equal to the number of identical positions between the two sequences obtained by dividing by the total number of positions in the query sequence over its entire length (or comparison window) and then multiplying by 100%. When the percentage of sequence identity is used in reference to a protein, it is recognized that non-identical residue positions are often distinguished by conservative amino acid substitutions, where the amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), and therefore does not change the functional properties of the molecule. If sequences differ in conservative substitutions, the percentage of sequence identity can be adjusted upward to compensate for the conservative nature of the substitutions. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity."

[0052] The term “percent sequence complementarity” or “percent complementarity” as used herein in reference to two nucleotide sequences is similar to the concept of percent identity, but refers to the percentage of nucleotides in the query sequence that optimally base-pair with or hybridize with the nucleotides of the target sequence, given that the query sequence and the target sequence are linearly arranged and optimally base-pair without secondary folding structures such as loops, stems, or hairpins. Such percent complementarity may be between two DNA strands, between two RNA strands, or between a DNA strand and an RNA strand. "Percent complementarity" can be calculated by (i) optimally base-pairing or hybridizing two nucleotide sequences in a linear and fully extended configuration (i.e., without folding or secondary structure) on a comparison window, (ii) determining the number of base-pairing positions between the two sequences on the comparison window to obtain 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 percentage complementarity of the two sequences. Optimal base-pairing of two sequences can be determined based on the pairing of known nucleotide bases such as GC, AT, and AU via hydrogen bonding. When "percent complementarity" is calculated with respect to a reference sequence without specifying a particular comparison window, percentage identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Therefore, for the purposes of this application, when the two sequences (query and target) optimally form base pairs (allowing for mismatches or nucleotides that do not form base pairs), the "percent complementarity" of the query sequence is equal to the number of base-paired positions between the two sequences, obtained by dividing the query sequence by the total number of positions in the query sequence over its entire length and then multiplying by 100%.

[0053] Various pairwise or multi-sequence alignment algorithms and programs, such as ClustalW or Basic Local Alignment Search Tool (BLAST®), are known in the art and can be used to compare the sequence identity or similarity between two or more nucleotide or protein sequences for their optimal alignment to calculate the percentage identity of sequences. Although other alignment and comparison methods are known in the art, the alignment and percentage identity (including the percentage identity range described above) between two sequences can be determined by the ClustalW algorithm. For example, 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: pp. 4673-4680 (1994); Larkin MA et al., "Clustal W and Clustal See 215:403–410 (1990) (the entire contents and disclosures of these are incorporated herein by reference).

[0054] As used herein, a first nucleic acid molecule can "hybridize" to a second nucleic acid molecule by non-covalent interactions (e.g., Watson-Crick base pairing) in a sequence-specific, antiparallel manner (i.e., nucleic acid specifically binds to complementary nucleic acid) under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base pairings include adenine (A) pairing with thymine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine. Furthermore, for hybridization between two RNA molecules (e.g., dsRNA), it is also known in the art that guanine bases pair with uracil. For example, G / U base pairs contribute to the degenerate (i.e., redundancy) of the genetic code in the context of tRNA anticodons that base-pair with codons in mRNA. In the context of this disclosure, guanine in the protein-binding segment (dsRNA double helix) of the target DNA-targeting RNA molecule is considered complementary to uracil, and vice versa. Therefore, if a G / U base pair can be constructed at a given nucleotide position on the protein-binding segment (dsRNA double helix) of the target DNA-targeting RNA molecule, that position is not considered non-complementary, but rather complementary.

[0055] Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly in Chapter 11 and Table 11.1; and in Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Temperature and ionic strength conditions determine the "stringency" of hybridization.

[0056] Hybridization requires that two nucleic acids contain complementary sequences, although mismatches between bases are acceptable. The appropriate conditions for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, which are well-known variables in the art. The higher the degree of complementarity between two nucleotide sequences, the higher the melting point (Tm) value for the hybrid of nucleic acids having these sequences. For hybridization between nucleic acids with a short complementarity range (e.g., complementarity over 35 nucleotides or less), the location of the mismatch becomes important (see Sambrook et al.). Typically, the length of a hybridizable nucleic acid is at least 10 nucleotides. Examples of minimum hybridizable nucleic acid lengths are 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. Furthermore, those skilled in the art will recognize that the temperature and salt concentration of the washing solution can be adjusted as needed depending on factors such as the length and degree of complementarity of the complementary region.

[0057] It is understood in the art that the sequence of a polynucleotide does not need to be specifically hybridizable or 100% complementary to the sequence of its target nucleic acid that is hybridizable. Furthermore, a polynucleotide may hybridize across one or more segments (e.g., loop or hairpin structures) such that intervening or adjacent segments do not participate in the hybridization event. For example, if 18 of the 20 nucleotides in an antisense compound are complementary to the target region, and therefore the antisense nucleic acid that specifically hybridizes represents 90 percent complementarity. In this example, the remaining non-complementary nucleotides may cluster with complementary nucleotides or be scattered, and do not need to be contiguous with each other or with complementary nucleotides. The percentage complementarity between specific ranges of nucleic acid sequences within a nucleic acid can be routinely determined using the BLAST® program (basic local alignment search tools) and the PowerBLAST program (see Altschul et al., J. Mol. Biol., 1990, 215, pp. 403-410; Zhang and Madden, Genome Res., 1997, 7, pp. 649-656), which are known in the art, or by using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, pp. 482-489) and the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) with default settings.

[0058] Edit generation Some embodiments described herein relate to compositions and methods for producing genetic editing in the genome of offspring plants by preferentially expressing DNA-modifying enzymes, such as inducible nucleases, in the eggs, meiotic cells, and / or embryonic cells of parent plants. In some embodiments, the parent plant expressing the DNA-modifying enzyme is female. In some embodiments, the parent plant expressing the DNA-modifying enzyme is male.

[0059] As used herein, the terms “genome editing” or “editing” refer to any modification of a nucleotide sequence in a site-specific manner. In this disclosure, genome editing techniques include the use of DNA-modifying enzymes such as endonucleases, recombinases, transposases, deaminases, methylases, helicases, and any combination thereof. In some embodiments, “modification” includes hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the sequence-specific editing system includes adenine deaminase. In some embodiments, “modification” includes hydrolytic deamination of adenine or adenosine. In some embodiments, “modification” includes hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, “modification” includes the insertion of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, 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 the insertion of one or more transgenes. In another embodiment, “modification” includes the deletion of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, 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 a further embodiment, “modification” includes the inversion of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, 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 embodiment, “modification” includes the 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 embodiment, “modification” includes duplication 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” includes substitution of “A” with “C”, “G”, or “T” in the nucleic acid sequence. In some embodiments, “modification” includes substitution of “C” with “A”, “G”, or “T” in the nucleic acid sequence. In some embodiments, “modification” includes substitution of “G” with “A”, “C”, or “T” in the nucleic acid sequence.In some embodiments, "modification" includes the substitution of "T" with "A", "C", or "G" in the nucleic acid sequence. In some embodiments, "modification" includes the substitution of "C" with "U" in the nucleic acid sequence. In some embodiments, "modification" includes the substitution of "G" with "A" in the nucleic acid sequence. In some embodiments, "modification" includes the substitution of "A" with "G" in the nucleic acid sequence. In some embodiments, "modification" includes the substitution of "T" with "C" in the nucleic acid sequence.

[0060] In some embodiments, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks in a double-stranded DNA molecule operably linked to a heterologous meiotic preferential 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 can hybridize to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one meiotic cell of the plant, and the ribonucleoprotein generates at least one double-strand break in a target sequence in the at least one meiotic cell.

[0061] In some embodiments, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks 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 a heterologous meiotic preferential promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one meiotic cell of the plant, and the ribonucleoprotein generates at least one double-strand break in a target sequence in the at least one meiotic cell.

[0062] In some embodiments, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks in a double-stranded DNA molecule operably linked to a heterologous oocyte-preferential 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 can hybridize to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one oocyte of the plant, and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one oocyte.

[0063] In some embodiments, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks 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 egg cell-preferential promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one egg cell of the plant, and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one egg cell.

[0064] In one embodiment, the present disclosure provides a method for editing the genome of a plant cell, comprising the steps of (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease capable of producing alternating breaks in a double-stranded DNA molecule operably linked to a heterologous embryo-preferential promoter, and 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 can hybridize to a target sequence in the genome; and (b) obtaining at least one embryo from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one embryo, and the ribonucleoprotein produces at least one double-strand break in a target sequence in at least one embryo.

[0065] In one embodiment, the present disclosure provides a method for editing the genome of a plant cell, comprising the steps of (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease capable of producing alternating breaks in a double-stranded DNA molecule operably linked to a heterologous promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to an embryo-tissue-preferential promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; and (b) obtaining at least one embryo from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one embryo, and the ribonucleoprotein produces at least one double-strand break in a target sequence in at least one embryo.

[0066] In one embodiment, the present disclosure provides a method for editing a plant genome, comprising the steps of (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks in a double-stranded DNA molecule operably linked to a heterologous embryo-preferential 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 can hybridize to a target sequence in the genome; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing at least one plant to produce at least one embryo, wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one embryo derived from step (c), and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one embryo.

[0067] In one embodiment, the present disclosure provides a method for editing a plant genome, comprising the steps of (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks 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-preferential promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing at least one plant to produce at least one embryo, wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one embryo derived from step (c), and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one embryo.

[0068] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell comprising: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous meiotic preferential promoter; and (ii) one or more guide nucleic acids capable of hybridizing to (A) a target sequence in the plant genome; and (B) a heterologous second promoter capable of hybridizing to a first and second site flanking the nucleic acid sequence encoding the gene of interest. The method comprises (iii) introducing a second nucleic acid sequence encoding one or more movably linked guide nucleic acids; and (iii) introducing a third nucleic acid sequence encoding a gene of interest; and (b) regenerating at least one plant from the plant cells of step (a); wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one meiotic cell of a plant, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest is incorporated into the target site in at least one egg cell.

[0069] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell comprising: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) one or more guide nucleic acids capable of hybridizing to (A) a target sequence in the plant genome; and (B) a meiotically preferential second promoter capable of hybridizing to a first and second site flanking the nucleic acid sequence encoding the gene of interest. The method provides comprising: (iii) introducing a second nucleic acid sequence encoding one or more guide nucleic acids linked to a gene of interest; and (iii) introducing a third nucleic acid sequence encoding a gene of interest; and (b) regenerating at least one plant from the plant cells of step (a); wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one meiotic cell of a plant, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest being incorporated into the target site in at least one meiotic cell.

[0070] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell containing (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous oocyte-preferential promoter; and (ii) one or more guide nucleic acids capable of hybridizing to (A) a target sequence in the plant genome; and (B) a heterologous second promoter capable of hybridizing to a first and second site flanking the nucleic acid sequence encoding the gene of interest. The method provides comprising: (iii) introducing a second nucleic acid sequence encoding one or more operably linked guide nucleic acids; and (iii) introducing a third nucleic acid sequence encoding a gene of interest; and (b) regenerating at least one plant from the plant cells of step (a); wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one egg cell of the plant, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest being incorporated into the target site in at least one egg cell.

[0071] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) one or more guide nucleic acids capable of hybridizing to (A) a target sequence in the plant genome; and (B) a first and second site flanking the nucleic acid sequence encoding the gene of interest, via an egg-cell-preferential second promoter. The method provides comprising: (iii) introducing a second nucleic acid sequence encoding one or more movably linked guide nucleic acids; and (iii) introducing a third nucleic acid sequence encoding a gene of interest; and (b) regenerating at least one plant from the plant cells of step (a); wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one egg cell of the plant, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest being incorporated into the target site in at least one egg cell.

[0072] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell containing (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous embryo-preferential promoter; and (ii) one or more guide nucleic acids operably linked to a heterologous second promoter, the guide nucleic acids being capable of (A) hybridizing to a target sequence in the plant genome; and (B) hybridizing to a first and second site flanking the nucleic acid sequence encoding the gene of interest. The method comprises (iii) introducing a second nucleic acid sequence encoding a target gene; (b) regenerating at least one plant from the plant cells of step (a); and (c) fertilizing at least one plant derived from step (b) to produce at least one embryo; wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one embryo, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the target gene is incorporated into the target site in at least one embryo.

[0073] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) one or more guide nucleic acids encoding one or more guide nucleic acids operably linked to an embryonic tissue-preferential promoter, which can (A) hybridize to a target sequence in the plant genome; and (B) hybridize to first and second sites flanking the nucleic acid sequence encoding the gene of interest. The method comprises (iii) introducing a second nucleic acid sequence; and (b) introducing a third nucleic acid sequence encoding the gene of interest; (c) regenerating at least one plant from the plant cells of step (a); and (b) fertilizing at least one plant derived from step (b) to produce at least one embryo; wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one embryo, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest is incorporated into the target site in at least one embryo.

[0074] Adjustment element Regulatory elements such as promoters, leaders (also known as 5'UTRs), enhancers, introns, and transcription termination regions (or 3'UTRs) play an integrated role in the overall expression of genes in living cells. As used herein, the term “regulatory element” refers to a DNA molecule having gene regulatory activity. As used herein, the term “gene regulatory activity” refers to the ability to influence the expression of a operably linked transcribed DNA molecule, for example, by influencing the transcription and / or translation of the operably linked transcribed DNA molecule. Regulatory elements such as promoters, leaders, enhancers, introns, and 3'UTRs that function in plants are useful for modifying plant phenotypes through genetic engineering. Regulatory elements may be characterized by their gene expression patterns, such as positive and / or negative effects, including constitutive expression or expression responsive to time, space, development, tissue, environment, physiology, pathology, cell cycle, and / or chemoresponsive expression, and any combination thereof, as well as quantitative or qualitative indicators. As used herein, “gene expression pattern” is the transcription of any pattern of operably linked DNA molecules into the RNA molecule to be transcribed. The RNA molecule to be transcribed can be translated to produce a protein molecule, or it can provide an antisense or other regulatory RNA molecule such as double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), or small interfering RNA (siRNA). As used herein, the term “protein expression” is the translation of any pattern of the RNA molecule to be transcribed into a protein molecule. Protein expression may be characterized by its temporal, spatial, developmental, or morphological quality, as well as quantitative or qualitative indicators.

[0075] As is commonly understood in the art, the term “promoter” refers to a DNA sequence that contains an RNA polymerase binding site, a transcription initiation site, and / or a TATA box, and that assists or promotes the transcription and expression of a bound transcriptable polynucleotide sequence and / or gene (or transgene). A promoter can initially be isolated from the 5' untranslated region (5'UTR) of a genomic copy of a gene. Promoters can be synthetically produced, known, or naturally occurring promoter sequences, or modified or induced from other promoter sequences. Promoters may also include chimeric promoters, which include combinations of two or more heterologous sequences. Therefore, the promoters of this application may include variants of promoter sequences that are similar in composition but not identical to known or other promoter sequences provided herein. Promoters can be classified according to various criteria related to the expression pattern of the associated code or transcriptable sequence or gene (including transgenes) operably linked to the promoter, such as constitutive, developmental, tissue-specific, cell cycle-specific, and inducible.

[0076] In some embodiments, the promoter is operably ligated to the 5' side of the leader sequence. As used herein, the term “leader” refers to a DNA molecule isolated from the untranslated 5' region (5'UTR) of a gene and generally defined as a nucleotide segment between the transcription start site (TSS) and the protein coding sequence start site. Alternatively, the leader may be a synthetically produced or engineered DNA element. The leader can be used as a 5' regulatory element to modulate the expression of an operably ligated transcriptable DNA molecule. The leader molecule can be used with a heterologous promoter or with its innate promoter.

[0077] As used herein, “operably linked” refers to a functional link between two or more elements. For example, an operably linked link between a polynucleotide of interest and a regulatory element (e.g., a promoter) is a functional link that enables the expression of the polynucleotide of interest. The operably linked elements may be continuous or discontinuous.

[0078] A promoter that is expressed in specific tissues of an organism but not in other tissues is called a “tissue-specific” promoter. A promoter that drives enhanced expression in certain tissues of an organism compared to other tissues of the organism is called a “tissue-preferential” promoter. Thus, a “tissue-preferential” promoter will cause relatively high or preferential expression in certain tissues of a plant, but will show lower levels of expression in other tissues of the plant. In another embodiment, the promoters provided herein are tissue-specific promoters. In yet another embodiment, the promoters provided herein are tissue-preferential promoters. In some embodiments, a tissue-preferential promoter includes a tissue-specific promoter.

[0079] A promoter that is expressed in meiotic cells of an organism but not in non-meiotic cells is referred to as a “meiotic cell-specific” or “meiosis-specific” promoter. A promoter that drives enhanced expression in meiotic cells of an organism compared to other cells of the organism is referred to as a “meiotic cell-preferential” or “meiosis-preferential” promoter. Therefore, a “meiotic cell-preferential” or “meiosis-preferential” promoter will cause relatively high or preferential expression in meiotic plant cells but will show lower levels of expression in other plant cells. In another embodiment, the promoters provided herein are meiosis-specific promoters. In yet another embodiment, the promoters provided herein are meiosis-preferential promoters. In some embodiments, a meiosis-preferential promoter includes a meiosis-specific promoter. A promoter that is expressed in a cell cycle-dependent manner is referred to as a “cell cycle-specific” promoter. In another embodiment, the promoters provided herein are cell cycle-specific promoters. In yet another embodiment, the promoters provided herein are cell cycle-preferential promoters. In some embodiments, the cell cycle-preferential promoter includes a cell cycle-specific promoter.

[0080] Deamination of promoter activity can be carried out using any standard method in the art. For example, but not limited to, the promoter of interest can be used to drive the expression of a fluorophore or other reported molecule, and the concentration of the expressed molecule can be used to determine the promoter activity in different cell or tissue types.

