WO2025221793A1

WO2025221793A1 - Modified cas proteins for nuclear localization and editing activity in plants

Info

Publication number: WO2025221793A1
Application number: PCT/US2025/024776
Authority: WO
Inventors: Matthew Bauer; Jitendra Kumar; Santiago Navarro; Shanshan Song; Christina Marie TAYLOR
Original assignee: Monsanto Technology LLC
Current assignee: Monsanto Technology LLC
Priority date: 2024-04-16
Filing date: 2025-04-15
Publication date: 2025-10-23
Anticipated expiration: 2026-10-16
Also published as: WO2025221793A9

Abstract

Provided are modified Cas proteins with alternative designs for attaching or embedding nuclear localization signals for eukaryotic cells, and uses thereof for editing in eukaryotic cells or organisms. Also provided are linker peptides useful in the methods described and methods and means for inserting heterologous peptides in Cas12a proteins without impacting the function thereof. Further provided are novel nuclear localization signals, including non-classic nuclear localization signals.

Description

TITLE OF THE INVENTION MODIFIED CAS PROTEINS FOR NUCLEAR LOCALIZATION AND EDITING ACTIVITY IN PLANTS FIELD [0001] The present disclosure relates to modified Cas proteins with alternative designs for nuclear localization in eukaryotic cells and uses thereof for editing in eukaryotic cells or organisms. CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims priority of U.S. Provisional Application Serial No.63/634654, filed April 16, 2024, the entire disclosure of which is incorporated herein by reference. INCORPORATION OF SEQUENCE LISTING [0003] A sequence listing contained in the file named “MONS597WO_ST26.xml” which is 146,006bytes (measured in MS-Windows®) and was created on April 15, 2025, containing 106 sequences, is filed electronically herewith and incorporated by reference in its entirety. BACKGROUND OF THE INVENTION [0004] Genome editing, using RNA guided nucleases, such as CRISPR/Cas proteins, holds great promises for medicines and agricultural applications. While some forms of genome editing, such as targeted indels, are of sufficient efficacy in most eukaryotic organisms, other forms, such as site-directed integration (SDI) of exogenous gene cassettes or precise edits by homology directed repair (HDR) or by alternative technologies, such as base editing (BE) or prime editing (PE), are still lacking in efficacy. Functional domains advantageous for performing these latter technologies require fusion or tethering to the Cas proteins, typically at the N-terminus and/or C-terminus of the Cas proteins. However, nuclear localization signals required for the translocation of Cas proteins to the nucleus of a eukaryotic cells to edit target sites in nuclear genomes of eukaryotic cells are also typically located at both termini of Cas proteins, potentially hampering the fusion of tethering the Cas proteins to additional functional domains. [0005] The present invention addresses these shortcomings in the art by providing new compositions of heterologous polypeptides that facilitate uptake of the CAS protein or a ribonucleoprotein complex comprising the Cas protein into the nucleus of a eukaryotic cell, which are added to only one of the termini, particularly only the C-terminus of the Cas protein, or embedded within the Cas protein, thereby freeing up at least one of the termini of the Cas protein for fusion or tethering of additional functional domains. Also provided are non-classic NLS heterologous polypeptides that facilitate uptake of the CAS protein or a ribonucleoprotein complex comprising the Cas protein into the nucleus of a plant cell or eukaryotic cell. SUMMARY OF THE INVENTION [0006] In summary, the invention is directed at the following numbered embodiments: [0007] Embodiment 1. A ribonucleoprotein complex comprising: a. an RNA guided polypeptide comprising: i. an effector polypeptide which is or is derived from a Crispr/CAS protein; ii. one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus of the effector polypeptide; b. at least one guide RNA; wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are connected to the effector polypeptide through a linker amino acid sequence comprising GGSG or GGGS. [0008] Embodiment 2. The ribonucleoprotein complex according to embodiment 1, wherein the linker further comprises one or more repeats of the amino acid sequence EAAAK (SEQ ID NO: 18). [0009] Embodiment 3. The ribonucleoprotein complex according to any one of embodiments 1 or 2, wherein the linker further comprises at least two repeats of the amino acid sequence EAAAK (SEQ ID NO: 18). [0010] Embodiment 4. The ribonucleoprotein complex according to any one of embodiments 1 to 3, wherein the linker further comprises at least four repeats of the amino acid sequence EAAAK (SEQ ID NO: 18). [0011] Embodiment 5. The ribonucleoprotein complex according to any one of embodiments 1 to 4, wherein the linker further comprises at least six repeats of the amino acid sequence EAAAK (SEQ ID NO: 18). [0012] Embodiment 6. The ribonucleoprotein complex according to any one of embodiments 1 or 2, wherein the linker comprises the amino acid sequence GGSG(EAAAK)n2GGSG (SEQ ID NO: 21). [0013] Embodiment 7. The ribonucleoprotein complex according to any one of embodiments 1 or 2, wherein the linker comprises the amino acid sequence GGSG(EAAAK)n4GGSG (SEQ ID NO: 22). [0014] Embodiment 8. The ribonucleoprotein complex according to any one of embodiments 1 or 2, wherein the linker comprises the amino acid sequence GGSG(EAAAK)n₆GGSG (SEQ ID NO: 23). [0015] Embodiment 9. The ribonucleoprotein complex according to embodiment 1, wherein the linker further comprises one or more repeats of the amino acid sequence YETKQ (SEQ ID NO: 19). [0016] Embodiment 10. The ribonucleoprotein complex according to embodiment 1 or 9, wherein the linker comprises the amino acid sequence GGGSGGGSYETKQGGGSG (SEQ ID NO: 24) [0017] Embodiment 11. The ribonucleoprotein complex according to embodiment 1, wherein the linker further comprises one or more repeats of the amino acid sequence PVTAT (SEQ ID NO: 20). [0018] Embodiment 12. The ribonucleoprotein complex according to embodiment 1 or 11, wherein the linker comprises the amino acid sequence GGGSGGGSPVTATGGGSGGGSG (SEQ ID NO: 25). [0019] Embodiment 13. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise a Class 1 nuclear localization signal (NLS) having the formula KR(K/R)R or K(K/R)RK. [0020] Embodiment 14. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise a Class 2 nuclear localization signal (NLS) having the formula (P/R)XXKR(^DE)(K/R). [0021] Embodiment 15. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise a Class 3 nuclear localization signal (NLS) having the formula KRX(W/F/Y)XXAF. [0022] Embodiment 16. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise a Class 4 nuclear localization signal (NLS) having the formula (R/P)XXKR(K/R)(^DE) or a Class 5 nuclear localization signal having the formula LGKR(K/R)(W/F/Y) or a Class 6 nuclear localization signal having the formula KRX[10- _12]K(KR)(KR). [0023] Embodiment 17. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise an NLS amino acid sequence from a tomato Heat- shock inducible protein HSFA1 (HsFA NLS), optionally comprising the amino acid sequence of SEQ ID No: 14 [ an HsFA NLS]. [0024] Embodiment 18. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise two copies of HsFA NLS, optionally comprising the amino acid sequence of SEQ ID No: 14. [0025] Embodiment 19. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise synthetic class 1 NLS, optionally comprising the amino acid sequence of SEQ ID No:15 [NLS1]. [0026] Embodiment 20. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise two copies of [NLS1], optionally comprising the amino acid sequence of SEQ ID No: 15. [0027] Embodiment 21. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID No: 16. [0028] Embodiment 22. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises the amino acid sequence of SEQ ID No: 89. [0029] Embodiment 23. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises the amino acid sequence of SEQ ID No: 90. [0030] Embodiment 24. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises the amino acid sequence of SEQ ID No: 91. [0031] Embodiment 25. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises the amino acid sequence of SEQ ID No: 92. [0032] Embodiment 26. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises the amino acid sequence of SEQ ID No: 93. [0033] Embodiment 27. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises the amino acid sequence of SEQ ID No: 94. [0034] Embodiment 28. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises an amino acid sequence encoded by SEQ ID No: 95. [0035] Embodiment 29. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises an amino acid sequence encoded by SEQ ID No: 96. [0036] Embodiment 30. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises an amino acid sequence encoded SEQ ID No: 97. [0037] Embodiment 31. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises an amino acid sequence encoded by SEQ ID No: 98. [0038] Embodiment 32. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises an amino acid sequence encoded by SEQ ID No: 99. [0039] Embodiment 33. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises the amino acid sequence of SEQ ID No: 100. [0040] Embodiment 34. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises the amino acid sequence of SEQ ID No: 101. [0041] Embodiment 35. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises the amino acid sequence of SEQ ID No: 102. [0042] Embodiment 36. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises the amino acid sequence of SEQ ID No: 103. [0043] Embodiment 37. The ribonucleoprotein complex according to any one of embodiments 1 to 12, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprises the amino acid sequence of SEQ ID No: 104. [0044] Embodiment 38. The ribonucleoprotein complex according to any one of embodiments 1 to 37, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are connected to the effector polypeptide at the N-terminus or the C-terminus. [0045] Embodiment 39. The ribonucleoprotein complex according to any one of embodiments 1 to 37, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are connected to the effector polypeptide at the C-terminus. [0046] Embodiment 40. The ribonucleoprotein complex according to any one of embodiments 1 to 37, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are connected to the effector polypeptide only at the C- terminus. [0047] Embodiment 41. The ribonucleoprotein complex according to any one of embodiments 1 to 40, wherein the effector polypeptide is or is derived from a CRISPR-Cas effector protein , selected from a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR- Cas system, a Type IV CRISPR-Cas system, Type V CRISPR-Cas system, or a Type VI CRISPR- Cas system, or a CRISPR-Cas effector protein derived therefrom. [0048] Embodiment 42. The ribonucleoprotein complex according to any one of embodiments 1 to 40, wherein the effector polypeptide is or is derived from a Type II or Type V Crispr/Cas protein. [0049] Embodiment 43. The ribonucleoprotein complex according to any one of embodiments 1 to 40 wherein the RNA guided endonuclease is a CRISPR-Cas effector protein selected from a Cas9, C2c1, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Csnl, Csx12, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, 30 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 nuclease, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b or Cas14c effector protein. [0050] Embodiment 44. The ribonucleoprotein complex according to any one of embodiments 1 to 40, wherein the effector polypeptide is or is derived from a Cas9 polypeptide or a Cas12a polypeptide. [0051] Embodiment 45. The ribonucleoprotein complex according to any one of embodiments 1 to 40, wherein the effector polypeptide is or is derived from a Type V Crispr/Cas protein. [0052] Embodiment 46. The ribonucleoprotein complex according to any one of embodiments 1 to 40, wherein the effector polypeptide is or is derived from a Cas12a polypeptide. [0053] Embodiment 47. The ribonucleoprotein complex according to embodiment 46, wherein the Cas12a effector protein is selected from FnCas12a, LbCas12a, ErCas12a (MAD7®) or AsCas12a or variants thereof. [0054] Embodiment 48. The ribonucleoprotein complex according to any one of embodiments 46 or 47, wherein the Cas12a effector protein has an amino acid sequence having at least 90% or at least 95% or at least 99% or 100% sequence identity to an amino acid selected from the group consisting of SEQ ID Nos: 33, 38 and 39. [0055] Embodiment 49. The ribonucleoprotein complex according to any one of embodiments 41 to 48, wherein the effector protein has double stranded DNA nuclease activity, single stranded DNA activity, or no DNA nuclease activity while retaining DNA binding capacity. [0056] Embodiment 50. The ribonucleoprotein complex according to any one of embodiments 41 to 49, wherein the effector protein is a fusion protein comprising a cleavage domain, a nuclease domain, a deaminase domain, a cytosine deaminase domain, an adenine deaminase domain, a transcription activator domain, a transcription repression domain, a reverse transcriptase domain, a uracil DNA glycolase inhibitor, a Dna2 polypeptide, and/or a 5’ flap endonuclease. [0057] Embodiment 51. The ribonucleoprotein complex according to any one of embodiments 1 to 50, wherein the guideRNA comprises a crRNA and a tracrRNA [0058] Embodiment 52. The ribonucleoprotein complex according to any one of embodiments 1 to 50, wherein the guideRNA is a single guide RNA comprising a crRNA portion and a tracrRNA portion. [0059] Embodiment 53. The ribonucleoprotein complex according to any one of embodiments 1 to 52, wherein the effector protein is or is derived from a Cas12a and the guide RNA comprises a direct repeat (or crRNA portion) and a spacer having complementarity to a target region in a prokaryotic or eukaryotic cell, optionally wherein the guideRNA comprise a direct repeat, followed by a spacer, followed by another copy of the direct repeat. [0060] Embodiment 54. The ribonucleoprotein complex according to any one of embodiments 1 to 53, wherein the RNA guided polypeptide has an amino acid sequence having at least 90% or at least 95% or at least 99% or at least 100% sequence identity to an amino acid selected from the group consisting of - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 17, SEQ ID NO:14 and SEQ ID NO:14; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:14, SEQ ID NO: 17:, SEQ ID NO:14; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:14, SEQ ID NO: 17:, SEQ ID NO:15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 21 and SEQ ID NO:15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 22 and SEQ ID NO:15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 23 and SEQ ID NO:15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 24 and SEQ ID NO:15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 25 and SEQ ID NO:15; - an amino acid sequence comprising in order SEQ ID NO: 35, SEQ ID NO:21, SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 35, SEQ ID NO:24, SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:21, SEQ ID NO: 15; - v an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 14; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 89; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 90; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 91; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 92; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 93; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 94; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 100; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 101; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 102; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 103; and - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 104. [0061] Embodiment 55. The ribonucleoprotein complex according to any one of embodiments 1 to 53, wherein the effector protein and the guide RNA do not naturally occur together. [0062] Embodiment 56. A ribonucleoprotein complex comprising: a. an RNA guided polypeptide comprising: i. an effector polypeptide derived from a Crispr/CAS protein; ii. one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell; b. at least one guide RNA; wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are embedded within the effector polypeptide. [0063] Embodiment 57. The ribonucleoprotein according to embodiment 56, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are embedded within the effector polypeptide in an exposed loop of the Crispr/Cas protein. [0064] Embodiment 58. The ribonucleoprotein according to embodiment 56 or 57, wherein the effector polypeptide is or is derived from a CRISPR-Cas effector protein , selected from a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR-Cas system, a Type IV CRISPR-Cas system, Type V CRISPR-Cas system, or a Type VI CRISPR-Cas system, or a CRISPR-Cas effector protein derived therefrom. [0065] Embodiment 59. The ribonucleoprotein complex according to any one of embodiments 56 to 58, wherein the effector polypeptide is or is derived from a Type II or Type V Crispr/Cas protein. [0066] Embodiment 60. The ribonucleoprotein complex according to any one of embodiments 56 to 58 wherein the RNA guided endonuclease is a CRISPR-Cas effector protein selected from a Cas9, C2c1, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Csnl, Csx12, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, 30 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 nuclease, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b or Cas14c effector protein. [0067] Embodiment 61. The ribonucleoprotein complex according to any one of embodiments 56 to 60, wherein the effector polypeptide is or is derived from a Cas9 polypeptide or a Cas12a polypeptide. [0068] Embodiment 62. The ribonucleoprotein complex according to any one of embodiments 56 to 60, wherein the effector polypeptide is or is derived from a Type V Crispr/Cas protein. [0069] Embodiment 63. The ribonucleoprotein complex according to any one of embodiments 56 to 60, wherein the effector polypeptide is or is derived from a Cas12a polypeptide. [0070] Embodiment 64. The ribonucleoprotein complex according to embodiment 63, wherein the Cas12a effector protein is selected from FnCas12a, LbCas12a, ErCas12a (MAD7®) or AsCas12a or variants thereof. [0071] Embodiment 65. The ribonucleoprotein complex according to any one of embodiments 63 or 64, wherein the Cas12a effector protein has an amino acid sequence having at least 90% or at least 95% or at least 99% or 100% sequence identity to an amino acid selected from the group consisting of SEQ ID Nos: 33, 38 and 39. [0072] Embodiment 66. The ribonucleoprotein complex according to any one of embodiments 56 to 65, wherein the effector protein has double stranded DNA nuclease activity, single stranded DNA activity, or no DNA nuclease activity while retaining DNA binding capacity. [0073] Embodiment 67. The ribonucleoprotein complex according to any one of embodiments 56 to 65, wherein the effector protein is a fusion protein comprising a cleavage domain, a nuclease domain, a deaminase domain, a cytosine deaminase domain, an adenine deaminase domain, a transcription activator domain, a transcription repression domain, a reverse transcriptase domain, a uracil DNA glycolase inhibitor, a Dna2 polypeptide, and/or a 5’ flap endonuclease. [0074] Embodiment 68. The ribonucleoprotein complex according to any one of embodiments 56 to 65, wherein the effector protein is or is derived from Cas12a and the exposed loop corresponds to the amino acid sequence from position 85 to 89, or the amino acid sequence from position 126- 137, or the amino acid sequence from position 1076-1085, or the amino acid sequence from position 370-379, or the amino acid sequence from position 437-460, or the amino acid sequence from position 485-490, or the amino acid sequence from position 449 to 461, or the amino acid sequence from position 487-496 of the amino acid sequence of SEQ ID NO: 33 [LbCas12a]. [0075] Embodiment 69. The ribonucleoprotein complex according to any one of embodiments 56 to 68, wherein the effector protein is or is derived from Cas12a and the exposed loop corresponds to the amino acid sequence from position 449 to 461 or the amino acid sequence from position 487-496 of the amino acid sequence of SEQ ID NO: 33[LbCas12a]. [0076] Embodiment 70. The ribonucleoprotein complex according to any one of embodiments 56 to 69 , wherein the heterologous polypeptide that facilitates uptake of the RNP complex into the nucleus of a eukaryotic cell is inserted into the exposed loop. [0077] Embodiment 71. The ribonucleoprotein complex according to any one of embodiments 56 to 69 , wherein the amino acid sequence of the exposed loop is substituted for the amino acid sequence of a heterologous polypeptide that facilitates uptake of the RNP complex into the nucleus of a eukaryotic cell are substituted into the exposed loop. [0078] Embodiment 72. The ribonucleoprotein complex according to any one of embodiments 56 to 71, wherein the heterologous polypeptide that facilitates uptake of the RNP complex into the nucleus of a eukaryotic cell comprises an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 75 or SEQ ID NO: 76. [0079] Embodiment 73. The ribonucleoprotein complex according to any one of embodiments 56 to 72, wherein the effector protein is or is derived from Cas12a, and the amino acid sequence corresponding to the amino acid sequence from position 449 to 461 of SEQ ID NO: 33 is replaced by the amino acid sequence of SEQ ID NO: 55. [0080] Embodiment 74. The ribonucleoprotein complex according to any one of embodiments 56 to 72, wherein the effector protein is or is derived from Cas12a, and the amino acid sequence corresponding to the amino acid sequence from position 487 to 496 of SEQ ID NO: 33 is replaced by the amino acid sequence of SEQ ID NO: 56. [0081] Embodiment 75. The ribonucleoprotein complex according to any one of embodiments 56 to 72, wherein the effector protein is or is derived from Cas12a, and the amino acid sequence corresponding to the amino acid sequence from position 487 to 496 of SEQ ID NO: 33 is replaced by the amino acid sequence of SEQ ID NO: 14. [0082] Embodiment 76. The ribonucleoprotein complex according to any one of embodiments 56 to 72, wherein the effector protein is or is derived from Cas12a, and 449 to 461 is replaced by the amino acid sequence of SEQ ID NO: 55 and the amino acid sequence corresponding to the amino acid sequence from position 487 to 496 of SEQ ID NO: 33 is replaced by the amino acid sequence of SEQ ID NO: 56. [0083] Embodiment 77. The ribonucleoprotein complex according to any one of embodiments 56 to 72, wherein the effector protein has an amino acid sequence having at least 90% or 95% or 99% or 100% sequence identity to the amino acid of SEQ ID NO: 34. [0084] Embodiment 78. The ribonucleoprotein complex according to any one of embodiments 56 to 72, wherein the effector protein has an amino acid sequence having at least 90% or 95% or 99% or 100% sequence identity to the amino acid of SEQ ID NO: 35. [0085] Embodiment 79. The ribonucleoprotein complex according to any one of embodiments 56 to 72, wherein the effector protein has an amino acid sequence having at least 90% or 95% or 99% or 100% sequence identity to the amino acid of SEQ ID NO: 36. [0086] Embodiment 80. The ribonucleoprotein complex according to any one of embodiments 56 to 72, wherein the effector protein has an amino acid sequence having at least 90% or 95% or 99% or 100% sequence identity to the amino acid of SEQ ID NO: 37. [0087] Embodiment 81. The ribonucleoprotein complex according to any one of embodiments 56 to 72, wherein the effector protein has an amino acid sequence having at least 90% or 95% or 99% or 100% sequence identity to the amino acid of SEQ ID NO: 67. [0088] Embodiment 82. The ribonucleoprotein complex according to any one of embodiments 56 to 72, wherein the effector protein has an amino acid sequence having at least 90% or 95% or 99% or 100% sequence identity to the amino acid of SEQ ID NO: 68. [0089] Embodiment 83. The ribonucleoprotein complex according to any one of embodiments 56 to 82, further comprising one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus of the effector polypeptide. [0090] Embodiment 84. The ribonucleoprotein complex according to embodiment 83, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus of the effector polypeptide are connected to the effector polypeptide through a linker amino acid sequence comprising GGSG or GGGS as described in any one of embodiments 1 to 12. [0091] Embodiment 85. The ribonucleoprotein complex according to embodiment 83 or 84, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus of the effector polypeptide comprise a nuclear localization signal having the formula or amino acid sequence as described in any one of embodiments 13 to 37. [0092] Embodiment 86. The ribonucleoprotein complex according to any one of embodiments 83 to 85, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus of the effector polypeptide are connected to the effector polypeptide at the C-terminus. [0093] Embodiment 87. The ribonucleoprotein complex according to any one of embodiments 83 to 85, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus of the effector polypeptide are connected to the effector polypeptide only at the C-terminus. [0094] Embodiment 88. A recombinant DNA molecule comprising the following operably linked DNA fragments: a. a promoter expressible in a eukaryotic cell; b. a DNA fragment encoding an effector polypeptide which is or is derived from a Crispr/CAS protein; c. a DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell; d. a DNA fragment encoding a linker amino acid sequence comprising GGSG (SEQ ID NO: 17); wherein at least one of the DNA fragment is heterologous to one of the other DNA fragments; and wherein the encoded one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell are located at or near or in proximity to a terminus of the encoded effector polypeptide upon expression of recombinant DNA molecule and are connected to the effector protein through the encoded linker amino acid sequence . [0095] Embodiment 89. The recombinant DNA molecule according to embodiment 88, wherein the linker further comprises on or more repeats of the amino acid sequence EAAAK (SEQ ID NO: 18). [0096] Embodiment 90. The recombinant DNA molecule according to embodiment 88 or 89, wherein the linker further comprises at least two repeats of the amino acid sequence EAAAK (SEQ ID NO: 18). [0097] Embodiment 91. The recombinant DNA molecule according to any one of embodiments 88 to 90, wherein the linker further comprises at least four repeats of the amino acid sequence EAAAK (SEQ ID NO: 18). [0098] Embodiment 92. The recombinant DNA molecule according to any one of embodiments 88 to 91, wherein the linker further comprises at least six repeats of the amino acid sequence EAAAK (SEQ ID NO: 18). [0099] Embodiment 93. The recombinant DNA molecule according to any one of embodiments 88 or 89, wherein the linker comprises the amino acid sequence GGSG(EAAAK)n2GGSG (SEQ ID NO: 21). [0100] Embodiment 94. The recombinant DNA molecule according to any one of embodiments 88 or 89, wherein the linker comprises the amino acid sequence GGSG(EAAAK)n4GGSG (SEQ ID NO: 22). [0101] Embodiment 95. The recombinant DNA molecule according to any one of embodiments 88 or 89, wherein the linker comprises the amino acid sequence GGSG(EAAAK)n6GGSG (SEQ ID NO: 23). [0102] Embodiment 96. The recombinant DNA molecule according to embodiment 88 wherein the linker further comprises one or more repeats of the amino acid sequence YETKQ (SEQ ID NO: 19). [0103] Embodiment 97. The recombinant DNA molecule according to embodiment 88 or 96, wherein the linker comprises the amino acid sequence GGGSGGGSYETKQGGGSG (SEQ ID NO: 24). [0104] Embodiment 98. The recombinant DNA molecule according to embodiment 88, wherein the linker further comprises one or more repeats of the amino acid sequence PVTAT (SEQ ID NO: 20). [0105] Embodiment 99. The recombinant DNA molecule according to embodiment 88 or 98, wherein the linker comprises the amino acid sequence GGGSGGGSPVTATGGGSGGGSG (SEQ ID NO: 25). [0106] Embodiment 100. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise a Class 1 nuclear localization signal (NLS) having the formula KR(K/R)R or K(K/R)RK. [0107] Embodiment 101. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise a Class 2 nuclear localization signal (NLS) having the formula (P/R)XXKR(^DE)(K/R). [0108] Embodiment 102. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise a Class 3 nuclear localization signal (NLS) having the formula KRX(W/F/Y)XXAF. [0109] Embodiment 103. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise a Class 4 nuclear localization signal (NLS) having the formula (R/P)XXKR(K/R)(^DE) or a Class 5 nuclear localization signal having the formula LGKR(K/R)(W/F/Y) or a Class 6 nuclear localization signal having the formula KRX[10- _12]K(KR)(KR). [0110] Embodiment 104. The recombinant DNA molecule according to any one of embodiments 88 to99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise an NLS amino acid sequence from a tomato Heat-shock inducible protein HSFA1 (HsFA NLS), optionally comprising the amino acid sequence of SEQ ID No: 14 [0111] Embodiment 105. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise two copies of HsFA NLS, optionally comprising the amino acid sequence of SEQ ID No: 14. [0112] Embodiment 106. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise synthetic class 1 NLS, optionally comprising the amino acid sequence of SEQ ID No: 15. [0113] Embodiment 107. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise two copies of [NLS1], optionally comprising the amino acid sequence of SEQ ID No: 15. [0114] Embodiment 108. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID No: 16. [0115] Embodiment 109. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID No:89. [0116] Embodiment 110. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID No:90. [0117] Embodiment 111. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID No: 91. [0118] Embodiment 112. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID No: 92. [0119] Embodiment 113. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID No: 93. [0120] Embodiment 114. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID No: 94. [0121] Embodiment 115. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise an amino acid sequence encoded by SEQ ID No: 95 or encoded by SEQ ID NO: 96.. [0122] Embodiment 116. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise an amino acid sequence encoded by SEQ ID NO: 97. [0123] Embodiment 117. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise an amino acid sequence encoded by SEQ ID NO: 98. [0124] Embodiment 118. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise an amino acid sequence encoded by SEQ ID NO: 99. [0125] Embodiment 119. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID NO: 100. [0126] Embodiment 120. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID NO: 101. [0127] Embodiment 121. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID NO: 102. [0128] Embodiment 122. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID NO: 103. [0129] Embodiment 123. The recombinant DNA molecule according to any one of embodiments 88 to 99, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise the amino acid sequence of SEQ ID NO: 104. [0130] Embodiment 124. The recombinant DNA molecule according to any one of embodiments 88 to 123, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are connected to the effector polypeptide at the N- terminus or the C-terminus. [0131] Embodiment 125. The recombinant DNA molecule according to any one of embodiments 88 to 123, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are connected to the effector polypeptide at the C- terminus. [0132] Embodiment 126. The recombinant DNA molecule according to any one of embodiments 88 to 123, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are connected to the effector polypeptide only at the C-terminus. [0133] Embodiment 127. The recombinant DNA molecule according to any one of embodiments 88 to 126, wherein the effector polypeptide is or is derived from a CRISPR-Cas effector protein , selected from a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR- Cas system, a Type IV CRISPR-Cas system, Type V CRISPR-Cas system, or a Type VI CRISPR- Cas system, or a CRISPR-Cas effector protein derived therefrom. [0134] Embodiment 128. The recombinant DNA molecule according to any one of embodiments 88 to126, wherein the effector polypeptide is or is derived from a Type II or Type V Crispr/Cas protein. [0135] Embodiment 129. The recombinant DNA molecule according to any one of embodiments 88 to 126 wherein the RNA guided endonuclease is a CRISPR-Cas effector protein selected from a Cas9, C2c1, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Csnl, Csx12, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, 30 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 nuclease, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b or Cas14c effector protein. [0136] Embodiment 130. The recombinant DNA molecule according to any one of embodiments 88 to 126, wherein the effector polypeptide is or is derived from a Cas9 polypeptide or a Cas12a polypeptide. [0137] Embodiment 131. The recombinant DNA molecule according to any one of embodiments 88 to 126, wherein the effector polypeptide is or is derived from a Type V Crispr/Cas protein. [0138] Embodiment 132. The recombinant DNA molecule according to any one of embodiments 88 to 126, wherein the effector polypeptide is or is derived from a Cas12a polypeptide. [0139] Embodiment 133. The recombinant DNA molecule according to embodiment 132, wherein the Cas12a effector protein is selected from FnCas12a, LbCas12a, ErCas12a (MAD7®) or AsCas12a or variants thereof. [0140] Embodiment 134. The recombinant DNA molecule according to any one of embodiments 131 or 132, wherein the Cas12a effector protein has an amino acid sequence having at least 90% or at least 95% or at least 99% or 100% sequence identity to an amino acid selected from the group consisting of SEQ ID Nos: 33, 38 and 39. [0141] Embodiment 135. The recombinant DNA molecule according to any one of embodiments 88 to 134 encoding a polypeptide having at least 90% or at least 95% or at least 99% or 100% sequence identity to an amino acid selected from the group consisting of - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 17, SEQ ID NO:14 and SEQ ID NO: 14; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:14, SEQ ID NO: 17, and SEQ ID NO:14; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:14, SEQ ID NO: 17:, SEQ ID NO:15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 21 and SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 22 and SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 23 and SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 24 and SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 25 and SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 35, SEQ ID NO:21, SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 35, SEQ ID NO:24, SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:21, SEQ ID NO: 15; - v an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 14; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 15; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 89; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 90; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 91; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 92; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 93; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 94; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 100; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 101; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 102; - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 103; and - an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 104. [0142] Embodiment 136. The recombinant DNA molecule according to any one of embodiments 88 to 135, wherein the DNA fragment encoding the linker comprises a DNA sequence according to SEQ ID NO: 5. [0143] Embodiment 137. The recombinant DNA molecule according to any one of embodiments 88 to 135, wherein the DNA fragment encoding the linker comprises a DNA sequence according to SEQ ID NO: 6. [0144] Embodiment 138. The recombinant DNA molecule according to any one of embodiments 88to 135, wherein the DNA fragment encoding the linker comprises a DNA sequence according to SEQ ID NO: 7. [0145] Embodiment 139. The recombinant DNA molecule according to any one of embodiments 88 to 135, wherein the DNA fragment encoding the linker comprises a DNA sequence according to SEQ ID NO: 8. [0146] Embodiment 140. The recombinant DNA molecule according to any one of embodiments 88 to 135, wherein the DNA fragment encoding the linker comprises a DNA sequence according to SEQ ID NO: 9. [0147] Embodiment 141. The recombinant DNA molecule according to any one of embodiments 88 to 135, wherein the DNA fragment encoding the linker comprises a DNA sequence according to SEQ ID NO: 10. [0148] Embodiment 142. The recombinant DNA molecule according to any one of embodiments 88 to 135, wherein the DNA fragment encoding the linker comprises a DNA sequence according to SEQ ID NO: 11. [0149] Embodiment 143. The recombinant DNA molecule according to any one of embodiments 88 to 135, wherein the DNA fragment encoding the linker comprises a DNA sequence according to SEQ ID NO: 12. [0150] Embodiment 144. The recombinant DNA molecule according to any one of embodiments 88 to 135, wherein the DNA fragment encoding the linker comprises a DNA sequence according to SEQ ID NO: 13. [0151] Embodiment 145. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 1. [0152] Embodiment 146. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 2. [0153] Embodiment 147. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 3. [0154] Embodiment 148. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 4. [0155] Embodiment 149. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 83. [0156] Embodiment 150. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 84. [0157] Embodiment 151. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 85. [0158] Embodiment 152. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 86. [0159] Embodiment 153. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 87. [0160] Embodiment 154. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 88. [0161] Embodiment 155. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 95. [0162] Embodiment 156. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 96. [0163] Embodiment 157. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 97. [0164] Embodiment 158. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 98. [0165] Embodiment 159. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence according to SEQ ID NO: 99. [0166] Embodiment 160. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence encoding the amino acid sequence of SEQ ID NO: 100. [0167] Embodiment 161. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence encoding the amino acid sequence of SEQ ID NO: 101. [0168] Embodiment 162. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence encoding the amino acid sequence of SEQ ID NO: 102. [0169] Embodiment 163. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence encoding the amino acid sequence of SEQ ID NO: 103. [0170] Embodiment 164. The recombinant DNA molecule according to any one of embodiments 88 to 144, wherein the DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell comprises a DNA sequence encoding the amino acid sequence of SEQ ID NO: 104. [0171] Embodiment 165. The recombinant DNA molecule according to any one of embodiments 88 to 164, wherein the DNA fragment encoding the effector polypeptide which is or is derived from a Crispr/CAS protein is codon-optimized for expression in a eukaryotic cell, optionally a eukaryotic cell selected from an animal cell, a plant cell, or a fungal cell, optionally a plant cell. [0172] Embodiment 166. The recombinant DNA molecule according to any one of embodiments 88 to 165, wherein the DNA fragment encoding the one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell is codon-optimized for expression in a eukaryotic cell, optionally a eukaryotic cell selected from an animal cell, a plant cell, or a fungal cell, optionally a plant cell. [0173] Embodiment 167. The recombinant DNA molecule according to any one of embodiments 88 to 166, wherein the DNA fragment encoding the linker amino acid sequence is codon-optimized for expression in a eukaryotic cell, optionally a eukaryotic cell selected from an animal cell, a plant cell, or a fungal cell, optionally a plant cell. [0174] Embodiment 168. The recombinant DNA molecule according to any one of embodiments 88 to 167, wherein the promoter expressible in a eukaryotic cell is a promoter recognized by an RNA dependent RNA polymerase II. [0175] Embodiment 169. The recombinant DNA molecule according to any one of embodiments 88 to 167, wherein the promoter expressible in a eukaryotic cell is a promoter recognized by an RNA dependent RNA polymerase III. [0176] Embodiment 170. The recombinant DNA molecule according to any one of embodiments 88 to 167, wherein the promoter expressible in a eukaryotic cell is a promoter recognized by an RNA dependent RNA polymerase I. [0177] Embodiment 171. The recombinant DNA molecule according to any one of embodiments 88 to 170, wherein the promoter is a plant-expressible promoter. [0178] Embodiment 172. The recombinant DNA molecule according to any one of embodiments 88 to 171, further comprising a DNA fragment having a polyadenylation signal. [0179] Embodiment 173. The recombinant DNA molecule according to any one of embodiments 88 to 172, further comprising a DNA fragment which is a transcription termination signal. [0180] Embodiment 174. A recombinant DNA molecule comprising the following operably linked DNA fragments: a. a promoter expressible in a eukaryotic cell; b. a DNA fragment encoding an effector polypeptide which is or is derived from a Crispr/CAS protein; c. a DNA fragment encoding one or more polypeptides that facilitate uptake of the effector polypeptide into the nucleus of a eukaryotic cell; wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are embedded within the effector polypeptide. [0181] Embodiment 175. The recombinant DNA molecule according to embodiment 174, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are embedded within the effector polypeptide in an exposed loop of the Crispr/Cas protein. [0182] Embodiment 176. The recombinant DNA molecule according to embodiment 174 or 175, wherein the effector polypeptide is or is derived from a CRISPR-Cas effector protein , selected from a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR-Cas system, a Type IV CRISPR-Cas system, Type V CRISPR-Cas system, or a Type VI CRISPR-Cas system, or a CRISPR-Cas effector protein derived therefrom. [0183] Embodiment 177. The recombinant DNA molecule according to any one of embodiments 174 to 176, wherein the effector polypeptide is or is derived from a Type II or Type V Crispr/Cas protein. [0184] Embodiment 178. The recombinant DNA molecule according to any one of embodiments 174 to 177, wherein the RNA guided endonuclease is a CRISPR-Cas effector protein selected from a Cas9, C2c1, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Csnl, Csx12, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, 30 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 nuclease, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b or Cas14c effector protein. [0185] Embodiment 179. The recombinant DNA molecule according to any one of embodiments 174 to 177, wherein the effector polypeptide is or is derived from a Cas9 polypeptide or a Cas12a polypeptide. [0186] Embodiment 180. The recombinant DNA molecule according to any one of embodiments 174 to 177, wherein the effector polypeptide is or is derived from a Type V Crispr/Cas protein. [0187] Embodiment 181. The recombinant DNA molecule according to any one of embodiments 174 to 177, wherein the effector polypeptide is or is derived from a Cas12a polypeptide. [0188] Embodiment 182. The recombinant DNA molecule according to embodiment 181, wherein the Cas12a effector protein is selected from FnCas12a, LbCas12a, ErCas12a (MAD7®) or AsCas12a or variants thereof. [0189] Embodiment 183. The recombinant DNA molecule according to any one of embodiments 181 or 182, wherein the Cas12a effector protein has an amino acid sequence having at least 90% or at least 95% or at least 99% or 100% sequence identity to an amino acid selected from the group consisting of SEQ ID NOs: 33, 38 and 39. [0190] Embodiment 184. The recombinant DNA molecule according to any one of embodiments 174 to 183, wherein the effector protein has double stranded DNA nuclease activity, single stranded DNA activity, or no DNA nuclease activity while retaining DNA binding capacity. [0191] Embodiment 185. The recombinant DNA molecule according to any one of embodiments 174 to 184, wherein the effector protein is a fusion protein comprising a cleavage domain, a nuclease domain, a deaminase domain, a cytosine deaminase domain, an adenine deaminase domain, a transcription activator domain, a transcription repression domain, a reverse transcriptase domain, a uracil DNA glycolase inhibitor, a Dna2 polypeptide, and/or a 5’ flap endonuclease. [0192] Embodiment 186. The recombinant DNA molecule according to any one of embodiments 174 to 185, wherein the effector protein is or is derived from Cas12a and the exposed loop corresponds to the amino acid sequence from position 85 to 89, or the amino acid sequence from position 126-137, or the amino acid sequence from position 1076-1085, or the amino acid sequence from position 1076-1085, or the amino acid sequence from position 370-379, or the amino acid sequence from position 437-460, or the amino acid sequence from position 485-490, or the amino acid sequence from position 449 to 461, or the amino acid sequence from position 487-496 of the amino acid sequence of reference SEQ ID NO: 33. [0193] Embodiment 187. The recombinant DNA molecule according to any one of embodiments 174 to 186, wherein the effector protein is or is derived from Cas12a and the exposed loop corresponds to the amino acid sequence from position 449 to 461 or the amino acid sequence from position 487-496 of the amino acid sequence of SEQ ID NO: 33. [0194] Embodiment 188. The recombinant DNA molecule according to any one of embodiments 174 to 187, wherein the heterologous polypeptide that facilitates uptake of the RNP complex into the nucleus of a eukaryotic cell is inserted into the exposed loop. [0195] Embodiment 189. The recombinant DNA molecule according to any one of embodiments 174 to 187 , wherein the amino acid sequence of the exposed loop is substituted for the amino acid sequence of a heterologous polypeptide that facilitates uptake of the RNP complex into the nucleus of a eukaryotic cell are substituted into the exposed loop. [0196] Embodiment 190.The recombinant DNA molecule according to any one of embodiments 174 to 187, wherein the heterologous polypeptide that facilitates uptake of the RNP complex into the nucleus of a eukaryotic cell comprises an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 75 or SEQ ID NO: 76. [0197] Embodiment 191. The recombinant DNA molecule according to any one of embodiments 174 to 187, wherein the effector protein is or is derived from Cas12a, and the amino acid sequence corresponding to the amino acid sequence from position 449 to 461 of SEQ ID NO: 33 is replaced by the amino acid sequence of SEQ ID NO: 55. [0198] Embodiment 192. The recombinant DNA molecule according to any one of embodiments 174 to 187, wherein the effector protein is or is derived from Cas12a, and the amino acid sequence corresponding to the amino acid sequence from position 487 to 496 of SEQ ID NO: 33 is replaced by the amino acid sequence of SEQ ID NO: 56. [0199] Embodiment 193. The recombinant DNA molecule according to any one of embodiments 174 to 187, wherein the effector protein is or is derived from Cas12a, and the amino acid sequence corresponding to the amino acid sequence from position 487 to 496 of SEQ ID NO: 33 is replaced by the amino acid sequence of SEQ ID NO: 14. [0200] Embodiment 194. The recombinant DNA molecule according to any one of embodiments 174 to 187, wherein the effector protein is or is derived from Cas12a, and the amino acid sequence corresponding to the amino acid sequence from position 449 to 461 of SEQ ID NO: 33 is replaced by the amino acid sequence of SEQ ID NO: 55 and the amino acid sequence corresponding to the amino acid sequence from position 487 to 496 of SEQ ID NO: 33 is replaced by the amino acid sequence of SEQ ID NO: 56. [0201] Embodiment 195. The recombinant DNA molecule according to any one of embodiments 174 to 187, wherein the effector protein has an amino acid sequence having at least 90% or at least 95% or at least 99% or 100% sequence identity to the amino acid of SEQ ID NO: 34. [0202] Embodiment 196. The recombinant DNA molecule according to any one of embodiments 174 to 187, wherein the effector protein has an amino acid sequence having at least 90% or at least 95% or at least 99% or 100% sequence identity to the amino acid of SEQ ID NO: 35. [0203] Embodiment 197. The recombinant DNA molecule according to any one of embodiments 174 to 187, wherein the effector protein has an amino acid sequence having at least 90% or at least 95% or at least 99% or 100% sequence identity to the amino acid of SEQ ID NO: 36. [0204] Embodiment 198. The recombinant DNA molecule according to any one of embodiments 174 to 187, wherein the effector protein has an amino acid sequence having at least 90% or at least 95% or at least 99% or 100% sequence identity to the amino acid of SEQ ID NO: 37. [0205] Embodiment 199. The recombinant DNA molecule according to any one of embodiments 174 to 187, wherein the effector protein has an amino acid sequence having at least 90% or at least 95% or at least 99% or 100% sequence identity to the amino acid of SEQ ID NO: 67. [0206] Embodiment 200. The recombinant DNA molecule according to any one of embodiments 174 to 187, wherein the effector protein has an amino acid sequence having at least 90% or at least 95% or at least 99% or at least 100% sequence identity to the amino acid of SEQ ID NO: 68. [0207] Embodiment 201. The recombinant DNA molecule according to any one of embodiments 174 to 200, further comprising one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus of the effector polypeptide. [0208] Embodiment 202. The recombinant DNA molecule according to embodiment201, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus of the effector polypeptide are connected to the effector polypeptide through a linker amino acid sequence comprising GGSG, as described in any one of embodiments 1 to 12. [0209] Embodiment 203. The recombinant DNA molecule according to embodiment 201 or 202, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus of the effector polypeptide comprise a nuclear localization signal having the formula or amino acid sequence as described in any one of embodiments 13 to 37. [0210] Embodiment 204. The recombinant DNA molecule according to any one of embodiments 201 to 203, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus of the effector polypeptide are connected to the effector polypeptide at the C-terminus. [0211] Embodiment 205. The recombinant DNA molecule according to any one of embodiments 201 to 203, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus of the effector polypeptide are connected to the effector polypeptide only at the C-terminus. [0212] Embodiment 206. The recombinant DNA molecule according to any one of embodiments 174 to 205 comprising the nucleotide sequence of SEQ ID NO: 27. [0213] Embodiment 207. The recombinant DNA molecule according to any one of embodiments 174 to 205 comprising the nucleotide sequence of SEQ ID NO: 28. [0214] Embodiment 208. The recombinant DNA molecule according to any one of embodiments 174 to 205 comprising the nucleotide sequence of SEQ ID NO: 29. [0215] Embodiment 209. The recombinant DNA molecule according to any one of embodiments 174 to 205 comprising the nucleotide sequence of SEQ ID NO: 30. [0216] Embodiment 210. The recombinant DNA molecule according to any one of embodiments 174 to 209, which is codon-optimized for expression in a eukaryotic cell, optionally a eukaryotic cell selected from an animal cell, a plant cell, or a fungal cell, optionally a plant cell. [0217] Embodiment 211. The recombinant DNA molecule according to any one of embodiments 174 to 210, wherein the promoter expressible in a eukaryotic cell is a promoter recognized by an RNA dependent RNA polymerase II. [0218] Embodiment 212. The recombinant DNA molecule according to any one of embodiments 174 to 210, wherein the promoter expressible in a eukaryotic cell is a promoter recognized by an RNA dependent RNA polymerase III. [0219] Embodiment 213. The recombinant DNA molecule according to any one of embodiments 174 to 210, wherein the promoter expressible in a eukaryotic cell is a promoter recognized by an RNA dependent RNA polymerase I. [0220] Embodiment 214. The recombinant DNA molecule according to any one of embodiments 174 to 210, wherein the promoter is a plant-expressible promoter. [0221] Embodiment 215. The recombinant DNA molecule according to any one of embodiments 174 to 214, further comprising DNA fragment having a polyadenylation signal. [0222] Embodiment 216. The recombinant DNA molecule according to any one of embodiments 174 to 215, further comprising a DNA fragment which is a transcription termination signal. [0223] Embodiment 217. The recombinant DNA molecule according to any one of embodiments 88 to 168 or 171 to 211 or 214 to 216, wherein the promoter is a plant-expressible promoter selected from constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue-specific promoters or is selected from a meiotic promoter, an egg cell-preferred or embryo-tissue preferred promoter such as a DSUL1 promoter, an EA1 promoter, an ES4 promoter, a DMC1 promoter, a Mps1 promoter, an Adf1 promoter or an EAL promoter, or is a floral-tissue preferred or floral cell-preferred promoter. [0224] Embodiment 218. A method for editing the genome of a eukaryotic cell at at least one target site in the eukaryotic cell comprising providing the eukaryotic cell with, or introducing into the eukaryotic cell, one or more ribonucleoprotein complexes according to any one of embodiments 1 to 55. [0225] Embodiment 219. A method for editing the genome of a eukaryotic cell at at least one target site in the eukaryotic cell comprising providing the eukaryotic cell with, or introducing into the eukaryotic cell, one or more ribonucleoprotein complexes according to any one of embodiments 56 to 87. [0226] Embodiment 220. A method for editing the genome of a eukaryotic cell at at least one target site in the eukaryotic cell comprising a. providing the eukaryotic cell with, or introducing into the cell, one or more recombinant DNA molecules according to any one of embodiments 88 to 173; b. providing the eukaryotic cell with, or introducing into the eukaryotic cell at least one guide RNA or a nucleic acid encoding at least one guide RNA comprising a complementarity region to the nucleotide sequence of the at least one target site of the eukaryotic cell . [0227] Embodiment 221. A method for editing the genome of a eukaryotic cell at at least one target site in the eukaryotic cell comprising a. providing the eukaryotic cell with, or introducing into the cell, one or more recombinant DNA molecules according to any one of embodiments 174 to 217; b. providing the eukaryotic cell with, or introducing into the eukaryotic cell at least one guide RNA or a nucleic acid encoding at least one guide RNA comprising a complementarity region to the nucleotide sequence of the at least one target site of the eukaryotic cell . [0228] Embodiment 222. The method according to any one of embodiments 218 to 221, wherein the guide RNA comprises two direct repeat sequences, flanking a spacer comprising a nucleotide sequence complementary to the target site. [0229] Embodiment 223. The method according to any one of embodiments 218 to 222, wherein the editing comprises a. inserting at least one nucleotide; b. deleting at least one nucleotide; or c. substituting at least one nucleotide. [0230] Embodiment 224. The method according to any one of embodiments 218 to 223, further comprising introducing or providing a donor template into the eukaryotic cell. [0231] Embodiment 225. The method according to any one of embodiments 218 to 224, wherein the eukaryotic cell is an in vitro cell. [0232] Embodiment 226. The method according to any one of embodiments 217 to 224, wherein the eukaryotic cell is an animal cell. [0233] Embodiment 227. The method according to embodiment 226 wherein the animal cell is a non-human cell. [0234] Embodiment 228. The method according to embodiment 226 or 227, which is not a method of treatment of the human or animal living body. [0235] Embodiment 229. The method according to any one of embodiments 218 to 225, wherein the eukaryotic cell is a fungal cell. [0236] Embodiment 230. The method according to any one of embodiments 218 to 225, wherein the eukaryotic cell is a plant cell. [0237] Embodiment 231. The method according to embodiment 230, wherein the plant cell is from a plant selected from a monocotyledonous species, a dicotyledonous species, an angiosperm species or a gymnosperm species. [0238] Embodiment 232. The method according to embodiment 230 or 231, wherein the plant cell is from a plant selected from a corn plant, a rice plant, a sorghum plant, a wheat plant, an alfalfa plant, a barley plant, a millet plant, a rye plant, a sugarcane plant, a cotton plant, a soybean plant, a canola plant, a tomato plant, an onion plant, a cucumber plant, an Arabidopsis plant, or a potato plant. [0239] Embodiment 233. The method according to any one of embodiments 230 to 232, further comprising the step of generating or regenerating a plant from the plant cell. [0240] Embodiment 234. A eukaryotic cell comprising one or more ribonucleoprotein complexes according to any one of embodiments 1 to 55. [0241] Embodiment 235. A eukaryotic cell comprising one or more ribonucleoprotein complexes according to any one of embodiments 56 to 87. [0242] Embodiment 236. A eukaryotic cell comprising one or more recombinant DNA molecules according to any one of 88 to 173. [0243] Embodiment 237. A eukaryotic cell comprising one or more recombinant DNA molecules according to any one of embodiment 174 to 217. [0244] Embodiment 238. The eukaryotic cell according to embodiment 236 or 237, further comprising at least one guide RNA or a nucleic acid encoding at least one guide RNA comprising a complementarity region to the nucleotide sequence of at least one target site within the eukaryotic cell. [0245] Embodiment 239. The eukaryotic cell according to any one of embodiments 234 to 238 , which is an in vitro cell. [0246] Embodiment 240. The eukaryotic cell according to any one of embodiments 234 to 239, which is an animal cell. [0247] Embodiment 241. The eukaryotic cell according to embodiment 240, which is a non-human animal cell. [0248] Embodiment 242. The eukaryotic cell according to any one of embodiments 234 to 239, which is a fungal cell. [0249] Embodiment 243. The eukaryotic cell according to any one of embodiments 234 to 239, which is a plant cell. [0250] Embodiment 244. The eukaryotic cell according to embodiment 243, wherein the plant cell is from a plant selected from a monocotyledonous species, a dicotyledonous species, an angiosperm species or a gymnosperm species. [0251] Embodiment 245. The eukaryotic cell according to embodiment 243 or 244, wherein the plant cell is from a plant selected from a corn plant, a rice plant, a sorghum plant, a wheat plant, an alfalfa plant, a barley plant, a millet plant, a rye plant, a sugarcane plant, a cotton plant, a soybean plant, a canola plant, a tomato plant, an onion plant, a cucumber plant, an Arabidopsis plant, or a potato plant. [0252] Embodiment 246. A plant comprising a cell or consisting essentially of cells according to any one of embodiments 243 to 245. [0253] Embodiment 247. A linker polypeptide comprising the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20. [0254] Embodiment 248. The linker polypeptide according to embodiment 247 comprising the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO:25. [0255] Embodiment 249. A fusion protein comprising two polypeptide domains linked by a linker polypeptide according to embodiment 247 or 248. [0256] Embodiment 250. A method for inserting a heterologous polypeptide sequence in a Cas12a protein comprising a. identifying an exposed loop in said Cas12a protein b. inserting a DNA sequence encoding said heterologous polypeptide into a DNA sequence encoding the exposed loop of said Cas12a protein to produce a modified Cas12a protein. [0257] Embodiment 251. The method according to embodiment 250, wherein the modified Cas12a protein retains DNA binding activity when combined with a guide RNA. [0258] Embodiment 252. The method according to embodiment 250 or 251, wherein the modified Cas12a protein retains nuclease activity when combined with a guide RNA. [0259] Embodiment 253. The method according to embodiment 250 or 251, wherein the modified Cas12a protein retains nickase activity when combined with a guide RNA. [0260] Embodiment 254. The method according to any one of embodiments 250 to 253, wherein the Cas12a protein has an amino acid sequence having at least 90% or at least 95% or at least 99% or 100% sequence identity to an amino acid selected from the group consisting of SEQ ID Nos: 33, 38 and 39. [0261] Embodiment 255. The method according to any one of embodiments 250 to 254, wherein said exposed loop corresponds to the amino acid sequence from position 449 to 461 or the amino acid sequence from position 487-496 or the amino acid sequence from position 370-379, or the amino acid sequence from position 1076-1085 of the amino acid sequence of SEQ ID NO: 33. [0262] Embodiment 256. The method according to any one of embodiments 250 to 255, wherein the heterologous polypeptide is inserted into the exposed loop. [0263] Embodiment 257. The method according to any one of embodiments 250 to 255, wherein the heterologous polypeptide replaces the exposed loop. [0264] Embodiment 258. The method according to any one of embodiments 250 to 257, wherein the heterologous polypeptide is a tethering motif, a tag, a nucleic acid binding motif, a DNA binding motif, an RNA binding motif, a protein binding motif. [0265] Embodiment 259. A modified Cas12a protein comprising a heterologous polypeptide in an exposed loop, obtainable by the methods of any one of embodiments 250 to 258. [0266] Embodiment 260. A modified Cas12a protein comprising a heterologous polypeptide at the amino acid sequence corresponding to the amino acid sequence from position 449 to 461 or the amino acid sequence from position 487-496 of the amino acid sequence of SEQ ID NO: 33. [0267] Embodiment 261. The modified Cas12a protein of embodiment 259 or 260, wherein the heterologous polypeptide is a tethering motif, a tag, a nucleic acid binding motif, a DNA binding motif, an RNA binding motif, a protein binding motif or a transposase. [0268] Embodiment 262. A heterologous polypeptide that facilitates uptake of a protein into the nucleus of a eukaryotic cell, such as a plant cell, comprising an amino acid sequence having at least 95% or at least 96% or at least 97% or at least 98% or at least 99 % or 100% sequence identity to an amino acid selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 90; SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 94. [0269] Embodiment 263. A heterologous polypeptide that facilitates uptake of a protein into the nucleus of a eukaryotic cell, such as a plant cell, comprising an amino acid sequence having at least 95% or at least 96% or at least 97% or at least 98% or at least 99 % or 100% sequence identity to an amino acid selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 101; SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104. [0270] Embodiment 264. A nucleic acid encoding the heterologous polypeptide of embodiment 262. [0271] Embodiment 265. The nucleic acid of embodiment 264 comprising a nucleotide sequence having at least 95% or at least 96% or at least 97% or at least 98% or at least 99 % or 100% sequence identity to a nucleic acid selected from the group consisting of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO: 88. [0272] Embodiment 266. A nucleic acid encoding the heterologous polypeptide of embodiment 263. [0273] Embodiment 267. The nucleic acid of embodiment 266 comprising a nucleotide sequence having at least 95% or at least 96% or at least 97% or at least 98% or at least 99 % or 100% sequence identity to a nucleic acid selected from the group consisting of SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99. [0274] Embodiment 268. A fusion protein comprising a polypeptide that facilitates uptake of a protein into the nucleus of a eukaryotic cell according to any one of embodiments 262 or 263, operably linked to a heterologous polypeptide of interest. [0275] Embodiment 269. A recombinant DNA molecule comprising: a. a promoter operably in a eukaryotic cell, such as a plant cell; b. a nucleic acid according to any one of embodiments 264 to 267; c. a nucleic acid encoding a heterologous polypeptide of interest; and d. optionally a transcription termination and/or polyadenylation signal. [0276] Embodiment 270. A method of facilitating uptake of a heterologous protein of interest into the nucleus of a eukaryotic cell, such as a plant cell, comprising providing to said eukaryotic cell a fusion protein according to embodiment 268 or a recombinant DNA molecule according to embodiment 269. [0277] Embodiment 271. A eukaryotic cell comprising a fusion protein according to embodiment 268 or comprising a recombinant DNA molecule according to embodiment 269. [0278] Embodiment 272. The eukaryotic cell according to embodiment 271, which is an in vitro cell. [0279] Embodiment 273. The eukaryotic cell according to any one of embodiments 271 or 272, which is an animal cell. [0280] Embodiment 274. The eukaryotic cell according to embodiment 273, which is a non-human animal cell. [0281] Embodiment 275. The eukaryotic cell according to embodiment 271 or 272, which is a fungal cell. [0282] Embodiment 276. The eukaryotic cell according to any one of embodiments 271 or 272, which is a plant cell. [0283] Embodiment 277. The eukaryotic cell according to embodiment 276, wherein the plant cell is from a plant selected from a monocotyledonous species, a dicotyledonous species, an angiosperm species or a gymnosperm species. [0284] Embodiment 278. The eukaryotic cell according to embodiment 276 or 277, wherein the plant cell is from a plant selected from a corn plant, a rice plant, a sorghum plant, a wheat plant, an alfalfa plant, a barley plant, a millet plant, a rye plant, a sugarcane plant, a cotton plant, a soybean plant, a canola plant, a tomato plant, an onion plant, a cucumber plant, an Arabidopsis plant, or a potato plant. [0285] Embodiment 279. A plant comprising a cell or consisting essentially of cells according to any one of embodiments 276 to 278.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING [0286] SEQ ID NO: 1: nucleotide sequence of the nuclear localization signal of HSFA protein from Lycoperiscon esculentum. [0287] SEQ ID NO: 2: codon optimized nucleotide sequence variant of the nuclear localization signal of HSFA protein from Lycoperiscon esculentum. [0288] SEQ ID NO: 3: nucleotide sequence of the synthetic nuclear localization signal NLS1. [0289] SEQ ID NO: 4: nucleotide sequence of the synthetic nuclear localization signal NLSC. [0290] SEQ ID NO: 5: nucleotide sequence of the short linker L1 (GGSG). [0291] SEQ ID NO: 6: nucleotide sequence of the short linker EAAAK. [0292] SEQ ID NO: 7: nucleotide sequence of the short linker YETKQ. [0293] SEQ ID NO: 8: nucleotide sequence of the short linker PVTAT. [0294] SEQ ID NO: 9: nucleotide sequence of the extended linker L2. [0295] SEQ ID NO: 10: nucleotide sequence of the extended linker L3. [0296] SEQ ID NO: 11: nucleotide sequence of the extended linker L4. [0297] SEQ ID NO: 12: nucleotide sequence of the extended linker L5. [0298] SEQ ID NO: 13: nucleotide sequence of the extended linker L6. [0299] SEQ ID NO: 14: amino acid sequence of the nuclear localization signal of HSFA protein from Lycoperiscon esculentum. [0300] SEQ ID NO: 15: amino acid sequence of the nuclear localization signal of NLS-C. [0301] SEQ ID NO: 16: amino acid sequence of the synthetic nuclear localization signal NLS1. [0302] SEQ ID NO: 17: amino acid sequence of the short linker L1 (GGSG). [0303] SEQ ID NO: 18: amino acid sequence of the short linker EAAAK. [0304] SEQ ID NO: 19: amino acid sequence of the short linker YETKQ. [0305] SEQ ID NO: 20: amino acid sequence of the short linker PVTAT. [0306] SEQ ID NO: 21: amino acid sequence of the extended linker L2. [0307] SEQ ID NO: 22: amino acid sequence of the extended linker L3. [0308] SEQ ID NO: 23: amino acid sequence of the extended linker L4. [0309] SEQ ID NO: 24: amino acid sequence of the extended linker L5. [0310] SEQ ID NO: 25: amino acid sequence of the extended linker L6. [0311] SEQ ID NO: 26: Lachnospiraceae bacterium Cas12a protein encoding nucleotide sequence. [0312] SEQ ID NO: 27: nucleotide sequence encoding LbCas12a_E1, comprising an embedded nuclear localization sequence at amino acid positions corresponding to position 449-461 of LbCas12a. [0313] SEQ ID NO: 28: nucleotide sequence encoding LbCas12a_E2, comprising an embedded nuclear localization sequence at amino acid positions corresponding to position 487-496 of LbCas12a. [0314] SEQ ID NO: 29: nucleotide sequence encoding LbCas12a_E1-E2, comprising an embedded nuclear localization sequence at amino acid positions corresponding to position 449-461 of LbCas12a and an embedded nuclear localization sequence at amino acid positions corresponding to position 487-496 of LbCas12a. [0315] SEQ ID NO: 30: nucleotide sequence encoding LbCas12a_E3, comprising an embedded nuclear localization sequence of HSFA protein from Lycoperiscon esculentum at amino acid positions corresponding to position 487- 496 of LbCas12a. [0316] SEQ ID NO: 31: nucleotide sequence encoding Cas12a protein from Franscisella novicida. [0317] SEQ ID NO: 32: nucleotide sequence encoding Cas12a protein from Acidaminococcus sp. [0318] SEQ ID NO: 33: amino acid sequence of Lachnospiraceae bacterium Cas12a protein. [0319] SEQ ID NO: 34: amino acid sequence of LbCas12a_E1, comprising an embedded nuclear localization sequence at amino acid positions corresponding to position 449-461 of LbCas12a. [0320] SEQ ID NO: 35: amino acid sequence of LbCas12a_E2, comprising an embedded nuclear localization sequence at amino acid positions corresponding to position 487-496 of LbCas12a. [0321] SEQ ID NO: 36: amino acid sequence of LbCas12a_E1-E2, comprising an embedded nuclear localization sequence at amino acid positions corresponding to position 449-461 of LbCas12a and an embedded nuclear localization sequence at amino acid positions corresponding to position 487-496 of LbCas12a. [0322] SEQ ID NO: 37: amino acid sequence of LbCas12a_E3 protein comprising an embedded nuclear localization sequence of HSFA protein from Lycoperiscon esculentum at amino acid positions corresponding to position 487- 496 of LbCas12a. [0323] SEQ ID NO: 38: amino acid sequence of a Cas12a protein from Franscisella novicida. [0324] SEQ ID NO: 39: amino acid sequence of a Cas12a protein from Acidaminococcus sp. [0325] SEQ ID NO: 40: nucleotide sequence of a Lachnospiraceae bacterium ND2006 direct repeat included in the guide RNAs of the examples. [0326] SEQ ID NO: 41: nucleotide sequence of a spacer SP1 targeting Zea mays genomic sequence ZmTS1. [0327] SEQ ID NO: 42: nucleotide sequence of a spacer SP2 targeting Zea mays genomic sequence ZmTS2 . [0328] SEQ ID NO: 43: nucleotide sequence of a spacer SP3 targeting Zea mays genomic sequence ZmTS3 . [0329] SEQ ID NO: 44: intentionally skipped sequence [0330] SEQ ID NO: 45: nucleotide sequence of a spacer SP6 targeting Zea mays genomic sequence ZmTS6. [0331] SEQ ID NO: 46: nucleotide sequence of a spacer SP4 targeting Zea mays genomic sequence ZmTS4. [0332] SEQ ID NO: 47: nucleotide sequence of a spacer SP5 targeting Zea mays genomic sequence ZmTS5. [0333] SEQ ID NO: 48: nucleotide sequence of the region of LbCas12a encoding the amino acid sequence from amino acid position 449 to amino acid position 461 of LbCas12a protein. [0334] SEQ ID NO: 49: nucleotide sequence of the region of LbCas12a encoding the amino acid sequence from amino acid position 487 to amino acid position 496 of LbCas12a protein. [0335] SEQ ID NO: 50: nucleotide sequence encoding the heterologous polypeptide replacing the amino acid sequence from amino acid position 449 to amino acid position 461of LbCas12a protein in LbCas12a_E1. [0336] SEQ ID NO: 51: nucleotide sequence encoding the heterologous polypeptide replacing the amino acid sequence from amino acid position 487 to amino acid position 496 of LbCas12a protein in LbCas12a_E2. [0337] SEQ ID NO: 52: nucleotide sequence encoding the heterologous polypeptide for the nuclear localization sequence of HSFA protein from Lycoperiscon esculentum replacing the amino acid sequence from amino acid position 487 to amino acid position 496 of LbCas12a protein in LbCas12a_E3. [0338] SEQ ID NO: 53: amino acid sequence of the region of LbCas12a from amino acid position 449 to amino acid position 461. [0339] SEQ ID NO: 54: amino acid sequence of the region of LbCas12a from amino acid position 487 to amino acid position 496. [0340] SEQ ID NO: 55: amino acid sequence of nuclear localization signal embedded in LbCas12a_E1. [0341] SEQ ID NO: 56: amino acid sequence of nuclear localization signal embedded in LbCas12a_E2. [0342] SEQ ID NO: 57: nucleotide sequence of enhanced CaMV35S promoter. [0343] SEQ ID NO: 58: nucleotide sequence of Zea mays genomic target sequence ZmTS1. [0344] SEQ ID NO: 59: nucleotide sequence of Zea mays genomic target sequence ZmTS2 . [0345] SEQ ID NO: 60: nucleotide sequence of Zea mays genomic target sequence ZmTS3 . [0346] SEQ ID NO: 61: nucleotide sequence of GSP2273, a synthetic POL III promoter. [0347] SEQ ID NO: 62: nucleotide sequence encoding enhanced Yellow Fluorescence protein. [0348] SEQ ID NO: 63: amino acid sequence of enhanced Yellow Fluorescence protein. [0349] SEQ ID NO: 64: nucleotide sequence of a termination and polyadenylation signal of nopaline synthase gene. [0350] SEQ ID NO: 65: nucleotide sequence of LbCas12a_E4 with embedded nuclear localization signal replacing the amino acid sequence corresponding to the LbCas12 region from amino acid position 1076 to 1085. [0351] SEQ ID NO: 66: nucleotide sequence of LbCas12a_E5 with embedded nuclear localization signal replacing the amino acid sequence corresponding to the LbCas12 region from amino acid position 370 to 379. [0352] SEQ ID NO: 67: amino acid sequence of LbCas12a_E4 with embedded nuclear localization signal replacing the amino acid sequence corresponding to the LbCas12 region from amino acid position 1076 to 1085. [0353] SEQ ID NO: 68: amino acid sequence of LbCas12a_E5 with embedded nuclear localization signal replacing the amino acid sequence corresponding to the LbCas12 region from amino acid position 370 to 390. [0354] SEQ ID NO: 69: nucleotide sequence encoding the amino acid sequence corresponding to the LbCas12a region from amino acid position 1076 to 1085. [0355] SEQ ID NO: 70: nucleotide sequence encoding the amino acid sequence corresponding to the LbCas12a region from amino acid position 370 to 379. [0356] SEQ ID NO: 71: nucleotide sequence encoding the embedded nuclear localization signal in LbCas12a_E4 replacing the region corresponding to the LbCas12a region from amino acid position 1076 to 1085. [0357] SEQ ID NO: 72: nucleotide sequence encoding the embedded nuclear localization signal in LbCas12a_E5 replacing the region corresponding to the LbCas12a region from amino acid position 370 to 379. [0358] SEQ ID NO: 73: amino acid sequence corresponding to the LbCas12a region from amino acid position 1076 to 1085. [0359] SEQ ID NO: 74: amino acid sequence corresponding to the LbCas12a region from amino acid position 370 to 379. [0360] SEQ ID NO: 75: amino acid sequence of the region of LbCas12a_E4 replacing the region corresponding to the LbCas12 region from amino acid position 1076 to 1085. [0361] SEQ ID NO: 76: amino acid sequence of the region of LbCas12a_E5 replacing the region corresponding to the LbCas12 region from amino acid position 370 to 379. [0362] SEQ ID NO: 77: nucleotide sequence of Zea mays Ubiquitin promoter P-ZmUbqM1. [0363] SEQ ID NO: 78: transcription termination and polyadenylation signal form Oryza sativa Lipid transfer protein (LPT) gene. [0364] SEQ ID NO: 79: nucleotide sequence of Zea mays genomic target sequence ZmTS4. [0365] SEQ ID NO: 80: nucleotide sequence of Zea mays genomic target sequence ZmTS5. [0366] SEQ ID NO: 81: nucleotide sequence of Zea mays genomic target sequence ZmTS6. [0367] SEQ ID NO: 82: nucleotide sequence of GSP2262, a synthetic POL III promoter. [0368] SEQ ID NO: 83: nucleotide sequence of the nuclear localization signal NLS2. [0369] SEQ ID NO: 84: nucleotide sequence of the nuclear localization signal NLS3. [0370] SEQ ID NO: 85: nucleotide sequence of the nuclear localization signal NLS4. [0371] SEQ ID NO: 86: nucleotide sequence of the nuclear localization signal NLS5. [0372] SEQ ID NO: 87: nucleotide sequence of the nuclear localization signal NLS6(10). [0373] SEQ ID NO: 88: nucleotide sequence of the nuclear localization signal NLS6(11). [0374] SEQ ID NO: 89: amino acid sequence of the synthetic nuclear localization signal NLS2. [0375] SEQ ID NO: 90: amino acid sequence of the synthetic nuclear localization signal NLS3. [0376] SEQ ID NO: 91: amino acid sequence of the synthetic nuclear localization signal NLS4. [0377] SEQ ID NO: 92: amino acid sequence of the synthetic nuclear localization signal NLS5. [0378] SEQ ID NO: 93: amino acid sequence of the synthetic nuclear localization signal NLS6(10). [0379] SEQ ID NO: 94: amino acid sequence of the synthetic nuclear localization signal NLS6(11). [0380] SEQ ID NO: 95: nucleotide sequence of the Zea mays non-classical nuclear localization signal ncNLS1. [0381] SEQ ID NO: 96: nucleotide sequence of the Zea mays non-classical nuclear localization signal ncNLS2. [0382] SEQ ID NO: 97: nucleotide sequence of the Zea mays non-classical nuclear localization signal ncNLS3. [0383] SEQ ID NO: 98: nucleotide sequence of the Zea mays non-classical nuclear localization signal ncNLS4. [0384] SEQ ID NO: 99: nucleotide sequence of the Zea mays non-classical nuclear localization signal ncNLS5 [0385] SEQ ID NO: 100: amino acid sequence of the Zea mays non-classical nuclear localization signal ncNLS1. [0386] SEQ ID NO: 101: amino acid sequence of the Zea mays non-classical nuclear localization signal ncNLS2. [0387] SEQ ID NO: 102: amino acid sequence of the Zea mays non-classical nuclear localization signal ncNLS3. [0388] SEQ ID NO: 103: amino acid sequence of the Zea mays non-classical nuclear localization signal ncNLS4. [0389] SEQ ID NO: 104: amino acid sequence of the Zea mays non-classical nuclear localization signal ncNLS5. [0390] SEQ ID NO: 105: nucleotide sequence encoding an optimized E.coli beta-glucuronidase gene. [0391] SEQ ID NO: 106: amino acid sequence of an optimized E.coli beta-glucuronidase (GUS) gene. BRIEF DESCRIPTION OF THE DRAWINGS [0393] Figure 1A: Graphic representation of the average nuclear YFP intensity per transfected protoplast using various designs of linkers coupled to nuclear localization signals at the N-terminus or C-terminus of LbCas12a-YFP fusion protein or embedded nuclear localization signals within LbCas12a fused to YFP, described in Example 2. [0394] Figure 1B: Graphic representation of the average number of protoplasts with positive YFP intensity in the nucleus per transfection experiment using various designs of linkers coupled to nuclear localization signals at the N-terminus or C-terminus of LbCas12a-YFP fusion protein or embedded nuclear localization signals within LbCas12a fused to YFP, described in Example 2. [0395] Figure 2. Graphic representation of editing rates (% Ind/Del formation) and the ZmTS1 target site in protoplasts after transfection experiment using two types of guide RNAs and various designs of linkers coupled to nuclear localization signals at the N-terminus or C-terminus of LbCas12a effector protein or embedded nuclear localization signals within LbCas12a effector protein, described in Example 3. The gray boxes represent results for co-transfection with guide RNA of the Direct Repeat-Spacer type, while the black boxes represent results for co-transfection with guide RNA of the Direct Repeat-Spacer-Direct Repeat type. [0396] Figure 3. Graphic representation of editing rates (% Ind/Del formation) and the ZmTS2 target site in protoplasts after transfection experiment using two types of guide RNAs and various designs of linkers coupled to nuclear localization signals at the N-terminus or C-terminus of LbCas12a effector protein or embedded nuclear localization signals within LbCas12a effector protein, described in Example 3. The gray boxes represent results for co-transfection with guide RNA of the Direct Repeat-Spacer type, while the black boxes represent results for co-transfection with guide RNA of the Direct Repeat-Spacer-Direct Repeat type. [0397] Figure 4. Graphic representation of editing rates (% Ind/Del formation) and the ZmTS3 target site in protoplasts after transfection experiment using two types of guide RNAs and various designs of linkers coupled to nuclear localization signals at the N-terminus or C-terminus of LbCas12a effector protein or embedded nuclear localization signals within LbCas12a effector protein, described in Example 3. The gray boxes represent results for co-transfection with guide RNA of the Direct Repeat-Spacer type, while the black boxes represent results for co-transfection with guide RNA of the Direct Repeat-Spacer-Direct Repeat type. [0398] Figure 5A: Graphic representation of the average nuclear YFP intensity per transfected protoplast using various classical nuclear localization signals at the C-terminus of YFP-LbCas12a fusion protein described in Example 5. [0399] Figure 5B: Graphic representation of the average number of protoplasts with positive YFP intensity in the nucleus per transfection experiment using various classical nuclear localization signals at the C-terminus of YFP-LbCas12a fusion protein described in Example 5. [0400] Figure 6: Graphic representation of the percentage of cells with fluorescent nuclei in protoplasts transfected with constructs encoding various Zea mays non-classic nuclear localization signals (ncNLS) at the C-terminus of YFP-LbCas12a fusion protein described in Example 7. Four replicate transformations were carried out for each construct. DETAILED DESCRIPTION OF THE INVENTION [0401] Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Where a term is provided in the singular, the inventors also contemplate aspects of the disclosure described by the plural of that term. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various art-specific dictionaries, for example, “The American Heritage® Science Dictionary” (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), the “McGraw-Hill Dictionary of Scientific and Technical Terms” (6th edition, 2002, McGraw-Hill, New York), or the “Oxford Dictionary of Biology” (6th edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to be limited to a mechanism or mode of action. Reference thereto is provided for illustrative purposes only. [0402] The practice of this disclosure includes, unless otherwise indicated, conventional techniques of biochemistry, chemistry, molecular biology, microbiology, cell biology, plant biology, genomics, biotechnology, and genetics, which are within the skill of the art. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition (2012); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); Plant Breeding Methodology (N.F. Jensen, Wiley-Interscience (1988)); the series Methods In Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; Animal Cell Culture (R. I. Freshney, ed. (1987)); Recombinant Protein Purification: Principles And Methods, 18-1142-75, GE Healthcare Life Sciences; C. N. Stewart, A. Touraev, V. Citovsky, T. Tzfira eds. (2011) Plant Transformation Technologies (Wiley-Blackwell); and R. H. Smith (2013) Plant Tissue Culture: Techniques and Experiments (Academic Press, Inc.). [0403] Any references cited herein, including, e.g., all patents, published patent applications, and non-patent publications, are incorporated herein by reference in their entirety. [0404] When a grouping of alternatives is presented, any and all combinations of the members that make up that grouping of alternatives is specifically envisioned. For example, if an item is selected from a group consisting of A, B, C, and D, the inventors specifically envision each alternative individually (e.g., A alone, B alone, etc.), as well as combinations such as A, B, and D; A and C; B and C; etc. [0405] As used herein, terms in the singular and the singular forms “a,” “an,” and “the,” for example, include plural referents unless the content clearly dictates otherwise. [0406] Any composition, nucleic acid molecule, polypeptide, cell, plant, etc. provided herein is specifically envisioned for use with any method provided herein. [0407] The current invention relates to methods for targeted gene modification in eukaryotic cells, using guide RNAs and RNA guided polypeptides comprising an effector protein which is or is derived from a Crispr/Cas protein and one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus, such as the C-terminus only of the effector polypeptide, or which are embedded within an exposed loop of the Crispr/Cas protein. Further provided are means for such methods including ribonucleoprotein complexes or recombinant DNA molecules encoding such ribonucleoprotein complexes as well as eukaryotic cells, including plant cells, or plants, comprising such means. Also provided are methods for producing modified Cas12a proteins comprising a heterologous peptide embedded within the Cas12a protein, without compromising its nuclease activity and/or DNA binding activity and modified Cas12a proteins resulting from such methods, all as described in the above mentioned embodiments and elsewhere in this document. Nucleic acids and amino acids [0408] The use of the term “polynucleotide” or “nucleic acid molecule” is not intended to limit the present disclosure to polynucleotides comprising deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also envisioned. Those of ordinary skill in the art will recognize that polynucleotides and nucleic acid molecules can comprise deoxyribonucleotides, ribonucleotides, or combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the present disclosure also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. In an aspect, a nucleic acid molecule provided herein is a DNA molecule. In another aspect, a nucleic acid molecule provided herein is an RNA molecule. In an aspect, a nucleic acid molecule provided herein is single-stranded. In another aspect, a nucleic acid molecule provided herein is double-stranded. [0409] As used herein, the term “recombinant construct” in reference to a nucleic acid (DNA or RNA) molecule, protein, construct, vector, etc., refers to a nucleic acid or amino acid molecule or sequence that is man-made and not normally found in nature, and/or is present in a context in which it is not normally found in nature, including a nucleic acid molecule (DNA or RNA) molecule, protein, construct, etc., comprising a combination of polynucleotide or protein sequences that would not naturally occur contiguously or in close proximity together without human intervention, and/or a polynucleotide molecule, protein, construct, etc., comprising at least two polynucleotide or protein sequences that are heterologous with respect to each other. [0410] As used herein, the term “recombinant cassette” refers to a nucleic acid comprising a promoter sequence, a sequence of interest, and optionally a terminator sequence. The term “recombinant construct” may encompass a “recombinant cassette”. A recombinant construct may also be used interchangeable with a recombinant cassette. [0411] As used herein, the term "heterologous" refers to a nucleotide/polypeptide that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. A "heterologous" or a "recombinant" nucleotide sequence is a nucleotide sequence not naturally associated with a host cell into which it is introduced, including non- naturally occurring multiple copies of a naturally occurring nucleotide sequence. The term “heterologous” can also be used to refer to elements not normally associated with each other. [0412] In one aspect, methods and compositions provided herein comprise a vector or a construct. As used herein, the term “vector” or “construct” refers to a DNA molecule used as a vehicle to carry exogenous genetic material into a cell. [0413] In an aspect, one or more polynucleotide sequences from a vector are stably integrated into a genome of a plant. In an aspect, one or more polynucleotide sequences from a vector are stably integrated into a genome of a plant cell. [0414] In an aspect, a first nucleic acid sequence and a second nucleic acid sequence are provided in a single vector. In another aspect, a first nucleic acid sequence is provided in a first vector, and a second nucleic acid sequence is provided in a second vector. [0415] As used herein, the term “polypeptide” refers to a chain of at least two covalently linked amino acids. Polypeptides can be encoded by polynucleotides provided herein. An example of a polypeptide is a protein. Proteins provided herein can be encoded by nucleic acid molecules provided herein. [0416] Nucleic acids can be isolated using techniques routine in the art. For example, nucleic acids can be isolated using any method including, without limitation, recombinant nucleic acid technology, and/or the polymerase chain reaction (PCR). General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides. Polypeptides can be purified from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. A polypeptide also can be purified, for example, by expressing a nucleic acid in an expression vector. In addition, a purified polypeptide can be obtained by chemical synthesis. The extent of purity of a polypeptide can be measured using any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. [0417] Without being limiting, nucleic acids can be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et. al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). [0418] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. An antibody provided herein can be a polyclonal antibody or a monoclonal antibody. An antibody having specific binding affinity for a polypeptide provided herein can be generated using methods well known in the art. An antibody provided herein can be attached to a solid support such as a microtiter plate using methods known in the art. [0419] The terms “percent identity” or “percent identical” as used herein in reference to two or more nucleotide or protein sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or protein) over a window of comparison, (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100% to yield the percent identity. If the “percent identity” is being calculated in relation to a reference sequence without a particular comparison window being specified, then the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Accordingly, for purposes of the present application, when two sequences (query and subject) are optimally aligned (with allowance for gaps in their alignment), the “percent identity” for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or a comparison window), which is then multiplied by 100%. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” [0420] The terms “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 of a query sequence that optimally base-pair or hybridize to nucleotides a subject sequence when the query and subject sequences are linearly arranged and optimally base paired without secondary folding structures, such as loops, stems or hairpins. Such a percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and a RNA strand. The “percent complementarity” can be calculated by (i) optimally base-pairing or hybridizing the two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structures) over a window of comparison, (ii) determining the number of positions that base-pair between the two sequences over the window of comparison to yield the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the window of comparison, and (iv) multiplying this quotient by 100% to yield the percent complementarity of the two sequences. Optimal base pairing of two sequences can be determined based on the known pairings of nucleotide bases, such as G- C, A-T, and A-U, through hydrogen binding. If the “percent complementarity” is being calculated in relation to a reference sequence without specifying a particular comparison window, then the percent identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Thus, for purposes of the present application, when two sequences (query and subject) are optimally base-paired (with allowance for mismatches or non-base-paired nucleotides), the “percent complementarity” for the query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions in the query sequence over its length, which is then multiplied by 100%. [0421] For optimal alignment of sequences to calculate their percent identity, various pair-wise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool (BLAST®), etc., that can be used to compare the sequence identity or similarity between two or more nucleotide or protein sequences. Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the percent identity ranges described above) can be as determined by the ClustalW algorithm, see, e.g., Chenna R. et. al., “Multiple sequence alignment with the Clustal series of programs,” Nucleic Acids Research 31: 3497-3500 (2003); Thompson JD et. al., “Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research 22: 4673-4680 (1994); Larkin MA et. al., “Clustal W and Clustal X version 2.0,” Bioinformatics 23: 2947-48 (2007); and Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410 (1990), the entire contents and disclosures of which are incorporated herein by reference. [0422] As used herein, a first nucleic acid molecule can “hybridize” a second nucleic acid molecule via non-covalent interactions (e.g., Watson-Crick base-pairing) in a sequence-specific, antiparallel manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base-pairing includes: adenine (A) pairing with thymine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). In addition, it is also known in the art that for hybridization between two RNA molecules (e.g., dsRNA), guanine base pairs with uracil. For example, G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. In the context of this disclosure, a guanine of a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to an uracil, and vice versa. As such, when a G/U base-pair can be made at a given nucleotide position a protein- binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary. [0423] Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. [0424] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of complementation between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g. complementarity over 35 or fewer nucleotides) the position of mismatches becomes important (see Sambrook et. al.). Typically, the length for a hybridizable nucleic acid is at least 10 nucleotides. Illustrative minimum lengths for a hybridizable nucleic acid 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, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation. [0425] It is understood in the art that the sequence of polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined routinely using BLAST® programs (basic local alignment search tools) and PowerBLAST programs known in the art (see Altschul et. al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Promoters [0426] As used herein, a "promoter" is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (e.g., a coding sequence) that is operably associated with the promoter. The coding sequence controlled or regulated by a promoter may encode a polypeptide and/or a functional RNA. A "promoter" may refer to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription. In general, promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence. A promoter may comprise other elements that act as regulators of gene expression; e.g., a promoter region. These include a TATA box consensus sequence, and often a CAAT box consensus sequence (Breathnach and Chambon (1981) Annu. Rev. Biochem.50:349). In plants, the CAAT box may be substituted by the AGGA box (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211- 227). [0427] Promoters useful with this invention can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules, e.g., "synthetic nucleic acid constructs" or "protein-RNA complex." These various types of promoters are known in the art. [0428] The choice of promoter may vary depending on the temporal and spatial requirements for expression, and also may vary based on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge present in the art, the appropriate promoter can be selected for the particular host organism of interest. Thus, for example, much is known about promoters upstream of highly constitutively expressed genes in model organisms and such knowledge can be readily accessed and implemented in other systems as appropriate. [0429] In some embodiments, a promoter functional in a plant may be used with the constructs of this invention. Non-limiting examples of a promoter useful for driving expression in a plant include the promoter of the RubisCo small subunit gene 1 (PrbcS1), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr) and the promoter of duplicated carbonic anhydrase gene 1 (Pdca1) (See, Walker et al. (2005) Plant Cell Rep. 23:727-735; Li et al. (2007) Gene 403:132-142; Li et al. (2010) Mol Biol. Rep. 37:1143-1154). PrbcS1 and Pactin are constitutive promoters and Pnr and Pdca1 are inducible promoters. Pnr is induced by nitrate and repressed by ammonium (Li et al. (2007) Gene 403:132-142) and Pdca1 is induced by salt (Li et al. (2010) Mol Biol. Rep. 37:1143-1154). In some embodiments, a promoter useful with this invention is RNA polymerase II (Pol II) promoter. [0430] Examples of constitutive promoters useful for plants include, but are not limited to, cestrum virus promoter (cmp) (US Patent No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; as well as US Patent No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci USA 84:5745-5749), Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84:6624-6629), sucrose synthase promoter (Yang & Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-4148), and the ubiquitin promoter. The constitutive promoter derived from ubiquitin accumulates in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, for example, sunflower (Binet et al. (1991) Plant Science 79: 87-94), maize (Christensen et al. (1989) Plant Molec. Biol. 12: 619-632), and Arabidopsis (Norris et al. (1993) Plant Molec. Biol. 21:895- 906). The maize ubiquitin promoter (UbiP) has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0342926. The ubiquitin promoter is suitable for the expression of the nucleotide sequences of the invention in transgenic plants, especially monocotyledons. Further, the promoter expression cassettes described by McElroy et al. ((1991) Mol. Gen. Genet. 