[0081] Some embodiments described herein relate to the preferential expression of DNA-modifying enzymes, such as inducible nucleases, in plant eggs, meiosis, and / or embryonic cells. Non-limiting examples of expression elements useful in the compositions and methods described herein are provided in Table 1.

[0082] [Table 1A]

[0083] Table 1B

[0084]

Table 1C

[0085] In one embodiment, a promoter sequence fragment disclosed in Table 1 is provided. The promoter fragment may include egg, embryo, and / or meiotic expression activity as described above, and may be useful alone, in the construction of a chimeric promoter, or in combination with other promoters and promoter fragments, such as in combination with other expression elements and expression element fragments. In some embodiments, a promoter fragment is provided comprising a DNA molecule having promoter activity disclosed herein, 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 thereof.In some embodiments, a TATA box 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 is provided herein, and has promoter activity as disclosed herein. A promoter fragment is provided, comprising a DNA sequence having at least approximately 85 percent identity to ~88, at least approximately 86 percent identity, at least approximately 87 percent identity, at least approximately 88 percent identity, at least approximately 89 percent identity, at least approximately 90 percent identity, at least approximately 91 percent identity, at least approximately 92 percent identity, at least approximately 93 percent identity, at least approximately 94 percent identity, at least approximately 95 percent identity, at least approximately 96 percent identity, at least approximately 97 percent identity, at least approximately 98 percent identity, at least approximately 99 percent identity, or at least approximately 100 percent identity. Methods for producing such fragments from a starting promoter molecule are well known in the art.

[0086] In some embodiments, the meiotic cell-preferential or meiotic cell-preferential promoter includes the DMC1 promoter. In some embodiments, the meiotic cell-preferential or meiotic cell-preferential promoter includes the Mps1 promoter. In some embodiments, the meiotic cell-preferential or meiotic cell-preferential promoter includes the Adf1 promoter. In some embodiments, the meiotic cell-preferential or meiotic cell-preferential promoter includes a promoter selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter.

[0087] In some embodiments, the expression elements listed in Table 1 are operably ligated to nucleic acids encoding DNA-modifying enzymes, such as inducible nucleases. In some embodiments, the expression elements listed in Table 1 are operably ligated to nucleic acids encoding Cas9 nuclease. In some embodiments, the expression elements listed in Table 1 are operably ligated to nucleic acids encoding Cas12a nuclease. In some embodiments, the expression elements listed in Table 1 are operably ligated to nucleic acids encoding CasX nuclease. In some embodiments, the expression elements listed in Table 1 are operably ligated to nucleic acids encoding guide nucleic acids. In some embodiments, the expression elements listed in Table 1 are operably ligated to nucleic acids encoding guide RNA. In some embodiments, the expression elements listed in Table 1 are operably ligated to nucleic acids encoding single guide RNA. In some embodiments, the guide RNA acid is flanked by self-cleaving ribozymes. In one embodiment, the expression elements listed in Table 1 are operably ligated to a nucleic acid encoding a recombinase (e.g., Cre recombinase). In another embodiment, the expression elements listed in Table 1 are operably ligated to a nucleic acid encoding TALE.

[0088] In some embodiments, the DMC1 promoter is operably ligated to a nucleic acid encoding an inducible nuclease. In some embodiments, the DMC1 promoter is operably ligated to a nucleic acid encoding a Cas9 nuclease. In some embodiments, the DMC1 promoter is operably ligated to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the DMC1 promoter is operably ligated to a nucleic acid encoding a CasX nuclease. In some embodiments, the DMC1 promoter is operably ligated to a nucleic acid encoding a guide nucleic acid. In some embodiments, the DMC1 promoter is operably ligated to a nucleic acid encoding a guide RNA. In some embodiments, the DMC1 promoter is operably ligated to a nucleic acid encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the DMC1 promoter is operably ligated to a nucleic acid encoding a recombinase (e.g., Cre recombinase). In some embodiments, the DMC1 promoter is operably ligated to a nucleic acid encoding TALE.

[0089] In some embodiments, the Mps1 promoter is operably ligated to a nucleic acid encoding an inducible nuclease. In some embodiments, the Mps1 promoter is operably ligated to a nucleic acid encoding a Cas9 nuclease. In some embodiments, the Mps1 promoter is operably ligated to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the Mps1 promoter is operably ligated to a nucleic acid encoding a CasX nuclease. In some embodiments, the Mps1 promoter is operably ligated to a nucleic acid encoding a guide nucleic acid. In some embodiments, the Mps1 promoter is operably ligated to a nucleic acid encoding a guide RNA. In some embodiments, the Mps1 promoter is operably ligated to a nucleic acid encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the Mps1 promoter is operably ligated to a nucleic acid encoding a recombinase (e.g., Cre recombinase). In some embodiments, the Mps1 promoter is operably ligated to a nucleic acid encoding TALE.

[0090] In some embodiments, the Adf1 promoter is operably ligated to a nucleic acid encoding an inducible nuclease. In some embodiments, the Adf1 promoter is operably ligated to a nucleic acid encoding a Cas9 nuclease. In some embodiments, the Adf1 promoter is operably ligated to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the Adf1 promoter is operably ligated to a nucleic acid encoding a CasX nuclease. In some embodiments, the Adf1 promoter is operably ligated to a nucleic acid encoding a guide nucleic acid. In some embodiments, the Adf1 promoter is operably ligated to a nucleic acid encoding a guide RNA. In some embodiments, the Adf1 promoter is operably ligated to a nucleic acid encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the Adf1 promoter is operably ligated to a nucleic acid encoding a recombinase (e.g., Cre recombinase). In some embodiments, the Adf1 promoter is operably ligated to a nucleic acid encoding TALE.

[0091] In some embodiments, the DMC1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the DMC1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 85% identical to SEQ ID NO: 4. In some embodiments, the DMC1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to SEQ ID NO: 4. In some embodiments, the DMC1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 95% identical to SEQ ID NO: 4. In some embodiments, the DMC1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 96% identical to SEQ ID NO: 4. In some embodiments, the DMC1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 97% identical to SEQ ID NO: 4. In some embodiments, the DMC1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 98% identical to SEQ ID NO: 4. In some embodiments, the DMC1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 99% identical to SEQ ID NO: 4. In one embodiment, the DMC1 promoter includes a nucleic acid sequence or a functional fragment thereof that is 100% identical to SEQ ID NO: 4.

[0092] In some embodiments, the Mps1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the Mps1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 85% identical to SEQ ID NO: 5. In some embodiments, the Mps1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to SEQ ID NO: 5. In some embodiments, the Mps1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the Mps1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 96% identical to SEQ ID NO: 5. In some embodiments, the Mps1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 97% identical to SEQ ID NO: 5. In some embodiments, the Mps1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 98% identical to SEQ ID NO: 5. In some embodiments, the Mps1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 99% identical to SEQ ID NO: 5. In one embodiment, the Mps1 promoter includes a nucleic acid sequence or a functional fragment thereof that is 100% identical to SEQ ID NO: 5.

[0093] In some embodiments, the Adf1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 80% identical to SEQ ID NO: 6. In some embodiments, the Adf1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 85% identical to SEQ ID NO: 6. In some embodiments, the Adf1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to SEQ ID NO: 6. In some embodiments, the Adf1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 95% identical to SEQ ID NO: 6. In some embodiments, the Adf1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 96% identical to SEQ ID NO: 6. In some embodiments, the Adf1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 97% identical to SEQ ID NO: 6. In some embodiments, the Adf1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 98% identical to SEQ ID NO: 6. In some embodiments, the Adf1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 99% identical to SEQ ID NO: 6. In one embodiment, the Adf1 promoter includes a nucleic acid sequence or a functional fragment thereof that is 100% identical to SEQ ID NO: 6.

[0094] In some embodiments, the meiosis-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the meiosis-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 85% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the meiosis-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the meiosis-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the meiosis-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 96% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the meiosis-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the meiosis-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the meiosis-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85. In some embodiments, the meiosis-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85.

[0095] As used herein, “egg cell-preferential promoter” refers to a promoter that exhibits higher or preferential expression in egg cells compared to other cell or tissue types of the plant. Egg cell-preferential promoters may, but are not limited to, exhibit expression in neighboring cells such as synergid cells, antipodal cells, centroid cells, cortical cells, stigma cells, and style cells. Egg cell-preferential promoters may also exhibit expression in other ovarian cells, such as ovules. Egg cell-preferential promoters may also exhibit expression in other plant tissues such as pollen cells, root cells, embryonic cells, stem cells, meristem cells, flower cells, and leaf cells, insofar as they exhibit higher or preferential expression in egg cells.

[0096] As used herein, “egg cell-specific promoter” refers to a promoter that is expressed only in egg cells. In some embodiments, an egg cell-preferential promoter includes an egg cell-specific promoter.

[0097] As used herein, “ovule-preferential promoter” refers to a promoter that exhibits higher or preferential expression in at least one or all ovule tissues compared to other cell or tissue types of the plant. In seed plants, the ovule is a structure that gives rise to and contains female germ cells. As used herein, the ovule consists of non-reducing tissue that initially gives rise to the haploid tissue of the female gametophyte. The female gametophyte further develops into a “mature ovum sac” containing four unique cell types: one egg cell, a central cell, two synergid cells, and three or more antipodal cells. As used herein, an ovule-preferential promoter may exhibit expression in the ovule before or after pollination. An ovule-preferential promoter may also exhibit expression in other ovary cells.

[0098] As used herein, “ovule tissue-specific promoter” refers to a promoter that is expressed only in the ovule. In some embodiments, an ovule-preferential promoter includes an ovule tissue-specific promoter.

[0099] As used herein, “embryonic promoter” refers to a promoter that exhibits higher or preferential expression in embryonic tissue compared to other cell or tissue types of the plant. Embryonic promoters may, but are not limited to, exhibit expression in neighboring cells such as endosperm cells, cotyledon cells, and seed coat cells. Embryonic promoters may also exhibit expression in other plant tissues such as pollen cells, root cells, egg cells, stem cells, meristem cells, flower cells, and leaf cells, insofar as they exhibit higher or preferential expression in embryonic tissue.

[0100] As used herein, “embryo-specific promoter” refers to a promoter that is expressed only in embryonic tissue. In some embodiments, an embryonic tissue-preferential promoter includes an embryonic tissue-specific promoter.

[0101] As used herein, “zygote cell-preferential promoter” refers to a promoter that exhibits higher or preferential expression in zygotes compared to other cell or tissue types of the plant. Diploid zygotes are formed when haploid pollen cells fertilize egg cells, giving rise to embryos. Zygote cell-preferential promoters may also be expressed in other plant cells, such as pollen cells, egg cells, stem cells, meristem cells, endosperm cells, cotyledon cells, flower cells, leaf cells, and embryonic tissues, as long as they exhibit higher or preferential expression in zygotes.

[0102] As used herein, “zygote cell-specific promoter” refers to a promoter that is expressed only in zygotes. In some embodiments, a zygote cell-preferential promoter includes a zygote cell-specific promoter.

[0103] Since the fertilized egg develops into a zygote upon pollination, which then produces embryonic tissue, it can be understood that both promoters may be egg cell-preferential, zygote cell-preferential, and embryonic tissue-preferential.

[0104] In some embodiments, the embryonic tissue-preferential or embryonic tissue-specific promoter includes the DSUL1 promoter. In some embodiments, the oocyte-preferential or embryonic tissue-preferential promoter includes the EA1 promoter. In some embodiments, the oocyte-preferential or embryonic tissue-preferential promoter includes the ES4 promoter. In some embodiments, the oocyte-preferential or embryonic tissue-specific promoter includes the EAL1 promoter. In some embodiments, the oocyte-preferential or embryonic tissue-preferential promoter includes a promoter selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter.

[0105] In some embodiments, the DSUL1 promoter is operably ligated to a nucleic acid encoding an inducible nuclease. In some embodiments, the DSUL1 promoter is operably ligated to a nucleic acid encoding a Cas9 nuclease. In some embodiments, the DSUL1 promoter is operably ligated to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the DSUL1 promoter is operably ligated to a nucleic acid encoding a CasX nuclease. In some embodiments, the DSUL1 promoter is operably ligated to a nucleic acid encoding a guide nucleic acid. In some embodiments, the DSUL1 promoter is operably ligated to a nucleic acid encoding a guide RNA. In some embodiments, the DSUL1 promoter is operably ligated to a nucleic acid encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the DSUL1 promoter is operably ligated to a nucleic acid encoding a recombinase (e.g., Cre recombinase). In some embodiments, the DSUL1 promoter is operably ligated to a nucleic acid encoding TALE.

[0106] In some embodiments, the EA1 promoter is operably ligated to a nucleic acid encoding an inducible nuclease. In some embodiments, the EA1 promoter is operably ligated to a nucleic acid encoding a Cas9 nuclease. In some embodiments, the EA1 promoter is operably ligated to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the EA1 promoter is operably ligated to a nucleic acid encoding a CasX nuclease. In some embodiments, the EA1 promoter is operably ligated to a nucleic acid encoding a guide nucleic acid. In some embodiments, the EA1 promoter is operably ligated to a nucleic acid encoding a guide RNA. In some embodiments, the EA1 promoter is operably ligated to a nucleic acid encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the EA1 promoter is operably ligated to a nucleic acid encoding a recombinase (e.g., Cre recombinase). In some embodiments, the EA1 promoter is operably ligated to a nucleic acid encoding TALE.

[0107] In some embodiments, the ES4 promoter is operably ligated to a nucleic acid encoding an inducible nuclease. In some embodiments, the ES4 promoter is operably ligated to a nucleic acid encoding a Cas9 nuclease. In some embodiments, the ES4 promoter is operably ligated to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the ES4 promoter is operably ligated to a nucleic acid encoding a CasX nuclease. In some embodiments, the ES4 promoter is operably ligated to a nucleic acid encoding a guide nucleic acid. In some embodiments, the ES4 promoter is operably ligated to a nucleic acid encoding a guide RNA. In some embodiments, the ES4 promoter is operably ligated to a nucleic acid encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the ES4 promoter is operably ligated to a nucleic acid encoding a recombinase (e.g., Cre recombinase). In some embodiments, the ES4 promoter is operably ligated to a nucleic acid encoding TALE.

[0108] In some embodiments, the EAL1 promoter is operably ligated to a nucleic acid encoding an inducible nuclease. In some embodiments, the EAL1 promoter is operably ligated to a nucleic acid encoding a Cas9 nuclease. In some embodiments, the EAL1 promoter is operably ligated to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the EAL1 promoter is operably ligated to a nucleic acid encoding a CasX nuclease. In some embodiments, the EAL1 promoter is operably ligated to a nucleic acid encoding a guide nucleic acid. In some embodiments, the EAL1 promoter is operably ligated to a nucleic acid encoding a guide RNA. In some embodiments, the EAL1 promoter is operably ligated to a nucleic acid encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the EAL1 promoter is operably ligated to a nucleic acid encoding a recombinase (e.g., Cre recombinase). In some embodiments, the EAL1 promoter is operably ligated to a nucleic acid encoding TALE.

[0109] In some embodiments, the DSUL1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the DSUL1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 85% identical to SEQ ID NO: 1. In some embodiments, the DSUL1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the DSUL1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the DSUL1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 96% identical to SEQ ID NO: 1. In some embodiments, the DSUL1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 97% identical to SEQ ID NO: 1. In some embodiments, the DSUL1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 98% identical to SEQ ID NO: 1. In some embodiments, the DSUL1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 99% identical to SEQ ID NO: 1. In one embodiment, the DSUL1 promoter includes a nucleic acid sequence or a functional fragment thereof that is 100% identical to SEQ ID NO: 1.

[0110] In some embodiments, the EA1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 80% identical to SEQ ID NO: 2. In some embodiments, the EA1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 85% identical to SEQ ID NO: 2. In some embodiments, the EA1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to SEQ ID NO: 2. In some embodiments, the EA1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 95% identical to SEQ ID NO: 2. In some embodiments, the EA1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 96% identical to SEQ ID NO: 2. In some embodiments, the EA1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 97% identical to SEQ ID NO: 2. In some embodiments, the EA1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 98% identical to SEQ ID NO: 2. In some embodiments, the EA1 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 99% identical to SEQ ID NO: 2. In one embodiment, the EA1 promoter includes a nucleic acid sequence or a functional fragment thereof that is 100% identical to SEQ ID NO: 2.

[0111] In some embodiments, the ES4 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 80% identical to SEQ ID NO: 3. In some embodiments, the ES4 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 85% identical to SEQ ID NO: 3. In some embodiments, the ES4 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to SEQ ID NO: 3. In some embodiments, the ES4 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 95% identical to SEQ ID NO: 3. In some embodiments, the ES4 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 96% identical to SEQ ID NO: 3. In some embodiments, the ES4 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 97% identical to SEQ ID NO: 3. In some embodiments, the ES4 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 98% identical to SEQ ID NO: 3. In some embodiments, the ES4 promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 99% identical to SEQ ID NO: 3. In one embodiment, the ES4 promoter includes a nucleic acid sequence or a functional fragment thereof that is 100% identical to SEQ ID NO: 3.

[0112] In some embodiments, an egg cell-preferential promoter, an ovule-preferential promoter, a zygote-preferential or embryonic-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 21-45, 65-69, 75-82, and 86-88. In some embodiments, an egg cell-preferential promoter or an embryonic-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 85% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 21-45, 65-69, 75-82, and 86-88. In some embodiments, an egg cell-preferential promoter or an embryonic-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 21-45, 65-69, 75-82, and 86-88. In some embodiments, the oocyte-preferential promoter or embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 21-45, 65-69, 75-82, and 86-88. In some embodiments, the oocyte-preferential promoter or embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 96% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 21-45, 65-69, 75-82, and 86-88. In some embodiments, the oocyte-preferential promoter or embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 21-45, 65-69, 75-82, and 86-88. In one embodiment, the egg cell-preferential promoter or embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 21-45, 65-69, 75-82, and 86-88. In another embodiment, the egg cell-preferential promoter or embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 21-45, 65-69, 75-82, and 86-88.In one embodiment, the egg cell-preferential promoter or embryonic tissue-preferential promoter includes a nucleic acid sequence or a functional fragment thereof that is 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 21-45, 65-69, 75-82, and 86-88.

[0113] It is understood in the art that fragments of promoter sequences can function to drive the transcription of operablely 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 can drive transcription, the 500 bp fragment is referred to as a “functional fragment.”

[0114] In some embodiments, the egg cell-preferential promoter is operably ligated to a nucleic acid sequence encoding a DNA-modifying enzyme, such as an inducible nuclease. In some embodiments, the egg cell-preferential promoter is operably ligated to a nucleic acid sequence encoding a Cas9 nuclease. In some embodiments, the egg cell-preferential promoter is operably ligated to a nucleic acid sequence encoding a Cas12a nuclease. In some embodiments, the egg cell-preferential promoter is operably ligated to a nucleic acid sequence encoding a CasX nuclease. In some embodiments, the egg cell-preferential promoter is operably ligated to a nucleic acid sequence encoding a guide nucleic acid. In some embodiments, the egg cell-preferential promoter is operably ligated to a nucleic acid sequence encoding a guide RNA. In some embodiments, the egg cell-preferential promoter is operably ligated to a nucleic acid sequence encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the egg cell-preferential promoter is operably ligated to a nucleic acid sequence encoding a recombinase (e.g., Cre recombinase). In one embodiment, the egg cell-preferential promoter is operably linked to a nucleic acid sequence encoding the TALE.

[0115] In some embodiments, the egg cell-specific promoter is operably ligated to a nucleic acid sequence encoding a DNA-modifying enzyme, such as an inducible nuclease. In some embodiments, the egg cell-specific promoter is operably ligated to a nucleic acid sequence encoding a Cas9 nuclease. In some embodiments, the egg cell-specific promoter is operably ligated to a nucleic acid sequence encoding a Cas12a nuclease. In some embodiments, the egg cell-specific promoter is operably ligated to a nucleic acid sequence encoding a CasX nuclease. In some embodiments, the egg cell-specific promoter is operably ligated to a nucleic acid sequence encoding a guide nucleic acid. In some embodiments, the egg cell-specific promoter is operably ligated to a nucleic acid sequence encoding a guide RNA. In some embodiments, the egg cell-specific promoter is operably ligated to a nucleic acid sequence encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the egg cell-specific promoter is operably ligated to a nucleic acid sequence encoding a recombinase (e.g., Cre recombinase). In one embodiment, the egg cell-specific promoter is operably linked to the nucleic acid sequence encoding the TALE.

[0116] In some embodiments, the embryonic tissue-preferential promoter is operably ligated to a nucleic acid sequence encoding a DNA-modifying enzyme, such as an inducible nuclease. In some embodiments, the embryonic tissue-preferential promoter is operably ligated to a nucleic acid sequence encoding a Cas9 nuclease. In some embodiments, the embryonic tissue-preferential promoter is operably ligated to a nucleic acid sequence encoding a Cas12a nuclease. In some embodiments, the embryonic tissue-preferential promoter is operably ligated to a nucleic acid sequence encoding a CasX nuclease. In some embodiments, the embryonic tissue-preferential promoter is operably ligated to a nucleic acid sequence encoding a guide nucleic acid. In some embodiments, the embryonic tissue-preferential promoter is operably ligated to a nucleic acid sequence encoding a guide RNA. In some embodiments, the embryonic tissue-preferential promoter is operably ligated to a nucleic acid sequence encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the embryonic tissue-preferential promoter is operably ligated to a nucleic acid sequence encoding a recombinase (e.g., Cre recombinase). In one embodiment, the embryonic tissue-preferential promoter is operably linked to the nucleic acid sequence encoding the TALE.

[0117] In some embodiments, the embryo tissue-specific promoter is operably ligated to a nucleic acid sequence encoding a DNA-modifying enzyme, such as an inducible nuclease. In some embodiments, the embryo tissue-specific promoter is operably ligated to a nucleic acid sequence encoding a Cas9 nuclease. In some embodiments, the embryo tissue-specific promoter is operably ligated to a nucleic acid sequence encoding a Cas12a nuclease. In some embodiments, the embryo tissue-specific promoter is operably ligated to a nucleic acid sequence encoding a CasX nuclease. In some embodiments, the embryo tissue-specific promoter is operably ligated to a nucleic acid sequence encoding a guide nucleic acid. In some embodiments, the embryo tissue-specific promoter is operably ligated to a nucleic acid sequence encoding a guide RNA. In some embodiments, the embryo tissue-specific promoter is operably ligated to a nucleic acid sequence encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the embryo tissue-specific promoter is operably ligated to a nucleic acid sequence encoding a recombinase (e.g., Cre recombinase). In one embodiment, the embryonic tissue-specific promoter is operably ligated to the nucleic acid sequence encoding the TALE.

[0118] Some embodiments described herein relate to methods and compositions for providing preferential or specific expression of DNA-modifying enzymes, such as inducible nucleases (e.g., CRISPR / Cas systems), from a constitutive promoter in egg, embryo, and / or meiotic plant tissue. In some embodiments, a transcribable polynucleotide encoding a DNA-modifying enzyme, such as an inducible nuclease (e.g., CRISPR / Cas system), is operably linked to a constitutive promoter by preferential or specific cleavage of an intervening polynucleotide sequence in egg, embryo, and / or meiotic plant tissue. In some embodiments, the intervening sequence is cleaved by a recombinase that is preferentially or selectively expressed in egg, embryo, and / or meiotic plant tissue. In some embodiments, the intervening sequence is cleaved by Cre-mediated cleavage of an intervening Cre expression cassette, in which Cre is preferentially or selectively expressed in egg, embryo, and / or meiotic plant tissue.

[0119] Promoter that drives expression in all or many tissues of a plant is called a “constitutive” promoter. Promoter that drives expression during a specific period or stage of development is called a “developmental” promoter. “Inducible” promoters are promoters that initiate transcription in response to environmental stimuli such as heat, cold, drought, or light, or other stimuli such as wounds or chemical application. Promoter can also be classified in terms of their origin, such as heterogeneous, homogeneous, chimeric, or synthetic.

[0120] As used herein, the term “heterogeneous” refers to a promoter that has a different origin from its associated transcriptable DNA sequence, coding sequence, or gene (or transgene) and / or is not naturally occurring in the plant to be transformed. More broadly, the term “heterogeneous” may refer to a promoter and a combination of two or more DNA molecules or sequences, such as an associated transcriptable DNA sequence, coding sequence, or gene, in which case such a combination is artificial and not typically found in nature.

[0121] In some embodiments, the promoters provided herein are constitutive promoters. In yet another embodiment, the promoters provided herein are inductive promoters. In some embodiments, the promoters provided herein are selected from the group consisting of constitutive promoters, tissue-specific promoters, tissue-preferential promoters, and inductive promoters.

[0122] RNA polymerase III (PolIII) promoters can be used to drive the expression of non-protein-coding RNA molecules, such as guide RNA. In one embodiment, the promoter provided herein is a PolIII promoter. In another embodiment, the PolIII promoter provided herein is operably ligated to a nucleic acid molecule encoding non-protein-coding RNA. In yet another embodiment, the PolIII promoter provided herein is operably ligated to a nucleic acid molecule encoding a guide nucleic acid. In yet another embodiment, the PolIII promoter provided herein is operably ligated to a nucleic acid molecule encoding a single guide RNA. In yet another embodiment, the PolIII promoter provided herein is operably ligated to a nucleic acid molecule encoding CRISPR RNA (crRNA). In yet another embodiment, the PolIII promoter provided herein is operably ligated to a nucleic acid molecule encoding tracer RNA (tracrRNA). In some embodiments, nucleic acid molecules encoding non-protein-coding RNA (e.g., gRNA, single guide RNA, crRNA, tracrRNA, etc.) are operably linked to the PolIII promoter by preferential or specific excision of an intervening polynucleotide sequence in eggs, embryos, and / or meiotic plant tissues. In some embodiments, the intervening sequence is excised by a recombinase 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, in which Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues.

[0123] Non-limiting examples of PolIII promoters include the U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. See, for example, Schramm and Hernandez, 2002, Genes & Development, 16:2593–2620, which is incorporated herein by reference in its entirety. In one embodiment, the PolIII promoter provided herein is selected from the group consisting of the U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another embodiment, the guide RNA provided herein is operably ligated to a promoter selected from the group consisting of the U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another embodiment, the single guide RNA provided herein is operably ligated to a promoter selected from the group consisting of the U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another embodiment, the CRISPR RNA provided herein is operably ligated to a promoter selected from the group consisting of the U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another embodiment, the tracer RNA provided herein is operably ligated to a promoter selected from the group consisting of the U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter.

[0124] In one embodiment, the promoter provided herein is a dahlia mosaic virus (DaMV) promoter. In another embodiment, the promoter provided herein is a U6 promoter. In yet another embodiment, the promoter provided herein is an actin promoter. In one embodiment, the promoter provided herein is a cauliflower mosaic virus (CaMV) 35S promoter. In one embodiment, the promoter provided herein is a ubiquitin promoter.

[0125] In one embodiment, the constitutive promoter is selected from the group consisting of the CaMV 35S promoter, the actin promoter promoter, and the ubiquitin promoter.

[0126] Examples of promoters that may be used herein include, but are not limited to, U.S. Patent No. 6,437,217 (Maize RS81 Promoter), U.S. Patent No. 5,641,876 (Comeactin Promoter), U.S. Patent No. 6,426,446 (Maize RS324 Promoter), U.S. Patent No. 6,429,362 (Maize PR-1 Promoter), U.S. Patent No. 6,232,526 (Maize A3 Promoter), U.S. Patent No. 6,177,611 (Constitutive Maize Promoter), U.S. Patents No. 5,322,938, No. 5,352,605, No. 5,359,142 and No. 5,530,196 (35S Promoter), U.S. Examples include Japanese Patent No. 6,433,252 (Maize L3 Oleosin Promoter), U.S. Patent No. 6,429,357 (Comeactin 2 Promoter and Comeactin 2 Intron), U.S. Patent No. 5,837,848 (Root-Specific Promoter), U.S. Patent No. 6,294,714 (Photo-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 (Phosphorus Deficiency-Inducible Promoter), U.S. Patent No. 6,635,806 (Gamma-Coixin Promoter), and U.S. Patent Application No. 09 / 757,089 (Maize Chloroplast Aldolase Promoter).Further promoters that may be useful include the nopalin synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (supported on the tumor-inducing plasmid of Agrobacterium tumefaciens), Kalimovirus promoters, such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Molecular Biology (1987) 9: pp. 315-324), the CaMV 35S promoter (Odell et al., Nature (1985) 313: pp. 810-812), the scrophularia mosaic virus 35S promoter (US Patent No. 6,051,753; US Patent No. 5,378,619), and the sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA (1990)). These include the promoters for the R gene complex (87: pp. 4144-4148), the chlorophyll a / b binding protein gene promoter, PC1SV (US Patent No. 5,850,019), and the AGRtu.nos promoter (GenBank accession number V00087; Depicker et al., Journal of Molecular and Applied Genetics (1982) 1: pp. 561-573; Bevan et al., 1983).

[0127] Promoter hybrids can also be used and constructed to enhance transcriptional activity (see U.S. Patent No. 5,106,739), or to combine desired transcriptional activity, inducibility, and tissue-specificity or developmental specificity. Promoter functional in plants includes, but is not limited to, inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, and spatial-temporal regulated promoters. Other tissue-enhancing, tissue-specific, or developmentally regulated promoters are also known in the art and are expected to be useful in the practice of this disclosure.

[0128] In one embodiment, the constitutive promoter is operably ligated to a nucleic acid sequence encoding an inducible nuclease by Cre-mediated excision of an intervening Cre expression cassette, in which Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In another embodiment, the constitutive promoter is operably ligated to a nucleic acid sequence encoding a Cas9 nuclease by Cre-mediated excision of an intervening Cre expression cassette, in which Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In yet another embodiment, the constitutive promoter is operably ligated to a nucleic acid sequence encoding a Cas12a nuclease by Cre-mediated excision of an intervening Cre expression cassette, in which Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In one embodiment, the constitutive promoter is operably ligated to a nucleic acid sequence encoding a CasX nuclease by Cre-mediated excision of an intervening Cre expression cassette, in which Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In another embodiment, the constitutive promoter is operably ligated to a nucleic acid sequence encoding a guide nucleic acid by Cre-mediated excision of an intervening Cre expression cassette, in which Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In yet another embodiment, the constitutive promoter is operably ligated to a nucleic acid sequence encoding a guide RNA by Cre-mediated excision of an intervening Cre expression cassette, in which Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues. In one embodiment, a constitutive promoter is operably ligated to a nucleic acid sequence encoding a single guide RNA by Cre-mediated excision of an intervening Cre expression cassette, in which Cre is preferentially or selectively expressed in eggs, embryos, and / or meiotic plant tissues.

[0129] In some embodiments, the inductive promoter is operably ligated to a nucleic acid sequence encoding an inductive nuclease. In some embodiments, the inductive promoter is operably ligated to a nucleic acid sequence encoding a Cas9 nuclease. In some embodiments, the inductive promoter is operably ligated to a nucleic acid sequence encoding a Cas12a nuclease. In some embodiments, the inductive promoter is operably ligated to a nucleic acid sequence encoding a CasX nuclease. In some embodiments, the inductive promoter is operably ligated to a nucleic acid sequence encoding a guide nucleic acid. In some embodiments, the inductive promoter is operably ligated to a nucleic acid sequence encoding a guide RNA. In some embodiments, the inductive promoter is operably ligated to a nucleic acid sequence encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the inductive promoter is operably ligated to a nucleic acid sequence encoding a recombinase (e.g., Cre recombinase). In some embodiments, the inductive promoter is operably ligated to a nucleic acid sequence encoding a TALE.

[0130] In some embodiments, the developmental promoter is operably ligated to a nucleic acid sequence encoding an inducible nuclease. In some embodiments, the developmental promoter is operably ligated to a nucleic acid sequence encoding a Cas9 nuclease. In some embodiments, the developmental promoter is operably ligated to a nucleic acid sequence encoding a Cas12a nuclease. In some embodiments, the developmental promoter is operably ligated to a nucleic acid sequence encoding a CasX nuclease. In some embodiments, the developmental promoter is operably ligated to a nucleic acid sequence encoding a guide nucleic acid. In some embodiments, the developmental promoter is operably ligated to a nucleic acid sequence encoding a guide RNA. In some embodiments, the developmental promoter is operably ligated to a nucleic acid sequence encoding a single guide RNA. In some embodiments, the guide RNA acid is flanked by a self-cleaving ribozyme. In some embodiments, the developmental promoter is operably ligated to a nucleic acid sequence encoding a recombinase (e.g., Cre recombinase). In some embodiments, the developmental promoter is operably ligated to a nucleic acid sequence encoding a TALE.

[0131] As used herein, the term “leader” refers to the nucleotide segment between the transcription start site (TSS) and the protein coding sequence start site of a gene. It is isolated from the untranslated 5' region of the gene’s genomic copy. Leaders can be used as 5' regulatory elements to modulate the expression of operably linked transcriptable polynucleotide molecules. Leader molecules can be used with heterologous promoters or with their native promoters.

[0132] As used herein, the term “3' transcription termination molecule” or “3'UTR” refers to a DNA sequence used during transcription to produce the 3' untranslated region (3'UTR) of an mRNA molecule. The 3' untranslated region of an mRNA molecule, also known as the poly-A tail, can be generated by specific cleavage and 3' polyadenylation. The 3'UTR can be operably ligated to a transcriptionable polynucleotide molecule and positioned downstream thereof, which may contain polynucleotides that provide a polyadenylation signal and other regulatory signals that can influence transcription, mRNA processing, or gene expression. The poly-A tail is thought to function in mRNA stability and translation initiation. Examples of 3' transcription termination molecules in the art include the nopalin synthase 3' region (see Fraley et al., Proc. Natl. Acad. Sci. USA, 80: pp. 4803-4807 (1983)); the wheat hsp17 3' region; the endouloviscosmall subunit 3' region; the cotton E6 3' region (US Patent No. 6,096,950); the 3' region disclosed in WO0011200A2; and the coixin 3'UTR (US Patent No. 6,635,806). 3'UTRs are typically beneficial for the recombinant expression of specific genes. 3'UTRs can be used as 3' regulatory elements to modulate the expression of operably linked, transcriptable polynucleotide molecules. 3'UTRs can be used as 3' regulatory elements to modulate the tissue / cell preferential expression of operably linked, transcriptable polynucleotide molecules. 3'UTRs can be used with heterologous promoters or their native promoters. Non-limiting examples of 3'UTRs useful for carrying out the various embodiments described herein include Sequence IDs 46-64, 70-74, and 89-102.

[0133] DNA modification enzyme Some embodiments relate to compositions and methods for the preferential or specific expression of one or more components of a genome editing system in eggs, embryos, and / or meiotic plant tissues. Some embodiments relate to gene regulatory elements listed in Table 1, which are operably ligated to heterologous transcriptable DNA molecules encoding one or more components of a genome editing system. A genome editing system can be used to introduce one or more insertions, deletions, substitutions, base alterations, translocations, or inversions into the genome of a host cell. In some embodiments, the gene regulatory elements listed in Table 1 are operably ligated to heterologous transcriptable DNA molecules encoding a sequence-specific DNA modifying enzyme, such as a CRISPR-Cas effector protein, a zinc finger protein, or a transcription activator (TAL) protein. In some embodiments, the sequence-specific DNA modifying enzyme may be a fusion protein. In some embodiments, the sequence-specific DNA modifying enzyme may be an inducible nuclease.