231: 150-160) can be easily modified for the expression of the nucleotide sequences of the invention and are particularly suitable for use in monocotyledonous hosts. [0431] In some embodiments, tissue specific/tissue preferred promoters can be used for expression of a heterologous polynucleotide in a plant cell. Tissue specific or preferred expression patterns include, but are not limited to, green tissue specific or preferred, root specific or preferred, stem specific or preferred, flower specific or preferred or pollen specific or preferred. Promoters suitable for expression in green tissue include many that regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons. In one embodiment, a promoter useful with the invention is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula (1989) Plant Molec. Biol. 12:579-589). Non-limiting examples of tissue-specific promoters include those associated with genes encoding the seed storage proteins (such as β-conglycinin, cruciferin, napin and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4, see, e.g., Kridl et al. (1991) Seed Sci. Res.1:209-219; as well as EP Patent No. 255378). Tissue-specific or tissue-preferential promoters useful for the expression of the nucleotide sequences of the invention in plants, particularly maize, include but are not limited to those that direct expression in root, pith, leaf, or pollen. Such promoters are disclosed, for example, in WO 93/07278, herein incorporated by reference in its entirety. Other non-limiting examples of tissue specific or tissue preferred promoters useful with the invention the cotton rubisco promoter disclosed in US Patent No. 6,040,504; the rice sucrose synthase promoter disclosed in US Patent No. 5,604,121; the root specific promoter described by de Framond ((1991) FEBS 290:103-106; EP 0452269 to Ciba- Geigy); the stem specific promoter described in US Patent No. 5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpA gene; the cestrum yellow leaf curling virus promoter disclosed in WO 01/73087; and pollen specific or preferred promoters including, but not limited to, ProOsLPS10 and ProOsLPS11 from rice (Nguyen et al. (2015) Plant Biotechnol. Reports 9(5):297-306), ZmSTK2_USP from maize (Wang et al. (2017) Genome 60(6):485-495), LAT52 and LAT59 from tomato (Twell et al. (1990) Development 109(3):705-713), Zm13 (US Patent No. 10,421,972), PLA2-δ promoter from Arabidopsis (US Patent No.7,141,424), and/or the ZmC5 promoter from maize (International PCT Publication No. WO1999/042587. [0432] Additional examples of plant tissue-specific/tissue preferred promoters include, but are not limited to, the root hair–specific cis-elements (RHEs) (Kim et al. (2006) The Plant Cell 18:2958- 2970), the root-specific promoters RCc3 (Jeong et al. (2010) Plant Physiol.153:185-197) and RB7 (US Patent No. 5459252), the lectin promoter (Lindstrom et al. (1990) Der. Genet. 11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), corn alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res. 12:3983-4000), S-adenosyl-L-methionine synthetase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology 37(8):1108-1115), corn light harvesting complex promoter (Bansal et al. (1992) Proc. Natl. Acad. Sci. USA 89:3654- 3658), corn heat shock protein promoter (O'Dell et al. (1985) EMBO J. 5:451-458; and Rochester et al. (1986) EMBO J. 5:451-458), pea small subunit RuBP carboxylase promoter (Cashmore, "Nuclear genes encoding the small subunit of ribulose-l,5-bisphosphate carboxylase" pp.29-39 In: Genetic Engineering of Plants (Hollaender ed., Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet.205:193-200), Ti plasmid mannopine synthase promoter (Langridge et al. (1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), Ti plasmid nopaline synthase promoter (Langridge et al. (1989) supra), petunia chalcone isomerase promoter (van Tunen et al. (1988) EMBO J. 7:1257- 1263), bean glycine rich protein 1 promoter (Keller et al. (1989) Genes Dev. 3:1639-1646), truncated CaMV 35S promoter (O'Dell et al. (1985) Nature 313:810-812), potato patatin promoter (Wenzler et al. (1989) Plant Mol. Biol. 13:347-354), root cell promoter (Yamamoto et al. (1990) Nucleic Acids Res. 18:7449), maize zein promoter (Kriz et al. (1987) Mol. Gen. Genet. 207:90- 98; Langridge et al. (1983) Cell 34:1015-1022; Reina et al. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic Acids Res. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354), globulin-1 promoter (Belanger et al. (1991) Genetics 129:863-872), α-tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet.215:431-440), PEPCase promoter (Hudspeth & Grula (1989) Plant Mol. Biol.12:579-589), R gene complex-associated promoters (Chandler et al. (1989) Plant Cell 1:1175-1183), and chalcone synthase promoters (Franken et al. (1991) EMBO J. 10:2605-2612). [0433] Useful for seed-specific expression is the pea vicilin promoter (Czako et al. (1992) Mol. Gen. Genet. 235:33-40; as well as the seed-specific promoters disclosed in US Patent No. 5,625,136. Useful promoters for expression in mature leaves are those that are switched at the onset of senescence, such as the SAG promoter from Arabidopsis (Gan et al. (1995) Science 270:1986-1988). [0434] Plant-expressible promoters useful for the methods and compositions herein described also include egg cell-preferred or embryo-tissue preferred promoters as described in WO2022/056139 (incorporated herein in its entirety), such as a DSUL1 promoter, an EA1 promoter, an ES4 promoter, a DMC1 promoter, a Mps1 promoter, an Adf1 promoter or an EAL promoter. [0435] Other plant-expressible promoters useful for the invention include floral-tissue preferred or floral cell-preferred promoter as described in PCT/US2023/065042 (incorporated herein in its entirety). [0436] In addition, promoters functional in chloroplasts can be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 95' UTR and other promoters disclosed in US Patent No.7,579,516. Other promoters useful with the invention include but are not limited to the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3). [0437] In some embodiments, promoters may be selected from RNA polymerase III (Pol III) promoters. In some aspects, the POL III promoter may be a U6 promoter, an H1 promoter, a 5S promoter, an Adenovirus 2 (Ad2) VAI promoter, a tRNA promoter, and a 7SK promoter. See, for example, Schramm and Hernandez, 2002, Genes & Development, 16:2593-2620, which is incorporated by reference herein in its entirety. [0438] In some aspects, the POL III promoters may be derived from small nuclear RNA (snRNA) encoding genes. In some aspects, the POL III promoters may be selected from the corn, tomato and soybean U6, U3, U2, U5 and 7SL snRNA promoters disclosed in WO2015/131101 (incorporated herein by reference in its entirety) including the snRNA promoter sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20; SEQ ID NOs: 146-149, SEQ ID NOs: 160-166, SEQ ID NOs: 201 or SEQ ID NO: 283, included therein in the accompanying sequence listing. [0439] In some aspects, the POL III promoters may be synthetic snRNA promoters, such as the snRNA promoters described in WO2022/232407 (incorporated herein by reference in its entirety) including the snRNA promoter sequences of SEQ ID Nos: 1-10 included therein in the accompanying sequence listing. [0440] In some aspects, the POL III promoters may be chimeric POL III promoters. In some aspects, the POL III promoters may be variants of the POL III promoters. Regulatory elements [0441] Additional regulatory elements useful with this invention include, but are not limited to, introns, enhancers, termination sequences and/or 5' and 3' untranslated regions. [0442] An intron useful with this invention can be an intron identified in and isolated from a plant and then inserted into an expression cassette to be used in transformation of a plant. As would be understood by those of skill in the art, introns can comprise the sequences required for self-excision and are incorporated into nucleic acid constructs/expression cassettes in frame. An intron can be used either as a spacer to separate multiple protein-coding sequences in one nucleic acid construct, or an intron can be used inside one protein-coding sequence to, for example, stabilize the mRNA. If they are used within a protein-coding sequence, they are inserted "in-frame" with the excision sites included. Introns may also be associated with promoters to improve or modify expression. As an example, a promoter/intron combination useful with this invention includes but is not limited to that of the maize Ubi1 promoter and intron. [0443] Non-limiting examples of introns useful with the present invention include introns from the ADHI gene (e.g., Adh1-S introns 1, 2 and 6), the ubiquitin gene (Ubi1), the RuBisCO small subunit (rbcS) gene, the RuBisCO large subunit (rbcL) gene, the actin gene (e.g., actin-1 intron), the pyruvate dehydrogenase kinase gene (pdk), the nitrate reductase gene (nr), the duplicated carbonic anhydrase gene 1 (Tdca1), the psbA gene, the atpA gene, or any combination thereof. Guide Nucleic Acids [0444] As used herein, a “guide nucleic acid” refers to a nucleic acid that forms a ribonucleoprotein (e.g., a complex) with a guided nuclease (e.g., without being limiting, Cas12a, CasX) and then 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 complementary sequences. In an aspect, a ribonucleoprotein provided herein comprises at least one guide nucleic acid. [0445] In an aspect, a guide nucleic acid comprises DNA. In another aspect, a guide nucleic acid comprises RNA. In an aspect, a guide nucleic acid comprises DNA, RNA, or a combination thereof. In an aspect, a guide nucleic acid is single-stranded. In another aspect, a guide nucleic acid is at least partially double-stranded. [0446] When a guide nucleic acid comprises RNA, it can be referred to as a “guide RNA.” In another aspect, a guide nucleic acid comprises DNA and RNA. In another aspect, a guide RNA is single-stranded. In another aspect, a guide RNA is double-stranded. In a further aspect, a guide RNA is partially double-stranded. [0447] A "guide nucleic acid," "guide RNA," "gRNA," "CRISPR RNA/DNA" "crRNA" or "crDNA" as used herein means a nucleic acid that comprises at least one spacer sequence, which is complementary to (and hybridizes to) a target DNA (e.g., protospacer), and at least one repeat sequence (e.g., a repeat of a Type V Cas12a CRISPR-Cas system, or a fragment or portion thereof; a repeat of a Type II Cas9 CRISPR-Cas system, or fragment thereof; a repeat of a Type V C2c1 CRISPR Cas system, or a fragment thereof; a repeat of a CRISPR-Cas system of, for example, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3’, Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), and/or Csf5, or a fragment thereof), wherein the repeat sequence may be linked to the 5’ end and/or the 3’ end of the spacer sequence. The design of a gRNA of this invention may be based on a Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR-Cas system. [0448] In some embodiments, a Cas12a gRNA may comprise, from 5’ to 3’, a repeat sequence (full length or portion thereof ("handle"); e.g., pseudoknot-like structure) and a spacer sequence. [0449] In some embodiments, a guide nucleic acid may comprise more than one repeat sequence- spacer sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeat-spacer sequences) (e.g., repeat- spacer-repeat, e.g., repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer, and the like). The guide nucleic acids of this invention are synthetic, human-made and not found in nature. A gRNA can be quite long and may be used as an aptamer (like in the MS2 recruitment strategy) or other RNA structures hanging off the spacer. A guide RNA may comprise a donor template for introducing specific modifications in the target sequence. [0450] A "repeat sequence" as used herein, refers to, for example, any repeat sequence of a wild- type CRISPR Cas locus (e.g., a Cas9 locus, a Cas12a locus, a C2c1 locus, etc.) or a repeat sequence of a synthetic crRNA that is functional with the CRISPR-Cas effector protein encoded by the nucleic acid constructs of the invention. A repeat sequence useful with this invention can be any known or later identified repeat sequence of a CRISPR-Cas locus (e.g., Type I, Type II, Type III, Type IV, Type V or Type VI) or it can be a synthetic repeat designed to function in a Type I, II, III, IV, V or VI CRISPR-Cas system. A repeat sequence may comprise a hairpin structure and/or a stem loop structure. In some embodiments, a repeat sequence may form a pseudoknot-like structure at its 5’ end (i.e., "handle"). Thus, in some embodiments, a repeat sequence can be identical to or substantially identical to a repeat sequence from wild-type Type I CRISPR-Cas loci, Type II, CRISPR-Cas loci, Type III, CRISPR-Cas loci, Type IV CRISPR-Cas loci, Type V CRISPR-Cas loci and/or Type VI CRISPR-Cas loci. A repeat sequence from a wild-type CRISPR- Cas locus may be determined through established algorithms, such as using the CRISPRfinder offered through CRISPRdb (see, Grissa et al. (2007) Nucleic Acids Res. 35(Web Server issue):W52-7). In some embodiments, a repeat sequence or portion thereof is linked at its 3’ end to the 5’ end of a spacer sequence, thereby forming a repeat-spacer sequence (e.g., guide nucleic acid, guide RNA/DNA, crRNA, crDNA). [0451] In some embodiments, a repeat sequence comprises, consists essentially of, or consists of at least 10 nucleotides depending on the particular repeat and whether the guide nucleic acid comprising the repeat is processed or unprocessed (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 to 100 or more nucleotides, or any range or value therein). In some embodiments, a repeat sequence comprises, consists essentially of, or consists of about 10 to about 20, about 10 to about 30, about 10 to about 45, about 10 to about 50, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 40, about 40 to about 80, about 50 to about 100 or more nucleotides. [0452] A repeat sequence linked to the 5’ end of a spacer sequence can comprise a portion of a repeat sequence (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous nucleotides of a wild type repeat sequence). In some embodiments, a portion of a repeat sequence linked to the 5’ end of a spacer sequence can be about five to about ten consecutive nucleotides in length (e.g., about 5, 6, 7, 8, 9, 10 nucleotides) and have at least 90% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the same region (e.g., 5’ end) of a wild type CRISPR Cas repeat nucleotide sequence. In some embodiments, a portion of a repeat sequence may comprise a pseudoknot-like structure at its 5’ end (e.g., "handle"). [0453] A "spacer sequence" as used herein is a nucleotide sequence that is complementary to portion of a target nucleic acid (e.g., target DNA) (e.g., protospacer).. A spacer sequence can be fully complementary or substantially complementary (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a target nucleic acid. In some embodiments, the spacer sequence can have one, two, three, four, or five mismatches as compared to the target nucleic acid, which mismatches can be contiguous or noncontiguous. In some embodiments, the spacer sequence can have 70% complementarity to a target nucleic acid. In other embodiments, the spacer nucleotide sequence can have 80% complementarity to a target nucleic acid. In still other embodiments, the spacer nucleotide sequence can have 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% complementarity, and the like, to the target nucleic acid (protospacer). In some embodiments, the spacer sequence is 100% complementary to the target nucleic acid. A spacer sequence may have a length from about 15 nucleotides to about 30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or any range or value therein). Thus, in some embodiments, a spacer sequence may have complete complementarity or substantial complementarity over a region of a target nucleic acid (e.g., protospacer) that is at least about 15 nucleotides to about 30 nucleotides in length. In some embodiments, the spacer is about 20 nucleotides in length. In some embodiments, the spacer is about 21, 22, or 23 nucleotides in length. In some embodiments, a spacer sequence may comprise any one of the sequences of SEQ ID NOs:7-8, or any combination thereof. [0454] In some embodiments, the 5’ region of a spacer sequence of a guide nucleic acid may be identical to a target DNA, while the 3’ region of the spacer may be substantially complementary to the target DNA (such as a spacer of a Type V CRISPR-Cas system), or the 3’ region of a spacer sequence of a guide nucleic acid may be identical to a target DNA, while the 5’ region of the spacer may be substantially complementary to the target DNA (such as a spacer of a Type II CRISPR- Cas system), and therefore, the overall complementarity of the spacer sequence to the target DNA may be less than 100%. Thus, for example, in a guide for a Type V CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 5’ region (i.e., seed region) of, for example, a 20 nucleotide spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 3’ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA. In some embodiments, the first 1 to 8 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, nucleotides, and any range therein) of the 5’ end of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 3’ region of the spacer sequence are substantially complementary (e.g., at least about 50% complementary (e.g., 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to the target DNA. [0455] As a further example, in a guide for a Type II CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 3’ region (i.e., seed region) of, for example, a 20 nucleotide spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5’ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA. In some embodiments, the first 1 to 10 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, and any range therein) of the 3’ end of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5’ region of the spacer sequence are substantially complementary (e.g., at least about 50% complementary (e.g., at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or any range or value therein)) to the target DNA. [0456] In some embodiments, a seed region of a spacer may be about 8 to about 10 nucleotides in length, about 5 to about 6 nucleotides in length, or about 6 nucleotides in length. [0457] In an aspect, a guide nucleic acid comprises a guide RNA. In another aspect, a guide nucleic acid comprises at least one guide RNA. In another aspect, a guide nucleic acid comprises at least two guide RNAs. In another aspect, a guide nucleic acid comprises at least three guide RNAs. In another aspect, a guide nucleic acid comprises at least five guide RNAs. In another aspect, a guide nucleic acid comprises at least ten guide RNAs. [0458] In another aspect, a guide nucleic acid comprises at least 10 nucleotides. In another aspect, a guide nucleic acid comprises at least 11 nucleotides. In another aspect, a guide nucleic acid comprises at least 12 nucleotides. In another aspect, a guide nucleic acid comprises at least 13 nucleotides. In another aspect, a guide nucleic acid comprises at least 14 nucleotides. In another aspect, a guide nucleic acid comprises at least 15 nucleotides. In another aspect, a guide nucleic acid comprises at least 16 nucleotides. In another aspect, a guide nucleic acid comprises at least 17 nucleotides. In another aspect, a guide nucleic acid comprises at least 18 nucleotides. In another aspect, a guide nucleic acid comprises at least 19 nucleotides. In another aspect, a guide nucleic acid comprises at least 20 nucleotides. In another aspect, a guide nucleic acid comprises at least 21 nucleotides. In another aspect, a guide nucleic acid comprises at least 22 nucleotides. In another aspect, a guide nucleic acid comprises at least 23 nucleotides. In another aspect, a guide nucleic acid comprises at least 24 nucleotides. In another aspect, a guide nucleic acid comprises at least 25 nucleotides. In another aspect, a guide nucleic acid comprises at least 26 nucleotides. In another aspect, a guide nucleic acid comprises at least 27 nucleotides. In another aspect, a guide nucleic acid comprises at least 28 nucleotides. In another aspect, a guide nucleic acid comprises at least 30 nucleotides. In another aspect, a guide nucleic acid comprises at least 35 nucleotides. In another aspect, a guide nucleic acid comprises at least 40 nucleotides. In another aspect, a guide nucleic acid comprises at least 45 nucleotides. In another aspect, a guide nucleic acid comprises at least 50 nucleotides. [0459] In another aspect, a guide nucleic acid comprises between 10 nucleotides and 50 nucleotides. In another aspect, a guide nucleic acid comprises between 10 nucleotides and 40 nucleotides. In another aspect, a guide nucleic acid comprises between 10 nucleotides and 30 nucleotides. In another aspect, a guide nucleic acid comprises between 10 nucleotides and 20 nucleotides. In another aspect, a guide nucleic acid comprises between 16 nucleotides and 28 nucleotides. In another aspect, a guide nucleic acid comprises between 16 nucleotides and 25 nucleotides. In another aspect, a guide nucleic acid comprises between 16 nucleotides and 20 nucleotides. [0460] In an aspect, a guide nucleic acid comprises at least 70% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 75% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 80% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 85% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 90% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 91% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 92% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 93% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 94% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 95% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 96% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 97% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 98% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 99% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises 100% sequence complementarity to a target site. In another aspect, a guide nucleic acid comprises between 70% and 100% sequence complementarity to a target site. In another aspect, a guide nucleic acid comprises between 80% and 100% sequence complementarity to a target site. In another aspect, a guide nucleic acid comprises between 90% and 100% sequence complementarity to a target site. [0461] In an aspect, a guide nucleic acid is capable of hybridizing to a target site. [0462] As noted above, some guided nucleases, such as CasX and Cas9, require another non- coding RNA component, referred to as a trans-activating crRNA (tracrRNA), to have functional activity. Guide nucleic acid molecules provided herein can combine a crRNA and a tracrRNA into one nucleic acid molecule in what is herein referred to as a “single guide RNA” (sgRNA). The gRNA guides the active CasX complex to a target site within a target sequence, where CasX can cleave the target site. In other embodiments, the crRNA and tracrRNA are provided as separate nucleic acid molecules. [0463] In an aspect, a guide nucleic acid comprises a crRNA. In another aspect, a guide nucleic acid comprises a tracrRNA. In a further aspect, a guide nucleic acid comprises a sgRNA. RNA guided nucleases [0464] Guided nucleases are nucleases that form a complex (e.g., a ribonucleoprotein) with a guide nucleic acid molecule (e.g., a guide RNA), which then guides the complex to a target site within a target sequence. One non-limiting example of guided nucleases are CRISPR nucleases. [0465] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) nucleases (e.g., Cas9, CasX, Cas12a (also referred to as Cpf1), CasY, MAD7®) are proteins found in bacteria that are guided by guide RNAs (“gRNAs”) to a target nucleic acid molecule, where the endonuclease can then cleave one or two strands the target nucleic acid molecule. Although the origins of CRISPR nucleases are bacterial, many CRISPR nucleases have been shown to function in eukaryotic cells. [0466] While not being limited by any particular scientific theory, a CRISPR nuclease forms a complex with a guide RNA (gRNA), which hybridizes with a complementary target site, thereby guiding the CRISPR nuclease to the target site. In class II CRISPR-Cas systems, CRISPR arrays, including spacers, are transcribed during encounters with recognized invasive DNA and are processed into small interfering CRISPR RNAs (crRNAs). The crRNA comprises a repeat sequence and a spacer sequence which is complementary to a specific protospacer sequence in an invading pathogen. The spacer sequence can be designed to be complementary to target sequences in a eukaryotic genome. [0467] CRISPR nucleases associate with their respective crRNAs in their active forms. CasX, similar to the class II endonuclease Cas9, requires another non-coding RNA component, referred to as a trans-activating crRNA (tracrRNA), to have functional activity. Nucleic acid molecules provided herein can combine a crRNA and a tracrRNA into one nucleic acid molecule in what is herein referred to as a “single guide RNA” (sgRNA). Cas12a or MAD7® do not require a tracrRNA to be guided to a target site; a crRNA alone is sufficient for Cas12a or MAD7®. The gRNA guides the active CRISPR nuclease complex to a target site, where the CRISPR nuclease can cleave the target site. [0468] When an RNA-guided CRISPR nuclease and a guide RNA form a complex, the whole system is called a “ribonucleoprotein.” Ribonucleoproteins provided herein can also comprise additional nucleic acids or proteins. [0469] A prerequisite for cleavage of the target site by a CRISPR ribonucleoprotein 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 (e.g., between 18-23 nucleotides for Cas12a) from the PAM site. PAM sites are only required for type I and type II CRISPR associated proteins, and different CRISPR endonucleases recognize different PAM sites. Without being limiting, 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 and MAD7® nuclease recognizes T-rich PAM sequences YTTN and seems to prefer TTTN to CTTN PAMs (where T is thymine; C is cytosine; A is adenine; Y is thymine or cytosine; and N is thymine, cytosine, guanine, or adenine). [0470] Cas12a is an RNA-guided nuclease of a class II, type V CRISPR/Cas system. Cas12a nucleases generate staggered cuts when cleaving a double-stranded DNA molecule. Staggered cuts of double-stranded DNA produce a single-stranded DNA overhang of at least one nucleotide. This is in contrast to a blunt-end cut (such as those generated by Cas9), which does not produce a single- stranded DNA overhang when cutting double-stranded DNA. [0471] In an aspect, a Cas12a nuclease provided herein is a Lachnospiraceae bacterium Cas12a (LbCas12a) nuclease. In another aspect, a Cas12a nuclease provided herein is a Francisella novicida Cas12a (FnCas12a) nuclease. In an aspect, a Cas12a nuclease is selected from the group consisting of LbCas12a and FnCas12a. [0472] In an aspect, a Cas12a nuclease, or a nucleic acid encoding a Cas12a nuclease, is derived from a bacteria genus selected from the group consisting of Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, Acidaminococcus, Peregrinibacteria, Butyrivibrio, Parcubacteria, Smithella, Candidatus, Moraxella, and Leptospira. [0473] In an aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 80% identical to a polynucleotide of SEQ ID NO: 26 or SEQ ID NO:31 or SEQ ID NO: 32. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 85% identical to a polynucleotide of SEQ ID NO: 26 or SEQ ID NO:31 or SEQ ID NO: 32. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 90% identical to a polynucleotide of SEQ ID NO: 26 or SEQ ID NO:31 or SEQ ID NO: 32. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 95% identical to a polynucleotide of SEQ ID NO: 26 or SEQ ID NO:31 or SEQ ID NO: 32. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 96% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 26 or SEQ ID NO:31 or SEQ ID NO: 32. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 97% identical to a polynucleotide of SEQ ID NO: 2. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 98% identical to a polynucleotide of SEQ ID NO: 26 or SEQ ID NO:31 or SEQ ID NO: 32. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 99% identical to a polynucleotide of SEQ ID NO: 26 or SEQ ID NO:31 or SEQ ID NO: 32. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence 100% identical to a polynucleotide of SEQ ID NO: 26 or SEQ ID NO:31 or SEQ ID NO: 32. [0474] In an aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 80% identical to an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 38 or SEQ ID NO: 39. In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 85% identical to an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 38 or SEQ ID NO: 39. In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 90% identical to an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 38 or SEQ ID NO: 39. In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 95% identical to an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 38 or SEQ ID NO: 39. In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 96% identical to an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 38 or SEQ ID NO: 39. In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 97% identical to an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 38 or SEQ ID NO: 39. In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 98% identical to an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 38 or SEQ ID NO: 39. In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 99% identical to an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 38 or SEQ ID NO: 39. In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at 100% identity to an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 38 or SEQ ID NO: 39. [0475] In an aspect, a Cas12a provided herein is a variant Lachnospiraceae bacterium Cas12a (LbCas12a) nuclease with enhanced DNA cleavage activities at non-canonical TTTT protospacer adjacent motifs such as described in US2021/0348144 (incorporated herein by reference in its entirety) In another aspect, a Cas12a provided herein is a variant Lachnospiraceae bacterium Cas12a (LbCas12a) nuclease with enhanced activity as described in US20230040148 (incorporated herein by reference in its entirety) such as the LbCas12a-ultra having an N527R and E795L substitution in its amino acid sequence (reference amino acid sequence is SEQ ID NO: 33) [0476] In an aspect, a Cas12a provided herein provided herein is a variant Lachnospiraceae bacterium Cas12a (LbCas12a) nuclease recognizing a PAM variant TYCV having a G532R and K595R substitution in its amino acid sequence (reference amino acid sequence is SEQ ID NO: 33) or a variant Lachnospiraceae bacterium Cas12a (LbCas12a) nuclease recognizing a PAM variant TATT having a G532R, K538R and Y524R substitution in its amino acid sequence (reference amino acid sequence is SEQ ID NO: 33) as disclosed in WO2016205711 (herein incorporated by reference in its entirety). [0477] CasX is a type of class II CRISPR-Cas nuclease that has been identified in the bacterial phyla Deltaproteobacteria and Planctomycetes. Similar to Cas12a, CasX nucleases generate staggered cuts when cleaving a double-stranded DNA molecule. However, unlike Cas12a, CasX nucleases require a crRNA and a tracrRNA, or a single-guide RNA, in order to target and cleave a target nucleic acid. [0478] In an aspect, a CasX nuclease provided herein is a CasX nuclease from the phylum Deltaproteobacteria. In another aspect, a CasX nuclease provided herein is a CasX nuclease from the phylum Planctomycetes. Without being limiting, additional suitable CasX nucleases are those set forth in WO 2019/084148, which is incorporated by reference herein in its entirety. [0479] MAD7® (also known as ErCas12a) is an engineered nuclease of the Class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) family with a low level of homology to canonical Cas12a nucleases. MAD7® nucleases generate staggered cuts when cleaving a double-stranded DNA molecule.MAD7® nuclease was initially identified in Eubacterium rectale. It only requires a crRNA like canonical Cas12a. An ErCas12a/MAD7® encoding nucleotide sequence can be found in the supplementary data (sequences S1) provided with Lin et al., 2021, Journal of Genetics and Genomics 48, pages 444-451) [0480] In an aspect, a guided nuclease capable of generating a staggered cut in a double-stranded DNA molecule is selected from the group consisting of Cas12a; MAD7® and CasX. In an aspect, a guided nuclease is selected from the group consisting of Cas12a, MAD7® and CasX. [0481] In an aspect, a guided nuclease is a RNA-guided nuclease. In another aspect, a guided nuclease is a CRISPR nuclease. In another aspect, a guided nuclease is a Cas12a nuclease. In another aspect, a guided nuclease is a CasX nuclease. In another aspect, a guided nuclease is a MAD7® nuclease. [0482] As used herein, a “nuclear localization signal” (NLS) refers to an amino acid sequence that “tags” a protein for import into the nucleus of a cell. In an aspect, a nucleic acid molecule provided herein encodes a nuclear localization signal. In another aspect, a nucleic acid molecule provided herein encodes two or more nuclear localization signals. [0483] There are two major import pathways into the nucleus of eukaryotic cells, mediated either by classic NLS (characterized by the involvement of importin α subunit) or non-classic NLS (characterized by involvement of importin β subunit). The size of nuclear localization signals varies considerably, although the majority of monopartite NLSs comprise 7 amino acids, while the majority of bipartite NLSs comprise 17 amino acids. More than 90% of naturally occurring proteins have only one NLS. [0484] There are six classes of classic nuclear localization signals characterized by the following formulas: Class 1: KR(K/R)R or K(K/R)RK Class 2: (P/R)XXKR(^DE)(K/R) Class 3: KRX(W/F/Y)XXAF Class 4: (R/P)XXKR(K/R)(^DE) Class 5: LGKR(K/R)(W/F/Y) Class 6: KRX_[10-12]K(KR)(KR) or KRX_[10-12]K(KR)X(KR) Class 1 and 2 NLS are predominant across organisms while Class 5 NLS are plant-specific NLS. Examples of classic NLS signals include the NLS sequence of HSFA protein from tomato (SEQ ID NO: 14) NLSC (SEQ ID NO: 16) or NLS1 (SEQ ID NO: 15) and the amino acid sequences of SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 94. [0485] Unlike classic nuclear localization signals, non-classic pathway NLS peptides engage the beta-importin (also known as karyopherin β) instead of the alpha-importin pathways for nuclear import. Diversity and complexity of signals recognized by Kapβs have prevented prediction of new Kapβ substrates and subsequent identification of the NLSs in candidate import substrates. The few characterized ncNLSs which are mostly from mammalian proteins, are diverse and encompass both structural domains and linear epitopes (see Lu, J., et al. Cell Commun Signal 19, 60 (2021). One of the best characterized Kapβ substrate is the human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). hnRNP A1 ncNLS belongs to the “proline-tyrosine” category, named PY-NLS. It assumes a disordered structure consisting of N-terminal hydrophobic or basic motifs and C-terminal R/K/H(X)2-5PY motifs (where X2-5 is any sequence of 2–5 residues). Other Kapβ substrates include ribosomal proteins, transcription factors and splicing factors. Characterized ncNLSs are longer than classic NLS’s, ranging from >20 amino acids and up to ~70 amino acids in length compared to 7-17 average length for classic NLS’s. [0486] Five mammalian Kapβ substrate proteins comprising ncNLS were sequence analysed against the corn genome database to identify putative homologs in corn. Factors like conserved amino acid residues and modelling was used to narrow down regions within the protein that could function as NLSs. The corn ncNLS sequences were extracted from the corn homologs and are disclosed in Table 14 and include SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102 SEQ ID NO: 103 and SEQ ID NO:104. [0487] Non-classic NLSs are different from classic NLSs canonical sequences and not rich in arginine or lysine residues as observed in classic NLSs. Argine-Glycine (-Glycine) or Arginine- serine-tyrosine regions act as non-classic NLS. [0488] Nuclear localization signals according to the present disclosure may be connected to one of the termini of Crispr/Cas proteins by a linker peptide. As used herein, “a linker” or “linker peptide” are relatively short amino acids sequences connecting separate domains in multidomain proteins. Two main types of linkers exists; helical and non-helical. Linkers may be flexible, such as Gly-rich linkers. Helical linkers usually act a rigid spacers separating two domains. Non-helical linkers are often rich in prolines, which also leads to structural rigidity and isolation of the linker from the attached domains. Linker databases intended for the rational design of linkers for domain fusion are available in the art. See e.g.http://mathbio.nimr.mrc.ac.uk.See also George and Heringa “An analysis of protein domain linkers: their classification and role in protein folding” Protein Engineering, Design and Selection, Volume 15, Issue 11, November 2002, Pages 871– 879, https://doi.org/10.1093/protein/15.11.871. Examples of linker peptides of varying lengths and rigidity used in the current document include: GGSG (SEQ ID NO 17), GGSG(EAAAK)n2GGSG (SEQ ID NO: 21), GGSG(EAAAK)n4GGSG (SEQ ID NO: 22), GGSG(EAAAK)n6GGSG (SEQ ID NO: 23), a linker comprising one or more repeats of YETKQ (SEQ ID NO: 19); a linker comprising one or more repeats of the amino acid sequence PVTAT (SEQ ID NO: 20); GGGSGYETKQGGGS (SEQ ID NO: 24); GGGSGPVTATGGGS (SEQ ID NO: 25) and the like. [0489] In an aspect, a Cas12a nuclease provided herein comprises a nuclear localization signal. In an aspect, a nuclear localization signal is positioned on the N-terminal end of a Cas12a nuclease. In a further aspect, a nuclear localization signal is positioned on the C-terminal end of a Cas12a nuclease. In yet another aspect, a nuclear localization signal is embedded within an exposed loop of a Cas12a nuclease. [0490] In an aspect, a CasX nuclease provided herein comprises a nuclear localization signal. In an aspect, a nuclear localization signal is positioned on the N-terminal end of a CasX nuclease. In a further aspect, a nuclear localization signal is positioned on the C-terminal end of a CasX nuclease. In yet another aspect, a nuclear localization signal is embedded within an exposed loop of a CasX nuclease. [0491] In an aspect, a MAD7® nuclease provided herein comprises a nuclear localization signal. In an aspect, a nuclear localization signal is positioned on the N-terminal end of a MAD7® nuclease. In a further aspect, a nuclear localization signal is positioned on the C-terminal end of a MAD7® nuclease. In yet another aspect, a nuclear localization signal embedded within an exposed loop of a MAD7® nuclease. [0492] As used herein “an exposed loop” of a Cas protein, such as a Cas12a protein, is a region of the Cas protein where prediction software of protein’s 3D structure, such as Alphafold, generates a less confident model or regions unresolved in the empirical crystal structures. Such regions are thought to be flexible and unstructured, and modification thereof may not have a significant impact on the function of the protein. Examples of exposed loops include the amino acid sequence from position 85 to 89, or the amino acid sequence from position 126-137, or the amino acid sequence from position 1071-1075, or the amino acid sequence from position 1076-1085, or the amino acid sequence from position 370-379, or the amino acid sequence from position 437-460, or the amino acid sequence from position 485-490, or the amino acid sequence from position 449 to 461, or the amino acid sequence from position 487-496 of the amino acid sequence of reference SEQ ID NO: 33 for LbCas12a. [0493] The exposed loop may also be used to insert a heterologous peptide therein or to replace the exposed loop with a heterologous peptide. In one embodiment, the heterologous peptide is nuclear localization signal. In another embodiment, the heterologous peptide is a tethering motif. In another embodiment, the heterologous peptide is a tag. In another embodiment, the heterologous peptide is a nucleic acid binding motif containing heterologous peptide. In another embodiment, the heterologous peptide is a DNA binding motif containing heterologous peptide. In another embodiment, the heterologous peptide is a an RNA binding motif containing heterologous peptide. In another embodiment, the heterologous peptide comprises a protein binding motif. In one embodiment. In another embodiment, the heterologous peptide is an enzyme. In another embodiment, the heterologous peptide is a DNA demethylase. In another embodiment, the heterologous peptide is a histone modifying enzyme. In another embodiment, the heterologous peptide is a transposase. In another embodiment, the heterologous peptide is an HUH endonuclease or HUH tag. In another embodiment, the heterologous peptide is a Gal4 transcription factor. [0494] In an aspect, a ribonucleoprotein comprises at least one nuclear localization signal. In another aspect, a ribonucleoprotein comprises at least two nuclear localization signals. [0495] Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database" available at www[dot]kazusa[dot]or[dot]jp[forwards slash]codon and these tables can be adapted in a number of ways. See Nakamura et al., 2000, Nucl. Acids Res. 28:292. Computer algorithms for codon optimizing a particular sequence for expression in a particular plant cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available. [0496] As used herein, “codon optimization” refers to a process of modifying a nucleic acid sequence for enhanced expression in a plant cell of interest by replacing at least one codon (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of a sequence with codons that are more frequently or most frequently used in the genes of the plant cell while maintaining the original amino acid sequence (e.g., introducing silent mutations). [0497] In an aspect, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a guided nuclease correspond to the most frequently used codon for a particular amino acid. In another aspect, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a Cas12a nuclease or a CasX nuclease or a MAD7® nuclease correspond to the most frequently used codon for a particular amino acid. As to codon usage in plants, reference is made to Campbell and Gowri, 1990, Plant Physiol., 92: 1-11; and Murray et al., 1989, Nucleic Acids Res., 17:477-98, each of which is incorporated herein by reference in their entireties. [0498] In an aspect, a nucleic acid molecule encodes a guided nuclease that is codon optimized for a plant. In an aspect, a nucleic acid molecule encodes a Cas12a nuclease that is codon optimized for a plant. In an aspect, a nucleic acid molecule encodes a CasX nuclease that is codon optimized for a plant. In an aspect, a nucleic acid molecule encodes a MAD7® nuclease that is codon optimized for a plant. [0499] In another aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a plant cell. In another aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a monocotyledonous plant species. In another aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a dicotyledonous plant species. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a gymnosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for an angiosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a corn cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a soybean cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a rice cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a wheat cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a cotton cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a sorghum cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for an alfalfa cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a sugarcane cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for an Arabidopsis cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a tomato cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a cucumber cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a potato cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for an onion cell. [0500] In another aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a plant cell. In another aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a monocotyledonous plant species. In another aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a dicotyledonous plant species. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a gymnosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for an angiosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a corn cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a soybean cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a rice cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a wheat cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a cotton cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a sorghum cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for an alfalfa cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a sugar cane cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for an Arabidopsis cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a tomato cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a cucumber cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a potato cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for an onion cell. [0501] In another aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a plant cell. In another aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a monocotyledonous plant species. In another aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a dicotyledonous plant species. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a gymnosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for an angiosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a corn cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a soybean cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a rice cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a wheat cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a cotton cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a sorghum cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for an alfalfa cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a sugar cane cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for an Arabidopsis cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a tomato cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a cucumber cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a potato cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for an onion cell. In another aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a plant cell. In another aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a monocotyledonous plant species. In another aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a dicotyledonous plant species. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a gymnosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for an angiosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a corn cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a soybean cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a rice cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a wheat cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a cotton cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a sorghum cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for an alfalfa cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a sugar cane cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for an Arabidopsis cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a tomato cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a cucumber cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a potato cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for an onion cell. [0502] In some aspects the guided nuclease may be selected from Cas9, C2c1, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Csnl, Csx12, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, 30 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 nuclease, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, Cas14c effector protein [0503] In some aspects, the guided nuclease , such as a CRISPR/Cas effector protein useful with the invention may comprise a mutation in its nuclease active site (e.g., RuvC, HNH, e.g., RuvC site of a Cas12a nuclease domain, e.g., RuvC site and/or HNH site of a Cas9 nuclease domain). A CRISPR-Cas effector protein having a mutation in its nuclease active site, and therefore, no longer comprising nuclease activity, is commonly 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 activity or reduced activity as compared to the same CRISPR-Cas effector protein without the mutation, e.g., a nickase, e.g., Cas9 nickase, Cas12a nickase. [0504] In some aspects, the guided nuclease may comprise another functional domain than a nuclease, such as a adenine deaminase domain or a cytosine deaminase domain or a reverse transcriptase domain. [0505] An adenine deaminase (or adenosine deaminase) useful with this invention may be any known or later identified adenine deaminase from any organism (see, e.g., U.S. Patent No. 10,113,163, which is incorporated by reference herein for its disclosure of adenine deaminases). An adenine deaminase can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the adenine deaminase may catalyze the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase may catalyze the hydrolytic deamination of adenine or adenosine in DNA. In some embodiments, an adenine deaminase encoded by a nucleic acid construct of the invention may generate an A→G conversion in the sense (e.g., "+"; template) strand of the target nucleic acid or a T→C conversion in the antisense (e.g., "˗", strand of the target nucleic acid. [0506] In some embodiments, an adenosine deaminase may be a variant of a naturally occurring adenine deaminase. Thus, in some embodiments, an adenosine deaminase may be about 70% to 100% identical to a wild type adenine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and any range or value therein, to a naturally occurring adenine deaminase). In some embodiments, the deaminase or deaminase does not occur in nature and may be referred to as an engineered, mutated or evolved adenosine deaminase. Thus, for example, an engineered, mutated or evolved adenine deaminase polypeptide or an adenine deaminase domain may be about 70% to 99.9% identical to a naturally occurring adenine deaminase polypeptide/domain (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical, and any range or value therein, to a naturally occurring adenine deaminase polypeptide or adenine deaminase domain). In some embodiments, the adenosine deaminase may be from a bacterium, (e.g., Escherichia coli, Staphylococcus aureus, Haemophilus influenzae, Caulobacter crescentus, and the like). In some embodiments, a polynucleotide encoding an adenine deaminase polypeptide/domain may be codon optimized for expression in a plant. [0507] In some embodiments, an adenine deaminase domain may be a wild type tRNA-specific adenosine deaminase domain, e.g., a tRNA-specific adenosine deaminase (TadA) and/or a mutated/evolved adenosine deaminase domain, e.g., mutated/evolved tRNA-specific adenosine deaminase domain (TadA*). In some embodiments, a TadA domain may be from E. coli. In some embodiments, the TadA may be modified, e.g., truncated, missing one or more N-terminal and/or C-terminal amino acids relative to a full-length TadA (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal and/or C terminal amino acid residues may be missing relative to a full length TadA. In some embodiments, a TadA polypeptide or TadA domain does not comprise an N-terminal methionine. In some embodiments, a polynucleotide encoding a TadA/TadA* may be codon optimized for expression in a plant. [0508] A cytosine deaminase catalyzes cytosine deamination and results in a thymidine (through a uracil intermediate), causing a C to T conversion, or a G to A conversion in the complementary strand in the genome. Thus, in some embodiments, the cytosine deaminase encoded by the polynucleotide of the invention generates a C→T conversion in the sense (e.g., "+"; template) strand of the target nucleic acid or a G →A conversion in antisense (e.g., strand of the target nucleic acid. [0509] In some embodiments, the adenine deaminase encoded by the nucleic acid construct of the invention generates an A→G conversion in the sense (e.g., "+"; template) strand of the target nucleic acid or a T→C conversion in the antisense (e.g., "˗", strand of the target nucleic acid. [0510] The nucleic acid constructs of the invention encoding a base editor comprising a sequence- specific DNA binding protein and a cytosine deaminase polypeptide, and nucleic acid constructs/expression cassettes/vectors encoding the same, may be used in combination with guide nucleic acids for modifying target nucleic acid including, but not limited to, generation of C→T or G →A mutations in a target nucleic acid including, but not limited to, a plasmid sequence; generation of C→T or G →A mutations in a coding sequence to alter an amino acid identity; generation of C→T or G →A mutations in a coding sequence to generate a stop codon; generation of C→T or G →A mutations in a coding sequence to disrupt a start codon; generation of point mutations in genomic DNA to disrupt transcription factor binding; and/or generation of point mutations in genomic DNA to disrupt splice junctions. [0511] The nucleic acid constructs of the invention encoding a base editor comprising a sequence- specific DNA binding protein and an adenine deaminase polypeptide, and expression cassettes and/or vectors encoding the same may be used in combination with guide nucleic acids for modifying a target nucleic acid including, but not limited to, generation of A→G or T→C mutations in a target nucleic acid including, but not limited to, a plasmid sequence; generation of A→G or T→C mutations in a coding sequence to alter an amino acid identity; generation of A→G or T→C mutations in a coding sequence to generate a stop codon; generation of A→G or T→C mutations in a coding sequence to disrupt a start codon; generation of point mutations in genomic DNA to disrupt function; and/or generation of point mutations in genomic DNA to disrupt splice junctions. Target sites [0512] As used herein, a “target sequence” refers to a selected sequence or region of a DNA molecule in which a modification (e.g., cleavage, site-directed integration) is desired. A target sequence comprises a target site. [0513] As used herein, a “target site” refers to the portion of a target sequence that is cleaved by a guided nuclease such as CRISPR nuclease. In contrast to a non-target nucleic acid (e.g., non-target ssDNA) or non-target region, a target site comprises significant complementarity to a guide nucleic acid or a guide RNA. [0514] In an aspect, a target site is 100% complementary to a guide nucleic acid. In another aspect, a target site is 99% complementary to a guide nucleic acid. In another aspect, a target site is 98% complementary to a guide nucleic acid. In another aspect, a target site is 97% complementary to a guide nucleic acid. In another aspect, a target site is 96% complementary to a guide nucleic acid. In another aspect, a target site is 95% complementary to a guide nucleic acid. In another aspect, a target site is 94% complementary to a guide nucleic acid. In another aspect, a target site is 93% complementary to a guide nucleic acid. In another aspect, a target site is 92% complementary to a guide nucleic acid. In another aspect, a target site is 91% complementary to a guide nucleic acid. In another aspect, a target site is 90% complementary to a guide nucleic acid. In another aspect, a target site is 85% complementary to a guide nucleic acid. In another aspect, a target site is 80% complementary to a guide nucleic acid. [0515] In an aspect, a target site comprises at least one PAM site. In an aspect, a target site is adjacent to a nucleic acid sequence that comprises at least one PAM site. In another aspect, a target site is within 5 nucleotides of at least one PAM site. In a further aspect, a target site is within 10 nucleotides of at least one PAM site. In another aspect, a target site is within 15 nucleotides of at least one PAM site. In another aspect, a target site is within 20 nucleotides of at least one PAM site. In another aspect, a target site is within 25 nucleotides of at least one PAM site. In another aspect, a target site is within 30 nucleotides of at least one PAM site. [0516] In an aspect, a target site is positioned within genic DNA. In another aspect, a target site is positioned within a gene. In another aspect, a target site is positioned within a gene of interest. In another aspect, a target site is positioned within an exon of a gene. In another aspect, a target site is positioned within an intron of a gene. In another aspect, a target site is positioned within the promoter of a gene. In another aspect, a target site is positioned within 5’-UTR of a gene. In another aspect, a target site is positioned within a 3’-UTR of a gene. In another aspect, a target site is positioned within intergenic DNA. [0517] A "protospacer sequence" refers to the target double stranded DNA and specifically to the portion of the target DNA (e.g., or target region in the genome) that is fully or substantially complementary (and hybridizes) to the spacer sequence of the CRISPR repeat-spacer sequences (e.g., guide nucleic acids, CRISPR arrays, crRNAs). [0518] In the case of Type V CRISPR-Cas (e.g., Cas12a) systems and Type II CRISPR-Cas (Cas9) systems, the protospacer sequence is flanked by (e.g., immediately adjacent to) a protospacer adjacent motif (PAM). For Type IV CRISPR-Cas systems, the PAM is located at the 5’ end on the non-target strand and at the 3’ end of the target strand (see below, as an example). 5'-NNNNNNNNNNNNNNNNNNN-3' RNA Spacer | | | | | | | | | | | | | | | | | | | | 3'AAANNNNNNNNNNNNNNNNNNN-5' Target strand | | | 5'TTTNNNNNNNNNNNNNNNNNNN-3' Non-target strand [0519] In the case of Type II CRISPR-Cas (e.g., Cas9) systems, the PAM is located immediately 3’ of the target region. The PAM for Type I CRISPR-Cas systems is located 5’ of the target strand. There is no known PAM for Type III CRISPR-Cas systems. Makarova et al. describes the nomenclature for all the classes, types and subtypes of CRISPR systems ((2015) Nature Reviews Microbiology 13:722–736). Guide structures and PAMs are described in by R. Barrangou ((2015) Genome Biol. 16:247). [0520] Canonical Cas12a PAMs are T rich. In some embodiments, a canonical Cas12a PAM sequence may be 5’-TTN, 5’-TTTN, or 5’-TTTV. In some embodiments, canonical Cas9 (e.g., S. pyogenes) PAMs may be 5’-NGG-3’. In some embodiments, non-canonical PAMs may be used but may be less efficient. [0521] Additional PAM sequences may be determined by those skilled in the art through established experimental and computational approaches. Thus, for example, experimental approaches include targeting a sequence flanked by all possible nucleotide sequences and identifying sequence members that do not undergo targeting, such as through the transformation of target plasmid DNA (Esvelt et al. (2013) Nat. Methods 10:1116-1121; Jiang et al. (2013) Nat. Biotechnol. 31:233-239). In some aspects, a computational approach can include performing BLAST searches of natural spacers to identify the original target DNA sequences in bacteriophages or plasmids and aligning these sequences to determine conserved sequences adjacent to the target sequence (Briner and Barrangou. (2014) Appl. Environ. Microbiol. 80:994- 1001; Mojica et al. (2009) Microbiology 155:733-740). [0522] In an aspect, a target DNA molecule is single-stranded. In another aspect, a target DNA molecule is double-stranded. [0523] In an aspect, a target sequence comprises genomic DNA. In an aspect, a target sequence is positioned within a nuclear genome. In an aspect, a target sequence comprises chromosomal DNA. In an aspect, a target sequence comprises plasmid DNA. In an aspect, a target sequence is positioned within a plasmid. In an aspect, a target sequence comprises mitochondrial DNA. In an aspect, a target sequence is positioned within a mitochondrial genome. In an aspect, a target sequence comprises plastid DNA. In an aspect, a target sequence is positioned within a plastid genome. In an aspect, a target sequence comprises chloroplast DNA. In an aspect, a target sequence is positioned within a chloroplast genome. In an aspect, a target sequence is positioned within a genome selected from the group consisting of a nuclear genome, a mitochondrial genome, and a plastid genome. [0524] In an aspect, a target sequence comprises genic DNA. As used herein, “genic DNA” refers to DNA that encodes one or more genes. In another aspect, a target sequence comprises intergenic DNA. In contrast to genic DNA, “intergenic DNA” comprises noncoding DNA, and lacks DNA encoding a gene. In an aspect, intergenic DNA is positioned between two genes. [0525] In an aspect, a target sequence encodes a gene. As used herein, a “gene” refers to a polynucleotide that can produce a functional unit (e.g., without being limiting, for example, a protein, or a non-coding RNA molecule). A gene can comprise a promoter, an enhancer sequence, a leader sequence, a transcriptional start site, a transcriptional 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” can comprise a polynucleotide sequence encoding a promoter, an enhancer sequence, a leader sequence, a transcriptional start site, a transcriptional 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 aspect, a gene encodes a non-protein-coding RNA molecule or a precursor thereof. In another aspect, a gene encodes a protein. In some embodiments, the target sequence is selected from the group consisting of: a promoter, an enhancer sequence, a leader sequence, a transcriptional start site, a transcriptional stop site, a polyadenylation site, an exon, an intron, a splice site, a 5’-UTR, a 3’-UTR, a protein coding sequence, a non-protein-coding sequence, a miRNA, a pre-miRNA and a miRNA binding site. [0526] Non-limiting examples of a non-protein-coding RNA molecule include a microRNA (miRNA), a miRNA precursor (pre-miRNA), a small interfering RNA (siRNA), a small RNA (18 to 26 nucleotides in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a CRISPR RNA (crRNA), a tracer RNA (tracrRNA), a guide RNA (gRNA), and a single guide RNA (sgRNA). In an aspect, a non-protein-coding RNA molecule comprises a miRNA. In an aspect, a non-protein-coding RNA molecule comprises a siRNA. In an aspect, a non-protein-coding RNA molecule comprises a ta- siRNA. In an aspect, a non-protein-coding RNA molecule is selected from the group consisting of a miRNA, a siRNA, and a ta-siRNA. [0527] As used herein, a “gene of interest” refers to a polynucleotide sequence encoding a protein or a non-protein-coding RNA molecule that is to be integrated into a target sequence, or, alternatively, an endogenous polynucleotide sequence encoding a protein or a non-protein-coding RNA molecule that is to be edited by a ribonucleoprotein. In an aspect, a gene of interest encodes a protein. In another aspect, a gene of interest encodes a non-protein-coding RNA molecule. In an aspect, a gene of interest is exogenous to a targeted DNA molecule. In an aspect, a gene of interest replaces an endogenous gene in a targeted DNA molecule. Mutations [0528] In an aspect, a ribonucleoprotein or method provided herein generates at least one mutation in a target sequence. [0529] In an aspect, a seed produced from a plant provided herein comprises at least one mutation in a gene of interest comprising a target site as compared to a seed of a control plant of the same line or variety that lacks a first nucleic acid sequence encoding a guided nuclease operably linked to a floral cell-preferred promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a heterologous second promoter. In an aspect, a seed produced from a plant provided herein comprises at least one mutation in a gene of interest comprising a target site as compared to a seed of a control plant of the same line or variety that lacks a first nucleic acid sequence encoding a guided nuclease operably linked to a floral tissue-preferred promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a heterologous second promoter. [0530] In an aspect, a seed produced from a plant provided herein comprises at least one mutation in a gene of interest comprising a target site as compared to a seed of a control plant of the same line or variety that lacks a first nucleic acid sequence encoding a guided nuclease operably linked to a heterologous promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a floral cell-preferred promoter. In an aspect, a seed produced from a plant provided herein comprises at least one mutation in a gene of interest comprising a target site as compared to a seed of a control plant of the same line or variety that lacks a first nucleic acid sequence encoding a guided nuclease operably linked to a heterologous promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a floral tissue-preferred promoter. [0531] As used herein, a “mutation” refers to a non-naturally occurring alteration to a nucleic acid or amino acid sequence as compared to a naturally occurring reference nucleic acid or amino acid sequence from the same organism. It will be appreciated that, when identifying a mutation, the reference sequence should be from the same nucleic acid (e.g, gene, non-coding RNA) or amino acid (e.g, protein). In determining if a difference between two sequences comprises a mutation, it will be appreciated in the art that the comparison should not be made between homologous sequences of two different species or between homologous sequences of two different varieties of a single species. Rather, the comparison should be made between the edited (e.g., mutated) sequence and the endogenous, non-edited (e.g., “wildtype”) sequence of the same organism. [0532] Several types of mutations are known in the art. In an aspect, a mutation comprises an insertion. An “insertion” refers to the addition of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation comprises a deletion. A “deletion” refers to the removal of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation comprises a substitution. A “substitution” refers to the replacement of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation comprises an inversion. An “inversion” refers to when a segment of a polynucleotide or amino acid sequence is reversed end- to-end. In an aspect, a mutation provided herein comprises a mutation selected from the group consisting of an insertion, a deletion, a substitution, and an inversion. [0533] In an aspect, a plant or seed comprises at least one mutation in a gene of interest, where the at least one mutation results in the deletion of one or more amino acids from a protein encoded by the gene of interest as compared to a wildtype protein. [0534] In an aspect, a plant or seed comprises at least one mutation in a gene of interest, where the at least one mutation results in the substitution of one or more amino acids within a protein encoded by the gene of interest as compared to a wildtype protein. [0535] In an aspect, a plant or seed comprises at least one mutation in a gene of interest, where the at least one mutation results in the insertion of one or more amino acids within a protein encoded by the gene of interest as compared to a wildtype protein. [0536] Mutations in coding regions of genes (e.g., exonic mutations) can result in a truncated protein or polypeptide when a mutated messenger RNA (mRNA) is translated into a protein or polypeptide. In an aspect, this disclosure provides a mutation that results in the truncation of a protein or polypeptide. As used herein, a “truncated” protein or polypeptide comprises at least one fewer amino acid as compared to an endogenous control protein or polypeptide. For example, if endogenous Protein A comprises 100 amino acids, a truncated version of Protein A can comprise between 1 and 99 amino acids. [0537] Without being limited by any scientific theory, one way to cause a protein or polypeptide truncation is by the introduction of a premature stop codon in an mRNA transcript of an endogenous gene. In an aspect, this disclosure provides a mutation that results in a premature stop codon in an mRNA transcript of an endogenous gene. As used herein, a “stop codon” refers to a nucleotide triplet within an mRNA transcript that signals a termination of protein translation. A “premature stop codon” refers to a stop codon positioned earlier (e.g., on the 5’-side) than the normal stop codon position in an endogenous mRNA transcript. Without being limiting, several stop codons are known in the art, including “UAG,” “UAA,” “UGA,” “TAG,” “TAA,” and “TGA.” [0538] In an aspect, a seed or plant comprises at least one mutation, where the at least one mutation results in the introduction of a premature stop codon in a messenger RNA encoded by the gene of interest as compared to a wildtype messenger RNA. [0539] In an aspect, a mutation provided herein comprises a null mutation. As used herein, a “null mutation” refers to a mutation that confers a complete loss-of-function for a protein encoded by a gene comprising the mutation, or, alternatively, a mutation that confers a complete loss-of-function for a small RNA encoded by a genomic locus. A null mutation can cause lack of mRNA transcript production, a lack of small RNA transcript production, a lack of protein function, or a combination thereof. [0540] A mutation provided herein can be positioned in any part of an endogenous gene. In an aspect, a mutation provided herein is positioned within an exon of an endogenous gene. In another aspect, a mutation provided herein is positioned within an intron of an endogenous gene. In a further aspect, a mutation provided herein is positioned within a 5’-untranslated region of an endogenous gene. In still another aspect, a mutation provided herein is positioned within a 3’- untranslated region of an endogenous gene. In yet another aspect, a mutation provided herein is positioned within a