[0134] Inducible nucleases are nucleases that form a complex (e.g., ribonucleoprotein) with a guide nucleic acid molecule (e.g., guide RNA) and then guide that complex to a target site within a target sequence. One non-limiting example of an inducible nuclease is the CRISPR nuclease.

[0135] CRISPR (clustered, short-interval, palindromic repeating structure) nucleases (e.g., Cas9, CasX, Cas12a (also known as Cpf1), CasY) are proteins found in bacteria that are guided to target nucleic acid molecules by guide RNA ("gRNA"), and the endonuclease can then cleave one or two strands of the target nucleic acid molecule. Although CRISPR nucleases originate in bacteria, many CRISPR nucleases have been shown to function in eukaryotic cells.

[0136] While not limited by any particular scientific theory, CRISPR nucleases form a complex with a guide RNA (gRNA) that hybridizes with a complementary target site, thereby guiding the CRISPR nuclease to the target site. In a Class II CRISPR-Cas system, a CRISPR array containing spacers is transcribed upon encounter with 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 protospacer sequence in the invading pathogen. The spacer sequence can be designed to be complementary to a target sequence in the eukaryotic genome.

[0137] In some embodiments, the gene regulatory elements described herein are operably ligated to heterologous transcriptionable DNA molecules encoding CRISPR-Cas effector proteins. In some embodiments, the CRISPR-Cas effector proteins are selected from the Type I CRISPR-Cas system, Type II CRISPR-Cas system, Type III CRISPR-Cas system, Type IV CRISPR-Cas system, Type V CRISPR-Cas system, or Type VI CRISPR-Cas system. Examples of CRISPR-Cas effector proteins, though not limited to them, include Cas9, C2c1, C2c3, C2c4, C2c5, C2c8, C2c9, C2c10, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3'', Cas4, Cas5, Cas6, Cas7, Cas8, and Cas9. Examples include 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, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), Csf5, Cas14a, Cas14b, and Cas14c effector proteins. In some embodiments, the gene regulatory elements described herein are operably ligated to CRISPR-Cas effector proteins that contain mutations in their nuclease active sites (e.g., RuvC, HNH, and / or NUC domains). CRISPR-Cas effector proteins that have mutations in their nuclease active sites and therefore no longer contain nuclease activity are generally referred to as "dead," e.g., dCas.In some embodiments, a CRISPR-Cas effector protein domain or polypeptide having a mutation in its nuclease active site may have impaired or reduced activity compared to the same CRISPR-Cas effector protein without the mutation. In some embodiments, the gene regulatory elements described herein are operably ligated to a CRISPR-Cas effector protein having a mutation in its nuclease active site to generate nickas activity operably ligated to reverse transcriptase.

[0138] CRISPR effector proteins bind to their corresponding crRNAs in their active form. Similar to the class II endonuclease Cas9, CasX requires another non-coding RNA component, called trans-activating crRNA (tracrRNA), to have functional activity. The nucleic acid molecules provided herein can combine crRNA and tracrRNA into a single nucleic acid molecule, referred herein to as “single guide RNA” (sgRNA). Cas12a does not require the tracrRNA to be guided to the target site; for Cas12a, crRNA alone is sufficient. The gRNA guides the active CRISPR nuclease complex to the target site, where the CRISPR nuclease can cleave the target site.

[0139] When a CRISPR effector protein and guide RNA form a complex, the entire system is called a "ribonucleoprotein." The ribonucleoproteins provided herein may also include further nucleic acids or proteins.

[0140] In one embodiment, the CRISPR effector protein and guide nucleic acid form a ribonucleoprotein in the egg cell. In another embodiment, the CRISPR effector protein and guide nucleic acid form a ribonucleoprotein in the embryonic tissue. In one embodiment, the Cas9 nuclease and guide nucleic acid form a ribonucleoprotein in the egg cell. In another embodiment, the Cas9 nuclease and guide nucleic acid form a ribonucleoprotein in the embryonic tissue. In one embodiment, the Cas12a nuclease and guide nucleic acid form a ribonucleoprotein in the egg cell. In another embodiment, the Cas12a nuclease and guide nucleic acid form a ribonucleoprotein in the embryonic tissue. In one embodiment, the CasX nuclease and guide nucleic acid form a ribonucleoprotein in the egg cell. In another embodiment, the CasX nuclease and guide nucleic acid form a ribonucleoprotein in the embryonic tissue. In one embodiment, the CRISPR effector protein and guide RNA form a ribonucleoprotein in the egg cell. In another embodiment, the CRISPR effector protein and guide RNA form ribonucleoprotein in embryonic tissue. In one embodiment, the Cas9 nuclease and guide RNA form ribonucleoprotein in egg cells. In another embodiment, the Cas9 nuclease and guide RNA form ribonucleoprotein in embryonic tissue. In one embodiment, the Cas12a nuclease and guide RNA form ribonucleoprotein in egg cells. In another embodiment, the Cas12a nuclease and guide RNA form ribonucleoprotein in embryonic tissue. In one embodiment, the CasX nuclease and guide RNA form ribonucleoprotein in egg cells. In another embodiment, the CasX nuclease and guide RNA form ribonucleoprotein in embryonic tissue. In one embodiment, the inducible nuclease and single guide RNA form ribonucleoprotein in egg cells. In another embodiment, the inducible nuclease and single guide RNA form ribonucleoprotein in embryonic tissue. In another embodiment, CasX nuclease and a single guide RNA form a ribonucleoprotein in embryonic tissue. In yet another embodiment, Cas9 nuclease and a single guide RNA form a ribonucleoprotein in embryonic tissue.

[0141] In one embodiment, the ribonucleoprotein generates at least one double-strand break at a target site in an egg cell. In another embodiment, the ribonucleoprotein generates at least one double-strand break at a target site in embryonic tissue. In another embodiment, the ribonucleoprotein generates at least one single-strand break at a target site in an egg cell. In another embodiment, the ribonucleoprotein generates at least one single-strand break at a target site in embryonic tissue.

[0142] A prerequisite for cleavage of a target site by CRISPR ribonucleoproteins is the presence of a conserved protospacer-adjacent motif (PAM) near the target site. Depending on the CRISPR nuclease, cleavage can occur within a certain number of nucleotides from the PAM site (e.g., between 18 and 23 nucleotides for Cas12a). PAM sites are required only for type I and type II CRISPR-related proteins, and different CRISPR endonucleases recognize different PAM sites. Cas12a can recognize at least the following PAM sites: TTTN and YTN; CasX can recognize at least the following PAM sites: TTCN, TTCA, and TTC (where T is thymine; C is cytosine; A is adenine; Y is thymine or cytosine; N is thymine, cytosine, guanine, or adenine).

[0143] Cas12a is a class II, type V CRISPR / Cas system RNA-induced nuclease. When Cas12a nucleases cleave double-stranded DNA molecules, they produce alternating breaks. These alternating breaks result in a single-stranded DNA overhang of at least one nucleotide. This is in contrast to blunt-end breaks (such as those produced by Cas9), which do not result in a single-stranded DNA overhang when cleaving double-stranded DNA.

[0144] In one embodiment, the Cas12a nuclease provided herein is the Cas12a (LbCas12a) nuclease of a Lachnospiraceae bacterium. In another embodiment, the Cas12a nuclease provided herein is the Cas12a (FnCas12a) nuclease of Francisella novicida. In one embodiment, the Cas12a nuclease is selected from the group consisting of LbCas12a and FnCas12a.

[0145] In one embodiment, Cas12a nuclease, or the nucleic acid encoding Cas12a nuclease, is derived from Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, and Azospirillum. Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridia Idium), Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfov ibrio), Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, Acidaminococcus, Peregrinibacteria, Butyrivibrio, Parcubacteria,It is derived from a bacterial genus selected from the group consisting of Smithella, Candidatus, Moraxella, and Leptospira.

[0146] In one embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 80% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 7. In another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 85% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 7. In yet another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 90% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 7. In yet another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 95% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 7. In yet another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 96% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 7. In yet another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 97% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 7. In another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 98% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 7. In another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 99% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 7. In yet another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is 100% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 7.

[0147] CasX is a type of class II CRISPR-Cas nuclease identified in the phylum Bacteria, specifically in the Deltaproteobacteria and Planctomyces. Similar to Cas12a, CasX nucleases produce alternating cleavage 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.

[0148] In one embodiment, the CasX nuclease provided herein is a CasX nuclease derived from the phylum Deltaproteobacteria. In another embodiment, the CasX nuclease provided herein is a CasX nuclease derived from the phylum Planctomyces. Further preferred CasX nucleases, though not limited thereto, are those described in WO 2019 / 084148, which are incorporated herein by reference in their entirety.

[0149] In one embodiment, the inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule is selected from the group consisting of Cas12a and CasX.

[0150] In one embodiment, the inducible nuclease is an RNA-inducible nuclease. In another embodiment, the inducible nuclease is a CRISPR nuclease. In yet another embodiment, the inducible nuclease is a Cas12a nuclease. In yet another embodiment, the inducible nuclease is a CasX nuclease.

[0151] As used herein, “nuclear localization signal” (NLS) refers to an amino acid sequence that “tags” a protein for transport to the nucleus of a cell. In one embodiment, the nucleic acid molecules provided herein encode a nuclear localization signal. In another embodiment, the nucleic acid molecules provided herein encode two or more nuclear localization signals.

[0152] In some embodiments, the CRISPR effector protein provided herein includes a nuclear localization signal. In some embodiments, the Cas9 effector protein provided herein includes a nuclear localization signal. In some embodiments, the nuclear localization signal is located on the N-terminus of the Cas12a nuclease. In further embodiments, the nuclear localization signal is located on the C-terminus of the Cas9 effector protein. In yet another embodiment, the nuclear localization signal is located on both the N-terminus and the C-terminus of the Cas9 effector protein.

[0153] In some embodiments, the Cas12a effector protein provided herein includes a nuclear localization signal. In some embodiments, the nuclear localization signal is located on the N-terminus of the Cas12a effector protein. In further embodiments, the nuclear localization signal is located on the C-terminus of the Cas12a effector protein. In yet another embodiment, the nuclear localization signal is located on both the N-terminus and the C-terminus of the Cas12a effector protein.

[0154] In some embodiments, the CasX effector protein provided herein includes a nuclear localization signal. In some embodiments, the nuclear localization signal is located on the N-terminus of the CasX effector protein. In further embodiments, the nuclear localization signal is located on the C-terminus of the CasX effector protein. In yet another embodiment, the nuclear localization signal is located on both the N-terminus and the C-terminus of the CasX effector protein.

[0155] In one embodiment, the ribonucleoprotein includes at least one nuclear localization signal. In another embodiment, the ribonucleoprotein includes at least two nuclear localization signals. In one embodiment, the nuclear localization signals provided herein are encoded by SEQ ID NO: 8 or 9.

[0156] Various species exhibit specific biases for certain codons of particular amino acids. Codon bias (differences in codon use between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, and subsequently, in particular, depends on the characteristics of the translated codon and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell is generally a reflection of the codons most frequently used in peptide synthesis. Therefore, genes can be tuned for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the "Codon Usage Database" available at www.kazusa.codon or www.codon.jp, and these tables can be adapted in several ways. See Nakamura et al., 2000, Nucl. Acids Res. 28:292. Computer algorithms are available for codon-optimizing specific sequences for expression in specific plant cells, such as Gene Forge (Aptagen; Jacobus, PA).

[0157] As used herein, “codon optimization” refers to the process of modifying a nucleic acid sequence to enhance expression in a target plant cell by replacing at least one codon in a given sequence (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with a codon that is more frequently or most frequently used in the gene of the plant cell, while maintaining the original amino acid sequence (e.g., by introducing silent mutations).

[0158] In one embodiment, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all of the codons) in the sequence encoding the inducible nuclease correspond to the most frequently used codon for a particular amino acid. In another embodiment, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all of the codons) in the sequence encoding the Cas9 effector protein, Cas12a effector protein, or CasX effector protein correspond to the most frequently used codon for a particular amino acid. For codon use in plants, see Campbell and Gowri, 1990, Plant Physiol., 92: pp. 1-11; and Murray et al., 1989, Nucleic Acids Res., 17: pp. 477-98, respectively, which are incorporated herein by reference in their entirety.

[0159] In one embodiment, the nucleic acid molecule encodes an inducible nuclease that is codon-optimized for plants. In another embodiment, the nucleic acid molecule encodes a Cas9 effector protein that is codon-optimized for plants. In another embodiment, the nucleic acid molecule encodes a Cas12a effector protein that is codon-optimized for plants. In another embodiment, the nucleic acid molecule encodes a CasX effector protein that is codon-optimized for plants.

[0160] In another embodiment, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for plant cells. In yet another embodiment, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for monocotyledonous plant species. In yet another embodiment, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for dicotyledonous plant species. In yet another embodiment, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for gymnosperm species. In yet another embodiment, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for angiosperm species. In yet another embodiment, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for maize cells. In yet another embodiment, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for soybean cells. In yet another embodiment, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for rice cells. In further embodiments, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for wheat cells. In further embodiments, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for cotton cells. In further embodiments, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for sorghum cells. In further embodiments, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for alfalfa cells. In further embodiments, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for sugarcane cells. In further embodiments, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for Arabidopsis cells. In further embodiments, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for tomato cells. In further embodiments, the nucleic acid molecules provided herein encode codon-optimized inducible nucleases for cucumber cells.In a further embodiment, the nucleic acid molecule provided herein encodes an inducible nuclease that is codon-optimized for potato cells. In a further embodiment, the nucleic acid molecule provided herein encodes an inducible nuclease that is codon-optimized for onion cells.

[0161] In another embodiment, the nucleic acid molecule provided herein encodes a Cas12a effector protein that is codon-optimized for plant cells. In yet another embodiment, the nucleic acid molecule provided herein encodes a Cas12a effector protein that is codon-optimized for monocotyledonous plant species. In yet another embodiment, the nucleic acid molecule provided herein encodes a Cas12a effector protein that is codon-optimized for dicotyledonous plant species. In yet another embodiment, the nucleic acid molecule provided herein encodes a Cas12a effector protein that is codon-optimized for gymnosperm species. In yet another embodiment, the nucleic acid molecule provided herein encodes a Cas12a effector protein that is codon-optimized for angiosperm species. In yet another embodiment, the nucleic acid molecule provided herein encodes a Cas12a effector protein that is codon-optimized for maize cells. In yet another embodiment, the nucleic acid molecule provided herein encodes a Cas12a effector protein that is codon-optimized for soybean cells. In further embodiments, the nucleic acid molecules provided herein encode a Cas12a effector protein that is codon-optimized for rice cells. In further embodiments, the nucleic acid molecules provided herein encode a Cas12a effector protein that is codon-optimized for wheat cells. In further embodiments, the nucleic acid molecules provided herein encode a Cas12a effector protein that is codon-optimized for cotton cells. In further embodiments, the nucleic acid molecules provided herein encode a Cas12a effector protein that is codon-optimized for sorghum cells. In further embodiments, the nucleic acid molecules provided herein encode a Cas12a effector protein that is codon-optimized for alfalfa cells. In further embodiments, the nucleic acid molecules provided herein encode a Cas12a effector protein that is codon-optimized for sugarcane cells. In further embodiments, the nucleic acid molecules provided herein encode a Cas12a effector protein that is codon-optimized for Arabidopsis thaliana cells.In a further embodiment, the nucleic acid molecule provided herein encodes a Cas12a effector protein that is codon-optimized for tomato cells. In a further embodiment, the nucleic acid molecule provided herein encodes a Cas12a effector protein that is codon-optimized for cucumber cells. In a further embodiment, the nucleic acid molecule provided herein encodes a Cas12a effector protein that is codon-optimized for potato cells. In a further embodiment, the nucleic acid molecule provided herein encodes a Cas12a effector protein that is codon-optimized for onion cells.

[0162] In another embodiment, the nucleic acid molecule provided herein encodes a CasX effector protein that is codon-optimized for plant cells. In yet another embodiment, the nucleic acid molecule provided herein encodes a CasX effector protein that is codon-optimized for monocotyledonous plant species. In yet another embodiment, the nucleic acid molecule provided herein encodes a CasX effector protein that is codon-optimized for dicotyledonous plant species. In yet another embodiment, the nucleic acid molecule provided herein encodes a CasX effector protein that is codon-optimized for gymnosperm species. In yet another embodiment, the nucleic acid molecule provided herein encodes a CasX effector protein that is codon-optimized for angiosperm species. In yet another embodiment, the nucleic acid molecule provided herein encodes a CasX effector protein that is codon-optimized for maize cells. In yet another embodiment, the nucleic acid molecule provided herein encodes a CasX effector protein that is codon-optimized for soybean cells. In yet another embodiment, the nucleic acid molecule provided herein encodes a CasX effector protein that is codon-optimized for rice cells. In further embodiments, the nucleic acid molecules provided herein encode a CasX effector protein that is codon-optimized for wheat cells. In further embodiments, the nucleic acid molecules provided herein encode a CasX effector protein that is codon-optimized for cotton cells. In further embodiments, the nucleic acid molecules provided herein encode a CasX effector protein that is codon-optimized for sorghum cells. In further embodiments, the nucleic acid molecules provided herein encode a CasX effector protein that is codon-optimized for alfalfa cells. In further embodiments, the nucleic acid molecules provided herein encode a CasX effector protein that is codon-optimized for sugarcane cells. In further embodiments, the nucleic acid molecules provided herein encode a CasX effector protein that is codon-optimized for Arabidopsis thaliana cells. In further embodiments, the nucleic acid molecules provided herein encode a CasX effector protein that is codon-optimized for tomato cells.In a further embodiment, the nucleic acid molecule provided herein encodes a CasX effector protein that is codon-optimized for cucumber cells. In a further embodiment, the nucleic acid molecule provided herein encodes a CasX effector protein that is codon-optimized for potato cells. In a further embodiment, the nucleic acid molecule provided herein encodes a CasX effector protein that is codon-optimized for onion cells.

[0163] Guide nucleic acids As used herein, “guide nucleic acid” refers to a nucleic acid that, after forming a ribonucleoprotein (e.g., a complex) with a CRISPR effector protein (e.g., Cas9, Cas12a, CasX, but not limited to), guides the ribonucleoprotein to a specific sequence in a target nucleic acid molecule, where the guide nucleic acid and the target nucleic acid molecule share a complementary sequence. In some embodiments, the ribonucleoprotein provided herein comprises at least one guide nucleic acid.

[0164] In one embodiment, the guide nucleic acid includes DNA. In another embodiment, the guide nucleic acid includes RNA. In one embodiment, the guide nucleic acid includes DNA, RNA, or a combination thereof. In one embodiment, the guide nucleic acid is single-stranded. In another embodiment, the guide nucleic acid is at least partially double-stranded.

[0165] If the guide nucleic acid contains RNA, it can also be called "guide RNA." In another embodiment, the guide nucleic acid contains both DNA and RNA. In another embodiment, the guide RNA is single-stranded. In yet another embodiment, the guide RNA is double-stranded. In yet another embodiment, the guide RNA is partially double-stranded.

[0166] In one embodiment, the guide nucleic acid includes guide RNA. In another embodiment, the guide nucleic acid includes at least one guide RNA. In another embodiment, the guide nucleic acid includes at least two guide RNAs. In another embodiment, the guide nucleic acid includes at least three guide RNAs. In another embodiment, the guide nucleic acid includes at least five guide RNAs. In another embodiment, the guide nucleic acid includes at least ten guide RNAs.

[0167] In another embodiment, the guide nucleic acid contains at least 10 nucleotides. In another embodiment, the guide nucleic acid contains at least 11 nucleotides. In another embodiment, the guide nucleic acid contains at least 12 nucleotides. In another embodiment, the guide nucleic acid contains at least 13 nucleotides. In another embodiment, the guide nucleic acid contains at least 14 nucleotides. In another embodiment, the guide nucleic acid contains at least 15 nucleotides. In another embodiment, the guide nucleic acid contains at least 16 nucleotides. In another embodiment, the guide nucleic acid contains at least 17 nucleotides. In another embodiment, the guide nucleic acid contains at least 18 nucleotides. In another embodiment, the guide nucleic acid contains at least 19 nucleotides. In another embodiment, the guide nucleic acid contains at least 20 nucleotides. In another embodiment, the guide nucleic acid contains at least 21 nucleotides. In another embodiment, the guide nucleic acid contains at least 22 nucleotides. In another embodiment, the guide nucleic acid contains at least 23 nucleotides. In another embodiment, the guide nucleic acid contains at least 24 nucleotides. In another embodiment, the guide nucleic acid contains at least 25 nucleotides. In another embodiment, the guide nucleic acid contains at least 26 nucleotides. In another embodiment, the guide nucleic acid contains at least 27 nucleotides. In another embodiment, the guide nucleic acid contains at least 28 nucleotides. In another embodiment, the guide nucleic acid contains at least 30 nucleotides. In another embodiment, the guide nucleic acid contains at least 35 nucleotides. In another embodiment, the guide nucleic acid contains at least 40 nucleotides. In another embodiment, the guide nucleic acid contains at least 45 nucleotides. In another embodiment, the guide nucleic acid contains at least 50 nucleotides.

[0168] In another embodiment, the guide nucleic acid contains 10 to 50 nucleotides. In another embodiment, the guide nucleic acid contains 10 to 40 nucleotides. In another embodiment, the guide nucleic acid contains 10 to 30 nucleotides. In another embodiment, the guide nucleic acid contains 10 to 20 nucleotides. In another embodiment, the guide nucleic acid contains 16 to 28 nucleotides. In another embodiment, the guide nucleic acid contains 16 to 25 nucleotides. In another embodiment, the guide nucleic acid contains 16 to 20 nucleotides.

[0169] In some embodiments, the guide nucleic acid includes at least 70% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 75% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 80% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 85% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 90% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 91% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 92% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 93% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 94% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 95% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 96% sequence complementarity to the target site. In one embodiment, the guide nucleic acid contains at least 97% sequence complementarity to the target site. In another embodiment, the guide nucleic acid contains at least 98% sequence complementarity to the target site. In another embodiment, the guide nucleic acid contains at least 99% sequence complementarity to the target site. In another embodiment, the guide nucleic acid contains 100% sequence complementarity to the target site. In yet another embodiment, the guide nucleic acid contains 70% to 100% sequence complementarity to the target site. In yet another embodiment, the guide nucleic acid contains 80% to 100% sequence complementarity to the target site. In yet another embodiment, the guide nucleic acid contains 90% to 100% sequence complementarity to the target site. In one embodiment, the guide nucleic acid can hybridize to the target site.

[0170] As described above, some inducible nucleases, such as CasX and Cas9, require another non-coding RNA component, referred to as transactivating crRNA (tracrRNA), to have functional activity. The guide nucleic acid molecules provided herein can combine crRNA and tracrRNA into a single nucleic acid molecule referred to herein as “single guide RNA” (sgRNA). The gRNA guides the active CasX complex to a target site in the target sequence, where CasX can cleave the target site. In other embodiments, crRNA and tracrRNA are provided as separate nucleic acid molecules. In one embodiment, the guide nucleic acid comprises crRNA. In another embodiment, the guide nucleic acid comprises tracrRNA. In yet another embodiment, the guide nucleic acid comprises sgRNA.

[0171] target site As used herein, “target sequence” refers to a selected sequence or region of a DNA molecule that is to be modified (e.g., cleavage, deamination, site-directed integration). The target sequence includes the target site.

[0172] As used herein, “target site” refers to the portion of a target sequence that is modified (e.g., cleaved) by a CRISPR effector protein. In contrast to non-target nucleic acids (e.g., non-target ssDNA) or non-target regions, a target site involves significant complementarity to a guide nucleic acid or guide RNA.

[0173] In one embodiment, the target site is 100% complementary to the guide nucleic acid. In another embodiment, the target site is 99% complementary to the guide nucleic acid. In another embodiment, the target site is 98% complementary to the guide nucleic acid. In another embodiment, the target site is 97% complementary to the guide nucleic acid. In another embodiment, the target site is 96% complementary to the guide nucleic acid. In another embodiment, the target site is 95% complementary to the guide nucleic acid. In another embodiment, the target site is 94% complementary to the guide nucleic acid. In another embodiment, the target site is 93% complementary to the guide nucleic acid. In another embodiment, the target site is 92% complementary to the guide nucleic acid. In another embodiment, the target site is 91% complementary to the guide nucleic acid. In another embodiment, the target site is 90% complementary to the guide nucleic acid. In another embodiment, the target site is 85% complementary to the guide nucleic acid. In another embodiment, the target site is 80% complementary to the guide nucleic acid.

[0174] In one embodiment, the target site includes at least one PAM site. In another embodiment, the target site is adjacent to a nucleic acid sequence including at least one PAM site. In yet another embodiment, the target site is within 5 nucleotides of at least one PAM site. In yet another embodiment, the target site is within 10 nucleotides of at least one PAM site. In yet another embodiment, the target site is within 15 nucleotides of at least one PAM site. In yet another embodiment, the target site is within 20 nucleotides of at least one PAM site. In yet another embodiment, the target site is within 25 nucleotides of at least one PAM site. In yet another embodiment, the target site is within 30 nucleotides of at least one PAM site.

[0175] In one embodiment, the target site is located within gene DNA. In another embodiment, the target site is located within a gene. In yet another embodiment, the target site is located within the target gene. In yet another embodiment, the target site is located within an exon of a gene. In yet another embodiment, the target site is located within an intron of a gene. In yet another embodiment, the target site is located within the 5'-UTR of a gene. In yet another embodiment, the target site is located within the 3'-UTR of a gene. In yet another embodiment, the target site is located within intergenetic DNA.

[0176] In one embodiment, the target DNA molecule is single-stranded. In another embodiment, the target DNA molecule is double-stranded.

[0177] In some embodiments, the target sequence includes genomic DNA. In some embodiments, the target sequence is located within the nuclear genome. In some embodiments, the target sequence includes chromosomal DNA. In some embodiments, the target sequence includes plasmid DNA. In some embodiments, the target sequence is located within a plasmid. In some embodiments, the target sequence includes mitochondrial DNA. In some embodiments, the target sequence is located within the mitochondrial genome. In some embodiments, the target sequence includes plastid DNA. In some embodiments, the target sequence is located within the plastid genome. In some embodiments, the target sequence includes chloroplast DNA. In some embodiments, the target sequence is located within the chloroplast genome. In some embodiments, the target sequence is located within a genome selected from the group consisting of the nuclear genome, the mitochondrial genome, and the plastid genome.

[0178] In one embodiment, the target sequence includes gene DNA. As used herein, “gene DNA” means DNA that codes for one or more genes. In another embodiment, the target sequence includes intergenetic DNA. In contrast to gene DNA, “intergenetic DNA” includes non-coding DNA and lacks DNA that codes for genes. In one embodiment, intergenetic DNA is located between two genes.

[0179] In one embodiment, the target sequence encodes a gene. As used herein, “gene” means a polynucleotide capable of producing a functional unit (e.g., a protein or a non-coding RNA molecule). A gene may 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, a 5'-UTR, a 3'-UTR, or any combination thereof. A “gene sequence” may 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, a 5'-UTR, a 3'-UTR, or any combination thereof. In one embodiment, the gene encodes a non-protein-coding RNA molecule or its precursor. In another embodiment, the gene encodes a protein. In some embodiments, the target sequence is selected from the group consisting of promoters, enhancer sequences, leader sequences, transcription start sites, transcription stop sites, polyadenylation sites, exons, introns, splice sites, 5'-UTR, 3'-UTR, protein-coding sequences, non-protein-coding sequences, miRNAs, pre-miRNAs, and miRNA-binding sites.

[0180] Non-protein-coding RNA molecules include, but are not limited to, microRNAs (miRNAs), miRNA precursors (premiRNAs), small interfering RNAs (siRNAs), small RNAs (18-26 nucleotides in length) and their encoding precursors, heterochromatin siRNAs (hc-siRNAs), Piwi-binding RNAs (piRNAs), hairpin double-stranded RNAs (hairpin dsRNAs), trans-acting siRNAs (ta-siRNAs), naturally occurring antisense siRNAs (nat-siRNAs), CRISPR RNAs (crRNAs), tracer RNAs (tracrRNAs), guide RNAs (gRNAs), and single guide RNAs (sgRNAs). In some embodiments, non-protein-coding RNA molecules include miRNAs. In some embodiments, non-protein-coding RNA molecules include siRNAs. In some embodiments, non-protein-coding RNA molecules include ta-siRNAs. In some embodiments, non-protein-coding RNA molecules are selected from the group consisting of miRNAs, siRNAs, and ta-siRNAs.

[0181] As used herein, “the gene of interest” means a polynucleotide sequence encoding a protein or non-protein-coding RNA molecule to be incorporated into a target sequence, or an endogenous polynucleotide sequence encoding a protein or non-protein-coding RNA molecule to be edited by a ribonucleoprotein. In some embodiments, the gene of interest encodes a protein. In other embodiments, the gene of interest encodes a non-protein-coding RNA molecule. In some embodiments, the gene of interest is exogenous with respect to the target DNA molecule. In some embodiments, the gene of interest replaces an endogenous gene in the target DNA molecule.

[0182] mutation In some embodiments, the ribonucleoprotein or method provided herein generates at least one mutation in a target sequence of an egg, embryo, and / or meiotic cell.

[0183] In one embodiment, seeds produced from the plants provided herein contain at least one mutation in the gene of interest, including the target site, compared to seeds from a control plant of the same lineage or variety that lacks a first nucleic acid sequence encoding an inducible nuclease operably linked to an egg cell-preferential promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter. In one embodiment, seeds produced from the plants provided herein contain at least one mutation in the gene of interest, including the target site, compared to seeds from a control plant of the same lineage or variety that lacks a first nucleic acid sequence encoding an inducible nuclease operably linked to an embryo tissue-preferential promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter.

[0184] In one embodiment, seeds produced from the plants provided herein contain at least one mutation in the gene of interest, including the target site, compared to seeds from a control plant of the same lineage or variety that lacks a first nucleic acid sequence encoding an inducible 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-preferential promoter. In one embodiment, seeds produced from the plants provided herein contain at least one mutation in the gene of interest, including the target site, compared to seeds from a control plant of the same lineage or variety that lacks a first nucleic acid sequence encoding an inducible nuclease operably linked to a heterologous promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to an embryo tissue-preferential promoter.

[0185] As used herein, “mutation” refers to a modification to a nucleic acid or amino acid sequence that does not exist in nature, compared to a naturally occurring reference nucleic acid or amino acid sequence from the same organism. When identifying mutations, it will be understood that the reference sequence should be derived from the same nucleic acid (e.g., gene, non-coding RNA) or amino acid (e.g., protein). It will be understood in the art that when determining whether a difference between two sequences constitutes a mutation, comparisons should not be made between homologous sequences of two different species or between homologous sequences of two different varieties of a single species. Rather, comparisons should be made between an edited (e.g., mutated) sequence and an endogenous, unedited (e.g., “wild-type”) sequence from the same organism.

[0186] Several types of mutations are known in the art. In some embodiments, a mutation includes an insertion. "Insertion" means the addition of one or more nucleotides or amino acids to a given polynucleotide or amino acid chain sequence, compared to an endogenous reference polynucleotide or amino acid sequence. In other embodiments, a mutation includes a deletion. "Deletion" means the removal of one or more nucleotides or amino acids to a given polynucleotide or amino acid chain sequence, compared to an endogenous reference polynucleotide or amino acid sequence. In other embodiments, a mutation includes a substitution. "Substitution" means the replacement of one or more nucleotides or amino acids to a given polynucleotide or amino acid chain sequence, compared to an endogenous reference polynucleotide or amino acid sequence. In other embodiments, a mutation includes an inversion. "Inversion" means that a segment of a polynucleotide or amino acid sequence is reversed from end to end. In some embodiments, the mutations provided herein include mutations selected from the group consisting of insertions, deletions, substitutions, and inversions.

[0187] In one embodiment, a plant or seed contains at least one mutation in the gene of interest, the at least one mutation resulting in the deletion of one or more amino acids from the protein encoded by the gene of interest, compared to the wild-type protein.

[0188] In one embodiment, a plant or seed contains at least one mutation in the gene of interest, the at least one mutation resulting in one or more amino acid substitutions in the protein encoded by the gene of interest compared to the wild-type protein.

[0189] In one embodiment, a plant or seed contains at least one mutation in the gene of interest, the at least one mutation resulting in the insertion of one or more amino acids into the protein encoded by the gene of interest, compared to the wild-type protein.

[0190] Mutations in the coding region of a gene (e.g., exon mutations) can result in a truncated protein or polypeptide when the mutated messenger RNA (mRNA) is translated into a protein or polypeptide. In some embodiments, this disclosure provides mutations that result in the truncation of a protein or polypeptide. As used herein, a “truncated” protein or polypeptide contains at least one fewer amino acid than an endogenous control protein or polypeptide. For example, if endogenous protein A contains 100 amino acids, the truncated protein A may contain 1 to 99 amino acids.

[0191] While not limited by any scientific theory, one method for causing truncation of a protein or polypeptide is by introducing an immature stop codon into the mRNA transcript of an endogenous gene. In some embodiments, this disclosure provides mutations that result in an immature stop codon in the mRNA transcript of an endogenous gene. As used herein, “stop codon” refers to a nucleotide triplet in an mRNA transcript that signals the termination of protein translation. “Immature stop codon” refers to a stop codon located earlier (e.g., 5') than the normal stop codon position in the endogenous mRNA transcript. Several stop codons are known in the art, including, but are not limited to, “UAG”, “UAA”, “UGA”, “TAG”, “TAA”, and “TGA”.

[0192] In one embodiment, the seed or plant contains at least one mutation, the mutation resulting in the introduction of an immature stop codon into the messenger RNA encoded by the gene of interest, compared to the wild-type messenger RNA.

[0193] In some embodiments, the mutations provided herein include null mutations. As used herein, “null mutation” means a mutation that results in complete loss of function for a protein encoded by the gene containing the mutation, or a mutation that results in complete loss of function for a small RNA encoded by a genomic locus. A null mutation may result in a lack of mRNA transcript production, a lack of small RNA transcript production, a lack of protein function, or a combination thereof.

[0194] The mutations provided herein may be located in any part of an endogenous gene. In one embodiment, the mutations provided herein are located within an exon of the endogenous gene. In another embodiment, the mutations provided herein are located within an intron of the endogenous gene. In yet another embodiment, the mutations provided herein are located within the 5' untranslated region of the endogenous gene. In yet another embodiment, the mutations provided herein are located within the 3' untranslated region of the endogenous gene. In yet another embodiment, the mutations provided herein are located within the promoter of the endogenous gene.

[0195] In some embodiments, mutations are located at splice sites within a gene. Mutations at splice sites can disrupt the splicing of exons during mRNA processing. Splicing can be perturbed if one or more nucleotides are inserted, deleted, or substituted at a splice site. Perturbed splicing can result in unspliced ​​introns, lost exons, or both, derived from the mature mRNA sequence. Typically, but not always, proper splicing requires a "GU" sequence at the 5' end of an intron and an "AG" sequence at the 3' end of an intron. If either of these splice sites is mutated, splicing can be perturbed.

[0196] In one embodiment, the seed or plant contains at least one mutation, the at least one mutation containing the deletion of one or more splice sites from the gene of interest. In another embodiment, the seed or plant contains at least one mutation, the at least one mutation located within one or more splice sites from the gene of interest.

[0197] In some embodiments, the mutation includes site-directed integration. In some embodiments, site-directed integration includes the insertion of all or part of a desired sequence into a target sequence.

[0198] As used herein, “site-directed integration” means all or part of a desired sequence (e.g., an exogenous gene, an edited endogenous gene) that is inserted into or integrated into a desired site or locus (e.g., a target sequence) in the plant genome. As used herein, “desired sequence” means a DNA molecule containing a nucleic acid sequence to be integrated into the genome of a plant or plant cell. The desired sequence may include a transgene or a construct. In some embodiments, the nucleic acid molecule containing the desired sequence includes one or two homology arms flanking the desired sequence to facilitate an insertion event targeted by homologous recombination and / or homology-directed repair.

[0199] In some embodiments, the methods provided herein include site-specific insertion of a desired sequence into a target sequence.

[0200] Any site or locus within the plant genome can be selected for site-specific integration of the transgene or construct of the disclosure. In some embodiments, the target sequence is located within B, or an extra chromosome.

[0201] For site-directed integration, a double-strand break (DSB) or nick can be first created at the target sequence via an inducible nuclease or ribonucleoprotein provided herein. Then, in the presence of the desired sequence, the DSB or nick can be repaired by homologous recombination (HR) between 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 and creating a targeted insertion event at the site of the DSB or nick.

[0202] In one embodiment, site-directed integration includes the use of an endogenous NHEJ repair mechanism for the cell. In another embodiment, site-directed integration includes the use of an endogenous HR repair mechanism for the cell.

[0203] In one embodiment, double-strand break repair generates at least one mutation in the gene of interest compared to a control plant of the same lineage or variety.

[0204] In some embodiments, the mutation includes the incorporation of at least five consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least ten consecutive nucleotides of the desired sequence molecule into the target sequence. In some embodiments, the mutation includes the incorporation of at least fifteen consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least twenty consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least twenty-five consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least fifty consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least one hundred consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least two hundred consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least two hundred consecutive nucleotides of the desired sequence into the target sequence. In one embodiment, the mutation includes the incorporation of at least 1,000 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation includes the incorporation of at least 2,000 consecutive nucleotides of the desired sequence into the target sequence.

[0205] In one embodiment, the mutation includes the incorporation of 5 to 3500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation includes the incorporation of 5 to 2500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation includes the incorporation of 5 to 1500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation includes the incorporation of 5 to 750 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation includes the incorporation of 5 to 500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation includes the incorporation of 5 to 250 consecutive nucleotides of the desired sequence into the target sequence. In yet another embodiment, the mutation includes the incorporation of 5 to 150 consecutive nucleotides of the desired sequence into the target sequence. In one embodiment, the mutation involves the incorporation of 25 to 2500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation involves the incorporation of 25 to 1500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation involves the incorporation of 25 to 750 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation involves the incorporation of 50 to 2500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation involves the incorporation of 50 to 1500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation involves the incorporation of 50 to 750 consecutive nucleotides of the desired sequence into the target sequence. In yet another embodiment, the mutation involves the incorporation of 100 to 2500 consecutive nucleotides of the desired sequence into the target sequence. In one embodiment, the mutation involves the incorporation of 100 to 1500 consecutive nucleotides of a desired sequence into a target sequence.In one embodiment, the mutation involves the incorporation of 100 to 750 consecutive nucleotides of a desired sequence into a target sequence.

[0206] In some embodiments, the methods provided herein include detecting edits or mutations in a target sequence. Screening and selection of mutagenic or edited plants or plant cells may be by any method known to those skilled in the art. Examples of screening and selection methods, but not limited to, include Southern spectroscopy, PCR amplification for the detection of polynucleotides, Northern blotting, RNase protection, primer extension, RT-PCR amplification for the detection of RNA transcripts, Sanger sequencing, next-generation sequencing techniques (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assays for detecting the enzymatic or ribozyme activity of polypeptides and polynucleotides, protein gel electrophoresis, Western blotting, immunoprecipitation, and enzyme-conjugated immunoassays for detecting polypeptides. The presence or expression of polypeptides and / or polynucleotides can also be detected using other techniques such as in situ hybridization, enzyme staining, and immunostaining. Methods for carrying out all the techniques referenced above are known in the art.

[0207] Recombinase Some embodiments described herein relate to methods and compositions for preferentially or specifically inducing site-directed recombination in eggs, embryos, and / or meiotic plant tissues. Some embodiments described herein relate to methods and compositions for providing preferential or specific expression of recombinases in eggs, embryos, and / or meiotic plant tissues. Some embodiments described herein relate to methods and compositions for preferentially or specifically expressing DNA-modifying enzymes, such as inducible nucleases (e.g., CRISPR / Cas systems), in eggs, embryos, and / or meiotic plant tissues by inducing site-directed recombination and operably linking a polynucleotide encoding a DNA-modifying enzyme to a constitutive promoter. In some embodiments, a transcribable polynucleotide encoding a DNA-modifying enzyme, such as an inducible nuclease (e.g., CRISPR / Cas system), is operably linked to a constitutive promoter by recombinase-mediated excision of a preferential or specific intervening polynucleotide sequence in eggs, embryos, and / or meiotic plant tissues.

[0208] Site-directed recombination occurs when DNA strands are exchanged between DNA segments that have at least some sequence homology to each other. Site-directed recombinases can also recognize and bind to “recombination sites”—short, specific DNA sequences that are cleaved by recombinases and enable DNA strand exchange, and subsequently, strand repair. Typically, each recombinase protein binds to a specific and unique recombination site. As used herein, “recombinase” refers to an enzyme capable of catalyzing site-directed recombination events within DNA. Recombinases can excise DNA, insert DNA, invert DNA, translocate DNA, and / or exchange DNA.

[0209] In some embodiments, the Disclosure provides methods and compositions for specifically or preferentially providing recombinases in eggs, embryos, and / or meiotic plant tissues. In some embodiments, the Disclosure provides nucleic acid sequences encoding recombinases operably ligated to promoters listed in Table 1. In some embodiments, a recombinant nucleic acid construct comprises sequences encoding at least one recombinase operably ligated to promoters listed in Table 1. In some embodiments, a recombinant nucleic acid construct comprising sequences encoding at least one recombinase operably ligated to promoters listed in Table 1 is provided to plant cells together with a recombinant nucleic acid construct comprising a DNA-modifying enzyme, an intervening sequence flanked by a recombination site, and a polynucleotide encoding a constitutive promoter, wherein the excision of the intervening sequence operably ligates the polynucleotide encoding the DNA-modifying enzyme to the constitutive promoter, preferentially in eggs, embryos, and / or meiotic tissues.

[0210] In one embodiment, the recombinase is a tyrosine recombinase. In another embodiment, the tyrosine recombinase is selected from the group consisting of Cre recombinase and Flp recombinase.

[0211] In one embodiment, the recombinase is Cre recombinase. Cre-lox is a site-directed recombination system derived from bacteriophage P1. Cre-lox can be used to invert, delete, or translocate nucleic acid sequences. In this system, Cre recombinase recombinates a pair of lox nucleic acid sequences. The lox site contains 34 nucleotides, with the first and last 13 nucleotides (arms) being palindromes. During recombination, the Cre recombinase protein binds to two lox sites on different nucleic acids and cleaves them at the lox sites. The cleaved nucleic acids are then spliced ​​together (mutual translocation), completing the recombination.

[0212] In one embodiment, the recombinase is flippase (Flp). The Flp-FRT site-specific recombination system is derived from a 2μ plasmid from the baker's yeast Saccharomyces cerevisiae and is similar to the Cre-lox system. Flp can induce recombination between flippase-recognition target (FRT) sites. The FRT site contains 34 nucleotides. Flp binds to the "arms" of the FRT site (one arm is oriented in opposite directions) and cleaves the FRT site at either end of the intervening nucleic acid sequence. After cleavage, Flp recombines the nucleic acid sequences between the two FRT sites.

[0213] In one embodiment, the recombinant site is a lox site. In one embodiment, the lox site is selected from the group consisting of loxP site, lox2272 site, loxN site, lox511 site, lox5171 site, lox71 site, lox66 site, loxLTR site, M2 site, M3 site, M7 site, and M11 site. In one embodiment, the recombinant site is an FRT site.

[0214] TALE Several embodiments provided herein relate to the use of TALE activators for preferential expression of DNA-modifying enzymes, such as inducible nucleases (e.g., CRISPR / Cas systems), in eggs, embryos, and / or meiotic plant tissues. In some embodiments, high levels of egg, embryo, and / or meiotic tissue-specific expression of DNA-modifying enzymes, such as inducible nucleases (e.g., CRISPR / Cas systems), are achieved by providing plant cells with an expression construct comprising 1) a promoter described in Table 1 operably linked to a sequence encoding TALE, and 2) an expression construct comprising a minimal promoter and one or more TALE binding sites (TBs) operably linked to a sequence encoding the DNA-modifying enzyme; and generating plants therefrom. In some embodiments, expression constructs encoding one or more guide nucleic acids are further provided. In some embodiments, the level of egg, embryo, and / or meiotic tissue-specific expression of the DNA-modifying enzyme can be modulated by changing the number of TBs.

[0215] As used herein, “TALE protein” refers to a transcription activator-like effector (TALE) protein or its homologue. TALE proteins were originally identified as virulence factors derived from the plant pathogenic bacteria Xanthomonas or Ralstonia. These proteins are secreted by plant pathogenic bacteria to alter the transcription of host genes in plant cells. TALE proteins bind to nuclear DNA via a DNA-binding repeat domain, where they act as transcription activators, thereby contributing to virulence. TALE translocates to the nucleus, where it recognizes and binds to specific DNA sequences in the regulatory regions of specific genes in the host genome. TALE has a central DNA-binding domain composed of 13–28 repeat monomers of 33–34 amino acids. The amino acids of each monomer are highly conserved, except for two highly variable amino acid residues at positions 12 and 13. These two variable amino acids are called repeat variable duos (RVDs). The amino acid pairs NI, NG, HD, and NN of RVD preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and modulation of RVD can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has enabled the manipulation of specific DNA-binding domains by selecting combinations of repeat segments containing the appropriate RVD. As used herein, “TALE binding site” (TBS) refers to a specific DNA sequence recognized and bound by the “TALE DNA-binding domain” of a TALE protein.

[0216] plant Any plant or plant cell can be used with the methods and compositions provided herein. In some embodiments, the plant is selected from the group consisting of maize plants, rice plants, sorghum plants, wheat plants, alfalfa plants, barley plants, millet plants, rye plants, sugarcane plants, cotton plants, soybean plants, canola plants, tomato plants, onion plants, cucumber plants, Arabidopsis thaliana plants, and potato plants. In some embodiments, the plant is an angiosperm. In some embodiments, the plant is a gymnosperm. In some embodiments, the plant is a monocotyledonous plant. In some embodiments, the plant is a dicotyledonous plant. In some cases, plants belong to the families Alliaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Brassicaceae, Caesalpiniaceae, Cucurbitaceae, Ericaceae, Fabaceae, Juglandaceae, and blue These are plants belonging to families selected from the group consisting of Malvaeaceae, Mimosaceae, Moraceae, Musaceae, Orchidaceae, Papilionaceae, Pinaceae, Poaceae, Rosaceae, Rutaceae, Rubiaceae, and Solanaceae.

[0217] In some embodiments, plant cells are selected from the group consisting of maize cells, rice cells, sorghum cells, wheat cells, alfalfa cells, barley cells, millet cells, rye cells, sugarcane cells, cotton cells, soybean cells, canola cells, tomato cells, onion cells, cucumber cells, Arabidopsis thaliana cells, and potato cells. In some embodiments, plant cells are angiosperm cells. In some embodiments, plant cells are gymnosperm cells. In some embodiments, plant cells are monocotyledonous plant cells. In some embodiments, plant cells are dicotyledonous plant cells. In one embodiment, the plant cells are plant cells of a family selected from the group consisting of the Alliaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Brassicaceae, Caesalpinaceae, Cucurbitaceae, Ericaceae, Fabaceae, Juglandaceae, Malvaceae, Mimoceae, Moraceae, Musaceae, Orchidaceae, Fabuideae, Pinaceae, Poaceae, Rosaceae, Rutaceae, Rubiaceae, and Solanaceae.

[0218] As used herein, “cultivar” refers to a group of plants within a species (but not limited to zea mays) that share certain genetic traits that separate them from other possible cultivars within that species. Cultivars may be inbred or hybrids, but commercially available plants are often hybrids to take advantage of hybrid vigor. Individuals within a hybrid cultivar are of the same species, nearly genetically identical, and many loci are heterozygous.

[0219] As used herein, “inbred” means a lineage bred for genetic uniformity. In some embodiments, the seeds provided herein are inbred seeds. In some embodiments, the plants provided herein are inbred plants.

[0220] As used herein, the term "hybrid" means the progeny of a cross between at least two genetically dissimilar parents. By way of example, and not limitation, mating schemes include single cross, modified single cross, double modified single cross, three-way cross, modified three-way cross, and double cross, where at least one parent in the modified cross is the progeny of a cross between sister lines. In one aspect, the seeds provided herein are hybrid seeds. In one aspect, the plants provided herein are hybrid plants.

[0221] Transformation The method may include transient transformation or stable integration of any nucleic acid molecule into any plant or plant cell provided herein.

[0222] As used herein, "stable integration" or "stably integrated" refers to the transfer of DNA into the genomic DNA of a target cell or plant that enables the target cell or plant to pass on the transferred DNA to the next generation of the transformed organism. Stable transformation requires the integration of the transferred DNA into the germ cells of the transformed organism. As used herein, "transiently transformed" or "transient transformation" refers to the transfer of DNA into a cell that does not transfer to the next generation of the transformed organism. In transient transformation, the DNA being transformed typically does not integrate into the genomic DNA of the cell being transformed. In one aspect, the method stably transforms a plant cell or plant with one or more nucleic acid molecules provided herein. In another aspect, the method transiently transforms a plant cell or plant with one or more nucleic acid molecules provided herein.

[0223] In some embodiments, the nucleic acid molecule encoding the inducible nuclease is stably integrated into the genome of the plant. In some embodiments, the nucleic acid molecule encoding the Cas12a nuclease is stably integrated into the genome of the plant. In some embodiments, the nucleic acid molecule encoding the CasX nuclease is stably integrated into the genome of the plant. In some embodiments, the nucleic acid molecule encoding the guide nucleic acid is stably integrated into the genome of the plant. In some embodiments, the nucleic acid molecule encoding the guide RNA is stably integrated into the genome of the plant. In some embodiments, the nucleic acid molecule encoding the single guide RNA is stably integrated into the genome of the plant.

[0224] Several 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. Any suitable method or technique for transforming cells known in the art can be used according to the methods of the present invention. Effective methods for transforming plants include bacterial-mediated transformation such as Agrobacterium-mediated or Rhizobium-mediated transformation and microprojectile bombardment-mediated transformation. After transforming explants with a transformation vector by bacterial-mediated transformation or microprojectile bombardment, various methods for culturing these explants to regenerate or generate transgenic plants are known in the art.

[0225] In some embodiments, the method includes providing nucleic acid molecules to cells by Agrobacterium-mediated transformation. In some embodiments, the method includes providing nucleic acid molecules to cells by polyethylene glycol-mediated transformation. In some embodiments, the method includes providing nucleic acid molecules to cells by bioristic transformation. In some embodiments, the method includes providing nucleic acid molecules to cells by liposome-mediated transfection. In some embodiments, the method includes providing nucleic acid molecules to cells by viral transduction. In some embodiments, the method includes providing nucleic acid molecules to cells by the use of one or more delivery particles. In some embodiments, the method includes providing nucleic acid molecules to cells by microinjection. In some embodiments, the method includes providing nucleic acid molecules to cells by electroporation.

[0226] In some embodiments, nucleic acid molecules are delivered to cells by a method selected from the group consisting of Agrobacterium-mediated transformation, polyethylene glycol-mediated transformation, bioristic transformation, liposome-mediated transfection, viral transduction, the use of one or more delivery particles, microinjection, and electroporation.

[0227] Other methods for transformation, such as vacuum immersion, pressure, sonication, and silicon carbide fiber agitation, are also known in the art and are intended for use in conjunction with any method provided herein.

[0228] Methods for transforming cells are well known to those skilled in the art. For example, specific instructions for transforming plant cells by microprojectile bombardment (e.g., bioristic transformation) using recombinant DNA-coated particles can be found in U.S. Patents 5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,153,812, and Agrobacterium-mediated transformation can be found in U.S. Patents 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871; 5,463,174; and 5,188,958, all of which are incorporated herein by reference. Further methods for transforming plants can be found, for example, in Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Plant cells can be transformed with any of the nucleic acid molecules provided herein using any suitable method known to those skilled in the art.

[0229] Lipofection is described, for example, in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Suitable cationic and neutral lipids for efficient receptor recognition lipofection of polynucleotides include those described in Felgner's WO91 / 17424; WO91 / 16024. Delivery may be to cells (e.g., in vitro or ex vivo administration) or target tissue (e.g., in vivo administration).

[0230] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for the expression of one or more elements of nucleic acid molecules are as used in WO2014 / 093622. In one embodiment, a method for delivering nucleic acid molecules or proteins to cells includes delivery by delivery particles. In one embodiment, a method for delivering nucleic acid molecules to plant cells or plants includes delivery by delivery vesicles. In one embodiment, delivery vesicles are selected from the group consisting of exosomes and liposomes. In one embodiment, a method for delivering nucleic acid molecules to plant cells or plants includes delivery by viral vectors. In one embodiment, viral vectors are selected from the group consisting of adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors. In another embodiment, a method for delivering nucleic acid molecules to plant cells or plants includes delivery by nanoparticles. In one embodiment, a method for delivering nucleic acid molecules to plant cells or plants includes microinjection. In one embodiment, a method for delivering nucleic acid molecules to plant cells or plants includes polycations. In one embodiment, a method for delivering nucleic acid molecules to plant cells or plants includes cationic oligopeptides.

[0231] In some embodiments, the delivery particles are selected from the group consisting of exosomes, adenovirus vectors, lentiviral vectors, adeno-associated virus vectors, nanoparticles, polycations, and cationic oligopeptides. In some embodiments, the methods provided herein include the use of one or more delivery particles. In other embodiments, the methods provided herein include the use of two or more delivery particles. In yet another embodiment, the methods provided herein include the use of three or more delivery particles.

[0232] Suitable agents for facilitating the transfer of nucleic acids into plant cells include agents that increase the permeability to the outside of the plant, or agents that increase the permeability of plant cells to oligonucleotides or polynucleotides. Such agents for facilitating the transfer of a composition into plant cells include chemical agents, physical agents, or combinations thereof. Chemical agents for conditioning include (a) surfactants, (b) organic solvents, aqueous solutions, or aqueous mixtures of organic solvents, (c) oxidizing agents, (e) acids, (f) bases, (g) oils, (h) enzymes, or combinations thereof.

[0233] Useful organic solvents for conditioning plants to permeate with polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, and other solvents that are miscible with water or dissolve phosphonucleotides in non-aqueous systems (such as those used in synthesis reactions). Natural or synthetic oils, with or without surfactants or emulsifiers, may be used, such as plant-derived oils, crop oils (such as those listed in the 9th Compendium of Herbicide Adjuvants, which is publicly available online at www.herbicide.dot.adjuvants.com), and oils having short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine.

[0234] Examples of useful surfactants include sodium or lithium salts of fatty acids (such as animal fats, animal fat amines, or phospholipids) and organosilicon surfactants. Other useful surfactants include nonionic organosilicon surfactants, such as organosilicon surfactants containing trisiloxane ethoxylate surfactants, or silicone polyether copolymers such as the copolymer of polyalkylene oxide-modified heptamethyltrisiloxane and allyloxypolypropylene glycol methyl ether (commercially available as Silwet® L-77).

[0235] Useful physical agents may include (a) abrasives such as carborundum, corundum, sand, calcite, pumice, and garnet; (b) nanoparticles such as carbon nanotubes; or (c) physical forces. Carbon nanotubes are disclosed by Kam et al. (2004) Am. Chem. Soc, 126 (22): pp. 6850-6851, Liu et al. (2009) Nano Lett, 9(3): pp. 1007-1010, and Khodakovskaya et al. (2009) ACS Nano, 3(10): pp. 3221-3227. Examples of physical force agents include heating, cooling, application of positive pressure, or sonication. Embodiments of the method may optionally include incubation steps, neutralization steps (e.g., to neutralize acids, bases, or oxidizing agents, or to inactivate enzymes), washing steps, or a combination thereof. The method of the present invention may further include the application of other agents that would have an enhanced effect due to the silencing of certain genes. For example, if a polynucleotide is designed to modulate a gene that provides herbicide resistance, subsequent application of the herbicide may have a dramatic effect on the efficacy of the herbicide.

[0236] Examples of agents used for laboratory conditioning of plant cells for polynucleotide permeability include the application of chemical agents, enzymatic treatment, heating or cooling, treatment using positive or negative pressure, or sonication. Examples of agents used for conditioning plants in the field include chemical agents such as surfactants and salts.

[0237] In one embodiment, the cells to be transformed or transfected are plant cells. Recipient plant cells or explant targets for transformation include, but are not limited to, seed cells, fruit cells, leaf cells, cotyledon cells, hypocotyl cells, meristem cells, embryo cells, endosperm cells, root cells, shoot cells, stem cells, sheath cells, flower cells, inflorescence cells, stalk cells, pedicel cells, style cells, stigma cells, receptacle cells, petal cells, sepal cells, pollen cells, anther cells, filament cells, ovary cells, ovule cells, pericarp cells, phloem cells, bud cells, or vascular tissue cells. In another embodiment, the disclosure provides plant chloroplasts. In a further embodiment, the disclosure provides epidermal cells, guard cells, trichome cells, root hair cells, storage root cells, or tuber cells. In another embodiment, the disclosure provides protoplasts. In another embodiment, the disclosure provides plant callus cells. Any cells capable of regenerating fertile plants are intended to be useful recipient cells for the implementation of this disclosure. Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or embryonic portions, seedling apical meristems, microspores, etc. These cells, capable of growing as callus, can serve as recipient cells for transformation. Practical transformation methods and materials for producing transgenic plants of this disclosure (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Patent Nos. 6,194,636 and 6,232,526 and U.S. Patent Application Publication 2004 / 0216189, all of which are incorporated herein by reference. Transformed explants, cells, or tissues can be subjected to further culture steps, such as callus induction, selection, and regeneration, as known in the art. Transformed cells, tissues, or explants containing recombinant DNA insertions can be propagated, developed, or regenerated into transgenic plants in cultures, plugs, or soil according to methods known in the art. In one embodiment, the disclosure provides plant cells that are not reproductive material and do not mediate the natural reproduction of plants. In another embodiment, the disclosure provides plant cells that are reproductive material and mediate the natural reproduction of plants. In yet another embodiment, the disclosure provides plant cells that cannot sustain themselves by photosynthesis.In another embodiment, the disclosure provides somatic plant cells. Somatic cells, in contrast to germline cells, do not mediate reproduction in plants. In one embodiment, the disclosure provides non-reproductive plant cells.

[0238] Use of haploid / haploid induction systems Some embodiments relate to the use of the methods and compositions described herein in combination with haploid induction techniques. As used herein, a “haploid” cell or nucleus contains a single set of unpaired chromosomes (x). In contrast, a “diploid” cell or nucleus contains two complete sets of chromosomes (2x) that can form homologous pairs. As used herein, a “haploid plant” describes a sporophyte containing multiple cells with a haploid nuclear genome. A haploid plant provided herein may be a maternal haploid plant, meaning it has lost its paternal nuclear genome while retaining its maternal nuclear genome. Alternatively, a haploid plant provided herein may be a paternal haploid plant, meaning it has lost its maternal nuclear genome while retaining its paternal nuclear genome. Typically, the maternal mitochondrial and plastid (e.g., chloroplast) genomes are retained in both maternal and paternal haploid plants.

[0239] In some embodiments, the "doubled haploid (DH)" method is used to rapidly produce homozygous plants. Offspring of DH plants are genetically homogeneous material, and breeders can evaluate the desired traits on genetically fixed material at an early stage of the breeding cycle, thus increasing breeding efficiency (see Gilles LM et al., Curr Biol. 2017 Oct 23;27(20):R1095~R1097). The DH technique relies on two main steps: (1) a haploid induction system for generating haploid embryos or plants, and (2) a chromosome doubling step to restore diploidy to these plants.

[0240] Haploid induction (HI) is a phenomenon in some plants characterized by the loss of parental induction chromosomes during embryonic development. As used herein, a "haploid induction (HI) plant" is a plant in which haploidy can be induced in offspring plants by removing one set of chromosomes. Maternal haploid induction is induced by the pollinating (male) parent. Paternal haploid induction is induced by the female parent.

[0241] Haploid-inducible lines are routinely used, particularly in plant breeding for maize. Several known haploid-inducible maize lines exist, including, but are not limited to, Stock 6, MHI (Moldovian Haploid Inducer), intermediate gametophyte (ig1) mutations, KEMS, RWK, ZEM, ZMS, and KMS. Haploid-inducible lines have also been produced in Arabidopsis thaliana, Brassica juncea, and maize by the use of engineered centromere histone 3 (CENH3) variants (see Ravi and Chan. 2010. Nature. 464:615-6190). In some embodiments, the haploid-inducible lines described herein include maize Stock 6 lines, maize plants carrying mutations at the ig-1 locus, MHI-inducible lines, KEMS-inducible lines, RWK-inducible lines, ZEM-inducible lines, ZMS-inducible lines, and KMS-inducible lines. In some embodiments, the haploid-inducible line contains a modified MATRILINEAL / NOT LIKE DAD / ZmPHOSPHOLIPASE-A1 (MATL / NLD / ZmPLA1) gene. In some embodiments, the haploid-inducible line described herein contains a modified CEN H3 variant.

[0242] Since the DH technology can be used with several different molecular techniques to overcome various constraints on crop improvement, the benefits brought by the haploid induction system to crop breeding programs are diverse. One example is the use of a haploid induction system to expand the application of genome editing technology to crops. After introducing genome editing components (e.g., regarding inducible nucleases) into a haploid induction line, it can be crossed with a non-induced maize line. Then, the haploid progeny are screened for nuclease-induced mutations, and then genome doubling is induced to produce diploid, genome-edited cultivars that do not contain the editing components. This method is described in detail in US20190169596 (application number US16 / 275200), the entire content of which is incorporated herein by reference.

[0243] The present disclosure will now be described with reference to the following examples. It should be understood that these examples are not intended to limit the claims of the disclosure, but rather are intended to be illustrative of certain specific embodiments. Any variations in the exemplified methods contemplated by those skilled in the art are intended to be within the scope of the present disclosure.

Examples

[0244] (Example 1) Expression of Cas12a in meiotic oocytes or embryonic tissues to generate germ cell mutations or targeted integration of template DNA To preferentially express Cas12a in maize oocytes and / or maize embryo cells (meiotic cells) undergoing meiosis, several Agrobacterium T-DNA vectors were generated. See Table 2.

[0245]

Table 2

[0246] The plant codon-optimized LbCas12a sequence (SEQ ID NO: 7) in these cassettes was flanked by 5' and 3' terminal NLS sequences (SEQ ID NO: 8 and SEQ ID NO: 9) and operably linked to a transcription termination sequence derived from the rice lipid transfer protein (LTP) gene (described as SEQ ID NO: 8 in US20200080096). Each vector also contained an expression cassette encoding Cas12a gRNA targeting a unique maize genomic site (ZmTS1) under the control of the PolIII ZmU6 promoter (SEQ ID NO: 10); an expression cassette flanked by the ZmTS1 target site containing a constitutive promoter to which the cassette was operably linked to the gene of interest (GOI); and an expression cassette for a select marker conferring resistance to the herbicide glyphosate. Maize 01DKD2 cultivar embryos were transformed using the above vectors by Agrobacterium-mediated transformation, and R0 plants were regenerated from the transformed maize cells. DNA was extracted from leaf samples derived from 59–153 R0 seedlings generated from each construct. Genomic target sites were sequenced, and the presence of target mutations was analyzed. Taqman assays were also performed to identify the copy number of constructs carrying Cas12a. No mutations were detected at the ZmTS1 site in the R0 generation for all reproductive promoters except ZmDSUL. ZmDSUL:LbCas12a (construct 1) showed a target site mutation rate of approximately 32% in R0 seedlings (see Table 3). The absence of mutations at the ZmTS1 target site in newly transformed (or R0) plants is expected, in which case LbCas12a expression will be limited to reproductive tissues.

[0247] Twenty R0 lines derived from each transformed construct were grown to maturity, and at least one ear from each transformed maize plant was self-pollinated. Ten R1 lines were selected, and up to 16 seedlings were grown per line. Mutations in ZmTS1 were screened, and the mutation rate (break rate) was calculated. Taqman assays were also performed to determine the presence and copy number of LbCas12a expression cassettes. The overall target site mutation rate across all lines generated from constructs 2-5 ranged from approximately 1.1% to 14%. The average mutation rate is shown in Table 3. No mutations were observed when four R1 seedlings expressing ZmDSUL::LbCas12a (construct 1) were tested. Co-expression of Cas12a and its recognition gRNA in cells undergoing meiosis is expected to generate double-strand breaks at the ZmTS1 target site, and subsequent incomplete DNA repair will generate unique mutations in egg cells and embryos produced by pollination.

[0248] [Table 3]

[0249] The mutation rates in R1 plants generated from individual R0 lines can vary, as shown in Tables 4-8. For example, with the promoter with the highest cleavage rate, ZmES4, some lines with a single copy of the Cpf1 cassette showed a mutation rate of 0%, while others showed a mutation rate of approximately 11%. The highest mutation rate observed was 46%, which came from R0 lines with two copies of ZmES4:LbCas12a. In ZmES4:Cas12a plants, R0 lines with two copies of Cas12a consistently had higher target mutation rates than Ro lines with one copy across all lines tested (see Table 7).

[0250] [Table 4]

[0251] [Table 5]

[0252] [Table 6]

[0253] [Table 7]

[0254] [Table 8]

[0255] [Table 9]

[0256] To increase the reliability of 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. Lines exhibiting mutation rates of 74.2% and 49.4% were R1 seeds derived from R0 lines containing two copies of ZmES4:Cas12a.

[0257] Unique Target Mutations in R1 Plants: Two R0 lines (ZM_S22321326 and ZM_S22321323) derived from ZmES4::LbCas12a, which is expected to selectively express LbCas12a in eggs and synergid cells, were analyzed to evaluate the types of mutations produced. Five mutant plants were produced from the ZM_S22321326 line. Sequencing from the ZmTS1 gRNA target locus showed that all five plants had unique mutations at the target site. 49 mutant seedlings were analyzed from the ZM_S22321323 R0 line, which contained two copies of ZmES4:LbCas12a. Of the 49 mutant seedlings, 56 target site mutations were identified. This suggests that some plants contained heterologous mutations. The 49 plants had a total of 23 unique mutations.

[0258] [Table 10]

[0259] [Table 11]

[0260] A study on site-specific integration of template DNA in plants expressing Cas12a. In addition to the Cas12a and gRNA expression cassettes, each vector also contained an expression cassette for a target gene (GOI) flanked by a ZmTS1 gRNA target sequence. Cas12a expression in germ tissue is expected to create double-strand breaks on both sides of the GOI cassette, releasing them from the T-DNA. This released DNA can then act as a donor for targeted insertion at the genomic ZmTS1 target site. If the CRISPR-Cpf1 complex cleaves the target site in the genome, the non-homologous end-joining (NHEJ) DNA repair pathway can insert the donor GOI cassette into the genomic target site. This form of SDI is also known as trans-fragment targeting (TFT). To test for SDI by TFT, a Frank PCR assay similar to that described in WO2019084148 was used to identify putative target insertions. Primers were designed to PCR amplify the expected insertion flanking sequences. Four separate PCR reactions were performed: left-Frank PCR and right-Frank PCR for potential inserts located in the sense direction, and left-Frank PCR and right-Frank PCR for inserts located in the antisense direction. In the initial screening of the R0 line, two plants showed Frank-positive PCR. Both were identified in the ZmES4:LbCas12a line and both produced only one Frank PCR product. One Frank-positive plant was in a line with an established mutation rate (11.11%), while the other Frank-positive plant was in a line with a mutation rate of 0%.

[0261] In summary, the data showed that reproductive editing can be achieved when LbCas12a is preferentially expressed in cells undergoing meiosis, or under the control of promoters DMC1, Mps1, or Adf1, which are expressed alone. Furthermore, reproductive editing can also be achieved when LbCas12a is expressed by eggs or embryos expressing promoters such as ZmES4 and ZmEA1. Additionally, the data demonstrate that a single R0 plant can produce many R1 offspring, each with a unique target site edit. This suggests that these promoters can be used to drive nuclease expression to increase the frequency of unique edits produced per transformed plant.

[0262] Germ cell mutations can be inherited by the F1 generation. Two R1 individuals derived from the ZmES4::LbCas12a R0 event ZM_S22321323 were propagated to maturity, and the crosses were pollinated using a wild-type 01DKD2 tester. R1 plant-1 contained a 7bp deletion at nucleotide 15 of the target site (see mutation 3 in Table 11), and R1 plant-2 contained a 9bp deletion at nucleotide 16 of the target site (see mutation 9 in Table 11). 32 seedlings were planted from each F1 line, and the inheritance of mutations in the ZmTS1 target site was screened. Of the 32 F1 seedlings derived from the plant-1 cross, 11 grew, and 6 of them inherited the R1 event-specific mutation. Of the 32 seedlings derived from the plant-2 cross, 5 grew, and 3 of them inherited the R1 event-specific mutation.

[0263] (Example 2) Cas12a expression in cells undergoing meiosis to generate germ cell mutations Several constructs are generated to preferentially express Cas12a in maize cells undergoing meiosis, specifically in maize egg cells and / or maize embryos. See Table 12.

[0264] [Table 12]

[0265] The plant codon-optimized LbCas12a sequence (SEQ ID NO: 7) in the expression cassette described in Table 12 is flanked by NLS sequences at the 5' and 3' ends (SEQ ID NOs: 8 and 9). Each of the constructs described in Table 12 is introduced into maize cells simultaneously with a construct encoding a gRNA complementary to the target site under the control of the PolIII ZmU6 promoter (SEQ ID NO: 10) ("gRNA construct") using biorhythmic transformation methods routinely used in the art. Alternatively, the constructs described in Table 12 can be used to transform cells containing the gRNA construct biorhythmically or via an Agrobacterium T-DNA vector. The resulting transformed maize cells contain one of the constructs 7-13, as well as the gRNA construct. Maize plants are regenerated from the transformed maize cells and grown to maturity. At least one ear from each transformed maize plant is pollinated. Seeds resulting from pollination will be screened for mutations at target sites, and the number and type of mutations produced using constructs 8–13 will be compared to transformed maize plants produced using construct 7. Co-expression of Cas12a from constructs 8–13 and its recognition guide RNA from the gRNA construct is expected to result in double-strand breaks in genomic DNA at target sites, with subsequent DNA repair generating one or more unique mutations.

[0266] (Example 3) gRNA expression in egg or embryonic tissue where Cas12a is constitutively expressed to generate germ cell mutations. Several constructs are generated to preferentially express guide RNA (gRNA) complementary to the target site under the control of the PolII promoter in meiotic cells, maize egg cells, and / or maize embryos. See Table 13. After transcription, Pol-II products are rapidly modified at the 5' cap and poly-A tail and exported from the nucleus. These modifications and alterations in localization can hinder the efficient use of gRNA. To optimize gRNA availability and performance, self-cleaving ribozymes are incorporated into the gRNA cassette design. Self-cleaving ribozymes have been reported to facilitate the cleavage / processing of gRNA transcripts from PolII-expressed transcripts, thereby producing precise guide molecules (see Wang et al., 2018, J. of Integrative Plant Biol, 60:8, pp. 626-631).

[0267] [Table 13]

[0268] The constructs listed in Table 13 are stably introduced into maize cells using transformation methods routinely used in the art. Furthermore, a construct ("Cas12a construct") containing a plant codon-optimized nucleic acid sequence (SEQ ID NO: 1) encoding the Cas12a protein flanked by 5' and 3' terminal NLS sequences (SEQ ID NO: 2 and SEQ ID NO: 3) under the control of a ubiquitous ZmUbqM1 promoter (SEQ ID NO: 11) is co-introduced together with each construct provided in Table 13. The resulting transformed maize cells contain one of constructs 14-20, as well as the Cas12a construct. Maize plants are regenerated from the transformed maize cells and grown to maturity. At least one ear from each transformed maize plant is pollinated. Seeds resulting from pollination are screened for mutations at target sites, and the number and type of mutations produced using constructs 15-20 are compared to those produced in transformed maize plants using construct 14. Selective expression of gRNA is expected to generate one or more unique mutations in each meiotic cell, egg cell, or embryo produced by pollination.

[0269] (Example 4) Expression of Cas12a and gRNA as single transcripts in cells undergoing meiosis, egg cells, or embryonic tissue to generate germ cell mutations. Several constructs are generated to preferentially express LbCas12a and a complementary guide RNA (gRNA) as a single transcript in cells undergoing meiosis, maize egg cells, and / or maize embryos. See Table 14.

[0270] [Table 14]

[0271] Each construct listed in Table 14 is stably introduced into maize cells using a bioristic transformation method or Agrobacterium transformation method routinely used in the art. The resulting transformed maize cells contain one of constructs 21-27. Maize plants are regenerated from the transformed maize cells and grown to maturity. At least one ear derived from each transformed maize plant is pollinated. LbCas12a and gRNA are transcribed as part of a single transcript in cells expressing the promoter. Subsequently, ribozyme-mediated cleavage occurs, releasing the gRNA segment. The LbCas12a protein transcribed from the transcript forms a ribonucleoprotein (RNP) with the gRNA. The RNP generates a double-strand break at the target site, and subsequent repair will produce one or more unique mutations in each meiotic cell, egg cell, or embryo produced by pollination. Seeds resulting from pollination are screened for mutations at target sites, and the number and types of mutations produced using constructs 22-27 are compared with those produced using construct 21 in transformed maize plants.

[0272] (Example 5) Generation of mutations through crossbreeding A transgenic maize plant containing one of the constructs 8-13 (see Example 2, Table 2) is generated and propagated until flowering. Further transgenic maize plants containing the gRNA construct from Example 2 are also generated and propagated until flowering. A maize plant containing one of the constructs 8-13 is crossed with a maize plant containing the gRNA construct to produce offspring maize plants containing Cas12a and gRNA expressed in the resulting embryos.

[0273] Alternatively, a transgenic maize plant containing one of the constructs 15-20 (see Example 3, Table 13) is produced and propagated until flowering. Further transgenic maize plants containing the Cas12a construct from Example 3 are also produced and propagated until flowering. A maize plant containing one of the constructs 15-20 is crossed with a maize plant containing the Cas12a construct to produce offspring maize plants containing Cas12a and gRNA expressed in the resulting embryos.

[0274] Co-expression of Cas12a and gRNA generates double-strand breaks within the target site, thereby creating unique mutations in each cell where both components of the CRISPR system are expressed. The resulting embryos, or plants derived from these embryos, are screened to identify mutations at the target site.

[0275] (Example 6) Cas12a expression during meiosis in forward and reverse F1 egg cells or embryonic tissue to generate different germ cell mutations. 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, were planted. See Table 15 for a list of constructs.

[0276] [Table 15]

[0277] Thirty R1 individuals derived from each transformed construct, representing 4 to 10 independent events per construct, were propagated to maturity, and forward and reverse crosses were attempted using all individuals. F1 seeds were collected from 6 to 29 crosses per construct and direction. Up to 72 seedlings were grown from each F1 spike, and mutations at the ZmTS1 target site were screened, and the mutation rate (trellization rate) was calculated. See Table 16.

[0278] [Table 16]

[0279] The Taqman assay was also performed to determine the copy number of the LbCas12a expression cassette. The overall target site mutation rate among all F1 generations ranged from 0 to 14%, indicating directed expression (Figure 1 and Table 16).

[0280] The F1 mutation rate was compared to the mutation rate derived from the event parent (R1) to determine how many new edits (present in F1 but not in the R1 parent) were generated. These data are summarized in Figure 1. The ZmES4:LbCas12a construct showed the highest cleavage rate from females (average 12% of samples containing new edits). ZmDMC1:LbCas12a showed the highest cleavage rate from males (average 14% of samples containing new edits).

[0281] When carried by the female parent, ZmES4:LbCas12a not only produced a high percentage of F1 plants with novel edits, but also produced numerous unique edits in the F1 plants (Figure 1, Table 17). When carried by the female parent, ZmES4:LbCas12a produced 39 different mutant types when detected in F1 plants. Other promoters tested produced up to four unique edit types in a single offspring (see Figure 1).

[0282] Novel edits observed in ZmES4:LbCas12a F1 plants were remarkably abundant. Sequencing of target sites revealed 40%–100% of sequencing reads containing mutant alleles, indicating that these edits are generated early in embryo / zygote development and are therefore fixed in the plant (Table 17). Edits that become fixed in a plant are present in the germline of that plant and are therefore inherited by its offspring.

[0283] ZmES4 maintained high embryo / zygote expression across multiple generations, and many independent events exhibited functional activity of LbCas12a. Nearly all edited plants in each event contained unique edits (Table 17). Furthermore, all but one ZmES4 event tested produced new edits, with a mutation rate of up to 20% per event (Table 17).

[0284] [Table 17]

[0285] (Example 7) Cas12a expression in maize egg cells, meiotic cells, or embryos to enable editing of target genomes from haploid-inducible lines. When crossed with another line, a vector is generated to preferentially express Cas12a in zygotes, embryos, eggs, and / or meiotic cells of haploid-inducing (HI) lines that produce haploids. Non-limiting examples of promoters and regulatory sequences useful for driving expression in embryos, eggs, and / or meiotic cells are provided in Table 1. An expression cassette is provided in which a plant codon-optimized LbCas12a sequence is flanked by 5' and 3' terminal NLS sequences and operably ligated to the promoters listed in Table 1. Each vector may also contain an expression cassette encoding one or more Cas12a gRNAs targeting unique maize genome target sites; and optionally, an expression cassette encoding a select marker that confers resistance to the herbicide glyphosate. The vector can be directly transformed into a haploid-inducing line, or the vector-containing event can be generated in a different germplasm before crossing into the haploid-inducing line. The resulting “editing inducer” will contain a haploid-inducing trait and an expression cassette encoding Cas12a and, optionally, a gRNA directed to a target site. In some embodiments, the gRNA can be delivered separately from the Cas12a-expressing cassette to the haploid-inducing line or WT germplasm.

[0286] Editing induced by DNA-modifying enzymes such as inducible nucleases (e.g., the CRISPR / Cas system) expressed from haploid-inducible genomes occurs shortly after fertilization, but is limited by the efficiency of editing that may occur before the removal of the genome contributed by the haploid-inducible line. In systems where haploid induction is contributed by the female parent, strong expression of genome editing components from the maternal genome immediately before or after fertilization is a desirable feature. Therefore, the identification and use of promoters that are expressed at high levels in maternal tissues (e.g., egg and embryonic tissue), such as the ZmES4 promoter (see Example 6), is highly desirable.

[0287] An inducing line (editing inducer) carrying the expression construct described above is crossed with a male wild-type germplasm as female. After pollen from the male wild-type parent co...

Claims

1. A method for editing the genome of a plant, (a) In plant cells, (i) A first nucleic acid sequence encoding a CRISPR effector protein operably ligated to a first heterologous promoter selected from the group consisting of a heterologous oocyte-preferential promoter, a heterologous embryonic tissue-preferential promoter, and a heterologous meiotic cell-preferential promoter; and (ii) A second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the genome. The process of introducing; and (b) A step of regenerating at least one plant from the plant cells of step (a). A method comprising a CRISPR effector protein and at least one guide nucleic acid forming a ribonucleoprotein in at least one egg cell, at least one embryonic cell or at least one meiotic cell of a plant, the ribonucleoprotein generating at least one modification within a target sequence in at least one egg cell, at least one embryonic cell or at least one meiotic cell.

2. The method according to claim 1, wherein the heterologous first promoter is a heterologous oocyte-preferential promoter selected from the group consisting of the EA1 promoter and the ES4 promoter.

3. The method according to claim 1, wherein the heterogeneous first promoter is a heterogeneous oocyte-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82.

4. The method according to claim 1, wherein the heterogeneous first promoter is an embryonic tissue-preferential promoter selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter.

5. The method according to claim 1, wherein the heterogeneous first promoter is an embryonic tissue-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88.

6. The method according to claim 1, wherein the heterogeneous first promoter is a meiotic cell-preferential promoter selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter.

7. The method according to claim 1, wherein the heterogeneous first promoter is a meiotic cell-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85.

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

9. The method according to claim 1, further comprising the step of crossbreeding plants to produce offspring plants.

10. The method according to claim 9, wherein the plant is a haploid-induced strain and the offspring plant is a haploid.

11. The method according to claim 10, further comprising the step of treating the cells of the offspring plant with colchicine to produce a doubled haploid plant.

12. A method for editing the genome of a plant, (a) A step of crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding a CRISPR effector protein operably ligated to a heterologous promoter selected from the group consisting of heterologous oocyte-preferential promoters, heterologous embryo-preferential promoters, and heterologous meiotic cell-preferential promoters, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably ligated to a heterologous second promoter, the at least one guide nucleic acid capable of hybridizing to a target sequence in the genome; and (b) A step of obtaining at least one embryo from the mating of step (a), wherein the CRISPR effector protein and at least one guide nucleic acid form a ribonucleoprotein in at least one egg cell, at least one embryonic cell, or at least one meiotic cell, and the ribonucleoprotein generates at least one modification within a target sequence in at least one egg cell, at least one embryonic cell, or at least one meiotic cell. Methods that include...

13. The method according to claim 12, wherein the heterologous first promoter is a heterologous oocyte-preferential promoter selected from the group consisting of the EA1 promoter and the ES4 promoter.

14. The method according to claim 12, wherein the heterogeneous first promoter is a heterogeneous oocyte-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82.

15. The method according to claim 12, wherein the heterogeneous first promoter is an embryonic tissue-preferential promoter selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter.

16. The method according to claim 12, wherein the heterogeneous first promoter is an embryonic tissue-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88.

17. The method according to claim 12, wherein the heterogeneous first promoter is a meiotic cell-preferential promoter selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter.

18. The method according to claim 12, wherein the heterogeneous first promoter is a meiotic cell-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85.

19. The method according to claim 12, wherein the CRISPR effector protein is selected from the group consisting of Cas9, Cas12a, Cas12b, and CasX.

20. The method according to claim 12, wherein the heterogeneous second promoter is a PolIII promoter.

21. A plant produced by the method of claim 1, comprising a modified target sequence.

22. A recombinant DNA construct comprising (a) a first nucleic acid sequence encoding a CRISPR effector protein operably ligated to one or more TALE binding sites and a minimal promoter; and (b) a second nucleic acid sequence encoding a TALE operably ligated to an egg cell-preferential promoter, a meiotic cell-preferential promoter, or an embryonic tissue-preferential promoter, wherein the minimal promoter does not drive the expression of a DNA-modifying enzyme in the absence of a TALE bound to one or more TALE binding sites.

23. The recombinant DNA construct according to claim 22, further comprising a third nucleic acid sequence encoding a guide nucleic acid operably linked to a third promoter.

24. The recombinant DNA construct according to claim 22, wherein one or more TALE binding sites consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TALE binding sites.

25. The recombinant DNA construct according to claim 22, wherein a second nucleic acid sequence encoding TALE is operably linked to a heterologous oocyte-preferential promoter selected from the group consisting of the EA1 promoter and the ES4 promoter.

26. The recombinant DNA construct according to claim 22, wherein a second nucleic acid sequence encoding TALE is operably linked to a heterologous oocyte-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 2-3, 21-38, 41-45, and 65-82.

27. The recombinant DNA construct according to claim 22, wherein a second nucleic acid sequence encoding TALE is operably linked to a heterologous embryonic tissue-preferential promoter selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter.

28. The recombinant DNA construct according to claim 22, wherein a second nucleic acid sequence encoding TALE is operably linked to a heterogeneous embryonic tissue-preferential promoter comprising a nucleic acid sequence or functional fragment thereof that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88.

29. The recombinant DNA construct according to claim 22, wherein a second nucleic acid sequence encoding TALE is operably linked to a meiotic cell-preferential promoter selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter.

30. The recombinant DNA construct according to claim 22, wherein a second nucleic acid sequence encoding TALE is operably linked to a meiotic cell-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 4-6 and 83-85.

31. The recombinant DNA construct according to claim 22, wherein the CRISPR effector protein is selected from the group consisting of Cas9, Cas12a, Cas12b, and CasX.

32. The recombinant DNA construct according to claim 22, wherein the minimum promoter is the 35S(-46) promoter.

33. A plant comprising the recombinant DNA construct described in claim 22 in its genome.

34. 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 operably linked to a heterogeneous second promoter selected from the group consisting of an egg cell-preferential promoter, a meiotic cell-preferential promoter, or an embryonic tissue-preferential promoter, wherein the third nucleic acid is located between the first and second nucleic acids, and the third nucleic acid includes a first target site for the DNA-modifying enzyme at its 5' end and a second target site for the DNA-modifying enzyme at its 5' end.

35. The recombinant DNA construct according to claim 34, further comprising a fourth nucleic acid sequence encoding a guide nucleic acid operably linked to a third promoter.

36. The recombinant DNA construct according to claim 34, wherein the heterogeneous second promoter is an egg cell-preferential promoter selected from the group consisting of the EA1 promoter and the ES4 promoter.

37. The recombinant DNA construct according to claim 34, wherein the heterogeneous second promoter is an egg cell-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82.

38. The recombinant DNA construct according to claim 34, wherein the heterogeneous second promoter is an embryonic tissue-preferential promoter selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter.

39. The recombinant DNA construct according to claim 34, wherein the heterogeneous second promoter is an embryonic tissue-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88.

40. The recombinant DNA construct according to claim 34, wherein the heterogeneous second promoter is a meiotic cell-preferential promoter selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter.

41. The recombinant DNA construct according to claim 34, wherein the heterogeneous second promoter is a meiotic cell-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85.

42. The recombinant DNA construct according to claim 34, wherein the CRISPR effector protein is selected from the group consisting of Cas9, Cas12a, Cas12b, and CasX.

43. The recombinant DNA construct according to claim 34, wherein the first promoter is selected from the group consisting of the OCS promoter, the CaMV 19S promoter, the CaMV 35S promoter, the actin promoter, and the ubiquitin promoter.

44. The recombinant DNA construct according to claim 34, wherein the DNA modifying enzyme is Cre recombinase.

45. The recombinant DNA construct according to claim 44, wherein the first target site and the second target site are lox sites.

46. The recombinant DNA construct according to claim 34, wherein the DNA modifying enzyme is a CRISPR effector protein.

47. The recombinant DNA construct according to claim 46, wherein the first target site and the second target site are target sites for a guide nucleic acid.

48. A plant comprising the recombinant DNA construct described in claim 34 in its genome.

49. A method for generating two or more offspring plants having unique editing from a single transformed plant cell, (a) In plant cells, (i) A first nucleic acid sequence encoding a CRISPR effector protein operably ligated to a heterogeneous first promoter selected from the group consisting of a meiosis-preferential promoter, an egg cell-preferential promoter, and an embryo cell-preferential promoter; and (ii) A second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the genome. The process of introducing; and (b) A step of regenerating a first plant from the plant cells of step (a), wherein the CRISPR effector protein and at least one nucleic acid form a ribonucleoprotein in at least one meiotic cell, egg cell, or embryonic cell of the first plant, and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one meiotic cell, egg cell, or embryonic cell; (c) The process of pollinating the first plant in process (b); (d) A process of germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing. Methods that include...

50. The method according to claim 49, wherein the heterologous first promoter is a heterologous oocyte-preferential promoter selected from the group consisting of the EA1 promoter and the ES4 promoter.

51. The method according to claim 49, wherein the heterogeneous first promoter is a heterogeneous oocyte-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-3, 21-38, 41-45, and 65-82.

52. The method according to claim 49, wherein the heterogeneous first promoter is an embryonic tissue-preferential promoter selected from the group consisting of the DSUL1 promoter, the EA1 promoter, the ES4 promoter, and the EAL1 promoter.

53. The method according to claim 49, wherein the heterogeneous first promoter is an embryonic tissue-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 29-40, 43-45, and 86-88.

54. The method according to claim 49, wherein the heterogeneous first promoter is a meiotic cell-preferential promoter selected from the group consisting of the DMC1 promoter, the Mps1 promoter, and the Adf1 promoter.

55. The method according to claim 49, wherein the heterogeneous first promoter is a meiotic cell-preferential promoter comprising a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4-6 and 83-85.

56. The method according to claim 49, wherein the CRISPR effector protein is selected from the group consisting of Cas9, Cas12a, Cas12b, and CasX.

57. The method according to claim 49, wherein the heterogeneous second promoter is a PolIII promoter.

58. The method according to claim 49, wherein the first plant is a haploid-induced strain.

59. The method according to claim 49, wherein the first plant is female and the heterologous first promoter is ES4.

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