promoter of an endogenous gene. [0541] In an aspect, a mutation is positioned at a splice site within a gene. A mutation at a splice site can interfere with the splicing of exons during mRNA processing. If one or more nucleotides are inserted, deleted, or substituted at a splice site, splicing can be perturbed. Perturbed splicing can result in unspliced introns, missing exons, or both, from a mature mRNA sequence. Typically, although not always, a “GU” sequence is required at the 5’ end of an intron and a “AG” sequence is required at the 3’ end of an intron for proper splicing. If either of these splice sites are mutated, splicing perturbations can occur. [0542] In an aspect, a seed or plant comprises at least one mutation, where the at least one mutation comprises the deletion of one or more splice sites from a gene of interest. In another aspect, a seed or plant comprises at least one mutation, where the at least one mutation is positioned within one or more splice sites from a gene of interest. [0543] In an aspect, a mutation comprises a site-directed integration. In an aspect, a site-directed integration comprises the insertion of all or part of a desired sequence into a target sequence. [0544] As used herein, “site-directed integration” refers to all, or a portion, of a desired sequence (e.g., an exogenous gene, an edited endogenous gene) being inserted or integrated at a desired site or locus within the plant genome (e.g., target sequence). As used herein, a “desired sequence” refers to a DNA molecule comprising a nucleic acid sequence that is to be integrated into a genome of a plant or plant cell. The desired sequence can comprise a transgene or construct. In an aspect, a nucleic acid molecule comprising a desired sequence comprises one or two homology arms flanking the desired sequence to promote the targeted insertion event through homologous recombination and/or homology-directed repair. [0545] In an aspect, a method provided herein comprises site-directed integration of a desired sequence into a target sequence. [0546] Any site or locus within the genome of a plant can be chosen for site-directed integration of a transgene or construct of the present disclosure. In an aspect, a target sequence is positioned within a B, or supernumerary, chromosome. [0547] For site-directed integration, a double-strand break (DSB) or nick may first be made at a target sequence via a guided nuclease or ribonucleoprotein provided herein. In the presence of a desired sequence, the DSB or nick can then be repaired by homologous recombination (HR) between the homology arm(s) of the desired sequence and the target sequence, or by non- homologous end joining (NHEJ), resulting in site-directed integration of all or part of the desired sequence into the target sequence to create the targeted insertion event at the site of the DSB or nick. [0548] In an aspect, site-directed integration comprises the use of NHEJ repair mechanisms endogenous to a cell. In another aspect, site-directed integration comprises the use of HR repair mechanisms endogenous to a cell. [0549] In an aspect, repair of a double-stranded break generates at least one mutation in a gene of interest as compared to a control plant of the same line or variety. [0550] In an aspect, a mutation comprises the integration of at least 5 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 10 contiguous nucleotides of a desired sequence molecule into a target sequence. In an aspect, a mutation comprises the integration of at least 15 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 20 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 25 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 50 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 100 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 250 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 1000 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 2000 contiguous nucleotides of a desired sequence into a target sequence. [0551] In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 3500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 2500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 1500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 750 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 250 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 150 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 25 contiguous nucleotides and 2500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 25 contiguous nucleotides and 1500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 25 contiguous nucleotides and 750 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 50 contiguous nucleotides and 2500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 50 contiguous nucleotides and 1500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 50 contiguous nucleotides and 750 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 100 contiguous nucleotides and 2500 contiguous nucleotides of a desired sequence into a target Sequence. In an aspect, a mutation comprises the integration of between 100 contiguous nucleotides and 1500 contiguous nucleotides of a desired sequence into a target Sequence. In an aspect, a mutation comprises the integration of between 100 contiguous nucleotides and 750 contiguous nucleotides of a desired sequence into a target Sequence. [0552] In an aspect, a method provided herein further comprises detecting an edit or a mutation in a target sequence. The screening and selection of mutagenized or edited plants or plant cells can be through any methodologies known to those having ordinary skill in the art. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PCR amplification for detection of a polynucleotide, Northern blots, RNase protection, primer- extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, Next Generation sequencing technologies (e.g., Illumina, PacBio, Ion Torrent, 454) enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the above-referenced techniques are known in the art. [0553] In an aspect, a sequence provided herein encodes at least one ribozyme. In an aspect, a sequence provided herein encodes at least two ribozymes. In an aspect, a ribozyme is a self- cleaving ribozyme. Self-cleaving ribozymes are known in the art. For example, see Jimenez et al., Trends Biochem. Sci., 40:648-661 (2015). [0554] In an aspect, a sequence encoding at least one guide nucleic acid is flanked by self-cleaving ribozymes. In an aspect, a sequence encoding at least one guide nucleic acid is immediately adjacent to a sequence encoding a ribozyme (e.g., the 5′-most nucleotide of the guide nucleic acid abuts the 3′-most nucleotide of the ribozyme or the 3′-most nucleotide of the guide nucleic acid abuts the 5′-most nucleotide of the ribozyme). In an aspect, a sequence encoding at least one guide nucleic acid is separated from a sequence encoding a ribozyme by at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 250, at least 500, or at least 10000 nucleotides. Plants [0555] Any plant or plant cell can be used with the methods and compositions provided herein. In an aspect, a plant is selected from the group consisting of a corn plant, a rice plant, a sorghum plant, a wheat plant, an alfalfa plant, a barley plant, a millet plant, a rye plant, a sugarcane plant, a cotton plant, a soybean plant, a canola plant, a tomato plant, an onion plant, a cucumber plant, an Arabidopsis plant, and a potato plant. In an aspect, a plant is an angiosperm. In an aspect, a plant is a gymnosperm. In an aspect, a plant is a monocotyledonous plant. In an aspect, a plant is a dicotyledonous plant. In an aspect, a plant is a plant of a family selected from the group consisting of Alliaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Brassicaceae, Caesalpiniaceae, Cucurbitaceae, Ericaceae, Fabaceae, Juglandaceae, Malvaceae, Mimosaceae, Moraceae, Musaceae, Orchidaceae, Papilionaceae, Pinaceae, Poaceae, Rosaceae, Rutaceae, Rubiaceae, and Solanaceae. [0556] In an aspect, a plant cell is selected from the group consisting of a corn cell, a rice cell, a sorghum cell, a wheat cell, an alfalfa cell, a barley cell, a millet cell, a rye cell, a sugarcane cell, a cotton cell, a soybean cell, a canola cell, a tomato cell, an onion cell, a cucumber cell, an Arabidopsis cell, and a potato cell. In an aspect, a plant cell is an angiosperm plant cell. In an aspect, a plant cell is a gymnosperm plant cell. In an aspect, a plant cell is a monocotyledonous plant cell. In an aspect, a plant cell is a dicotyledonous plant cell. In an aspect, a plant cell is a plant cell of a family selected from the group consisting of Alliaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Brassicaceae, Caesalpiniaceae, Cucurbitaceae, Ericaceae, Fabaceae, Juglandaceae, Malvaceae, Mimosaceae, Moraceae, Musaceae, Orchidaceae, Papilionaceae, Pinaceae, Poaceae, Rosaceae, Rutaceae, Rubiaceae, and Solanaceae. [0557] As used herein, a “variety” refers to a group of plants within a species (e.g., without being limiting Zea mays) that share certain genetic traits that separate them from other possible varieties within that species. Varieties can be inbreds or hybrids, though commercial plants are often hybrids to take advantage of hybrid vigor. Individuals within a hybrid cultivar are homogeneous, nearly genetically identical, with most loci in the heterozygous state. [0558] As used herein, the term “inbred” means a line that has been bred for genetic homogeneity. In an aspect, a seed provided herein is an inbred seed. In an aspect, a plant provided herein is an inbred plant. [0559] As used herein, the term “hybrid” means a progeny of mating between at least two genetically dissimilar parents. Without limitation, examples of mating schemes include single crosses, modified single cross, double modified single cross, three-way cross, modified three-way cross, and double cross wherein at least one parent in a modified cross is the progeny of a cross between sister lines. In an aspect, a seed provided herein is a hybrid seed. In an aspect, a plant provided herein is a hybrid plant. [0560] In some jurisdictions, products obtained exclusively by essentially biological processes, such as plant products are excluded from patent protection. Accordingly, the claimed plants, plant parts and cells and their progeny can be defined as directed only to those plants, plant parts and cells and their progeny which are obtained by technical intervention (regardless of any further propagation through crossing and selection). An embodiment of the invention is directed at plants, or plant parts or progeny produced or obtainable using gene editing technology herein described. Alternatively, the subject matter excluded from patentability may be disclaimed. An embodiment of the invention is directed at plants, part of plants or progeny thereof comprising the genomic alterations as elsewhere herein described, provided that the plants, parts or plants or progeny are not obtained exclusively through essentially biological processes, wherein essentially biological processes are processes for the production of plants or animals if they consist entirely of natural phenomena such as crossing or selection. Transformation [0561] Methods can involve transient transformation or stable integration of any nucleic acid molecule into any plant or plant cell provided herein. [0562] As used herein, “stable integration” or “stably integrated” refers to a transfer of DNA into genomic DNA of a targeted cell or plant that allows the targeted cell or plant to pass the transferred DNA to the next generation of the transformed organism. Stable transformation requires the integration of transferred DNA within the reproductive cell(s) of the transformed organism. As used herein, “transiently transformed” or “transient transformation” refers to a transfer of DNA into a cell that is not transferred to the next generation of the transformed organism. In a transient transformation the transformed DNA does not typically integrate into the transformed cell’s genomic DNA. In one aspect, a method stably transforms a plant cell or plant with one or more nucleic acid molecules provided herein. In another aspect, a method transiently transforms a plant cell or plant with one or more nucleic acid molecules provided herein. [0563] In an aspect, a nucleic acid molecule encoding a guided nuclease is stably integrated into a genome of a plant. In an aspect, a nucleic acid molecule encoding a Cas12a nuclease is stably integrated into a genome of a plant. In an aspect, a nucleic acid molecule encoding a CasX nuclease is stably integrated into a genome of a plant. In an aspect, a nucleic acid molecule encoding a guide nucleic acid is stably integrated into a genome of a plant. In an aspect, a nucleic acid molecule encoding a guide RNA is stably integrated into a genome of a plant. In an aspect, a nucleic acid molecule encoding a single-guide RNA is stably integrated into a genome of a plant. [0564] Numerous methods for transforming cells with a recombinant nucleic acid molecule or construct are known in the art, which can be used according to methods of the present application. Any suitable method or technique for transformation of a cell known in the art can be used according to present methods. Effective methods for transformation of plants include bacterially mediated transformation, such as Agrobacterium-mediated or Rhizobium-mediated transformation and microprojectile bombardment-mediated transformation. A variety of methods are known in the art for transforming explants with a transformation vector via bacterially mediated transformation or microprojectile bombardment and then subsequently culturing, etc., those explants to regenerate or develop transgenic plants. [0565] In an aspect, a method comprises providing a cell with a nucleic acid molecule via Agrobacterium-mediated transformation. In an aspect, a method comprises providing a cell with a nucleic acid molecule via polyethylene glycol-mediated transformation. In an aspect, a method comprises providing a cell with a nucleic acid molecule via biolistic transformation. In an aspect, a method comprises providing a cell with a nucleic acid molecule via liposome-mediated transfection. In an aspect, a method comprises providing a cell with a nucleic acid molecule via viral transduction. In an aspect, a method comprises providing a cell with a nucleic acid molecule via use of one or more delivery particles. In an aspect, a method comprises providing a cell with a nucleic acid molecule via microinjection. In an aspect, a method comprises providing a cell with a nucleic acid molecule via electroporation. [0566] In an aspect, a nucleic acid molecule is provided to a cell via a method selected from the group consisting of Agrobacterium-mediated transformation, polyethylene glycol-mediated transformation, biolistic transformation, liposome-mediated transfection, viral transduction, the use of one or more delivery particles, microinjection, and electroporation. [0567] Other methods for transformation, such as vacuum infiltration, pressure, sonication, and silicon carbide fiber agitation, are also known in the art and envisioned for use with any method provided herein. [0568] Methods of transforming cells are well known by persons of ordinary skill in the art. For instance, specific instructions for transforming plant cells by microprojectile bombardment with particles coated with recombinant DNA (e.g., biolistic transformation) are found in U.S. Patent Nos. 5,550,318; 5,538,880 6,160,208; 6,399,861; and 6,153,812 and Agrobacterium-mediated transformation is described in U.S. Patent Nos. 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871; 5,463,174; and 5,188,958, all of which are incorporated herein by reference. Additional methods for transforming plants can be found in, for example, Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Any appropriate method known to those skilled in the art can be used to transform a plant cell with any of the nucleic acid molecules provided herein. [0569] Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). [0570] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a nucleic acid molecule are as used in WO 2014/093622. In an aspect, a method of providing a nucleic acid molecule or a protein to a cell comprises delivery via a delivery particle. In an aspect, a method of providing a nucleic acid molecule to a plant cell or plant comprises delivery via a delivery vesicle. In an aspect, a delivery vesicle is selected from the group consisting of an exosome and a liposome. In an aspect, a method of providing a nucleic acid molecule to a plant cell or plant comprises delivery via a viral vector. In an aspect, a viral vector is selected from the group consisting of an adenovirus vector, a lentivirus vector, and an adeno-associated viral vector. In another aspect, a method providing a nucleic acid molecule to a plant cell or plant comprises delivery via a nanoparticle. In an aspect, a method providing a nucleic acid molecule to a plant cell or plant comprises microinjection. In an aspect, a method providing a nucleic acid molecule to a plant cell or plant comprises polycations. In an aspect, a method providing a nucleic acid molecule to a plant cell or plant comprises a cationic oligopeptide. [0571] In an aspect, a delivery particle is selected from the group consisting of an exosome, an adenovirus vector, a lentivirus vector, an adeno-associated viral vector, a nanoparticle, a polycation, and a cationic oligopeptide. In an aspect, a method provided herein comprises the use of one or more delivery particles. In another aspect, a method provided herein comprises the use of two or more delivery particles. In another aspect, a method provided herein comprises the use of three or more delivery particles. [0572] Suitable agents to facilitate transfer of nucleic acids into a plant cell include agents that increase permeability of the exterior of the plant or that increase permeability of plant cells to oligonucleotides or polynucleotides. Such agents to facilitate transfer of the composition into a plant cell include a chemical agent, or a physical agent, or combinations thereof. Chemical agents for conditioning includes (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. [0573] Organic solvents useful in conditioning a plant to permeation by polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, other solvents miscible with water or that will dissolve phosphonucleotides in non-aqueous systems (such as is used in synthetic reactions). Naturally derived or synthetic oils with or without surfactants or emulsifiers can be used, e. g. , plant-sourced oils, crop oils (such as those listed in the 9^th Compendium of Herbicide Adjuvants, publicly available on line at www(dot)herbicide(dot)adjuvants(dot)com) can be used, e. g. , paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine. [0574] Examples of useful surfactants include sodium or lithium salts of fatty acids (such as tallow or tallowamines or phospholipids) and organosilicone surfactants. Other useful surfactants include organosilicone surfactants including nonionic organosilicone surfactants, e. g. , trisiloxane ethoxylate surfactants or a silicone polyether copolymer such as a copolymer of polyalkylene oxide modified heptamethyl trisiloxane and allyloxypolypropylene glycol methylether (commercially available as Silwet® L-77). [0575] Useful physical agents can include (a) abrasives such as carborundum, corundum, sand, calcite, pumice, garnet, and the like, (b) nanoparticles such as carbon nanotubes or (c) a physical force. Carbon nanotubes are disclosed by Kam et. al. (2004) Am. Chem. Soc, 126 (22):6850-6851, Liu et. al. (2009) Nano Lett, 9(3): 1007-1010, and Khodakovskaya et. al. (2009) ACS Nano, 3(10):3221-3227. Physical force agents can include heating, chilling, the application of positive pressure, or ultrasound treatment. Embodiments of the method can optionally include an incubation step, a neutralization step (e.g., to neutralize an acid, base, or oxidizing agent, or to inactivate an enzyme), a rinsing step, or combinations thereof. The methods of the invention can further include the application of other agents which will have enhanced effect due to the silencing of certain genes. For example, when a polynucleotide is designed to regulate genes that provide herbicide resistance, the subsequent application of the herbicide can have a dramatic effect on herbicide efficacy. [0576] Agents for laboratory conditioning of a plant cell to permeation by polynucleotides include, e.g., application of a chemical agent, enzymatic treatment, heating or chilling, treatment with positive or negative pressure, or ultrasound treatment. Agents for conditioning plants in a field include chemical agents such as surfactants and salts. [0577] In an aspect, a transformed or transfected cell is a plant cell. Recipient plant cell or explant targets for transformation include, but are not limited to, a seed cell, a fruit cell, a leaf cell, a cotyledon cell, a hypocotyl cell, a meristem cell, an embryo cell, an endosperm cell, a root cell, a shoot cell, a stem cell, a pod cell, a flower cell, an inflorescence cell, a stalk cell, a pedicel cell, a style cell, a stigma cell, a receptacle cell, a petal cell, a sepal cell, a pollen cell, an anther cell, a filament cell, an ovary cell, an ovule cell, a pericarp cell, a phloem cell, a bud cell, or a vascular tissue cell. In another aspect, this disclosure provides a plant chloroplast. In a further aspect, this disclosure provides an epidermal cell, a guard cell, a trichome cell, a root hair cell, a storage root cell, or a tuber cell. In another aspect, this disclosure provides a protoplast. In another aspect, this disclosure provides a plant callus cell. Any cell from which a fertile plant can be regenerated is contemplated as a useful recipient cell for practice of this disclosure. Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, and the like. Those cells which are capable of proliferating as callus can serve as recipient cells for transformation. Practical transformation methods and materials for making 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. Patents 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 additional culturing steps, such as callus induction, selection, regeneration, etc., as known in the art. Transformed cells, tissues or explants containing a recombinant DNA insertion can be grown, developed or regenerated into transgenic plants in culture, plugs or soil according to methods known in the art. In one aspect, this disclosure provides plant cells that are not reproductive material and do not mediate the natural reproduction of the plant. In another aspect, this disclosure also provides plant cells that are reproductive material and mediate the natural reproduction of the plant. In another aspect, this disclosure provides plant cells that cannot maintain themselves via photosynthesis. In another aspect, this disclosure provides somatic plant cells. Somatic cells, contrary to germline cells, do not mediate plant reproduction. In one aspect, this disclosure provides a non-reproductive or non-regenerable plant cell. [0578] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure. [0579] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. [0580] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples. [0581] The following examples are included to demonstrate embodiments of the disclosure. It should be appreciated by those of skill in the art that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims. EXAMPLES Example 1: Nuclear localization Signals and linker for Cas12a [0582] This example describes the design of nuclear localization signals (NLS's) and optimized linkers that could be used to engineer targeting of RNA guided nucleases like Cas12a to nuclei of plant cells. NLS’s are generally short peptides that mediate the transport of proteins from the cytoplasm into the nucleus. There are two major import pathways into the nucleus: the classical pathway wherein the NLS peptide is recognized by the importin α subunit and the non-classical pathway wherein the NLS peptide is recognized by the importin β subunit. Many studies on nuclear localization of Cas12a have primarily used a classical NLS at the N-terminus and at the C- terminus. Recent articles propose stacking three to six NLS sequences to improve localization and cleavage activity (see Gier, R.A., Budinich, K.A., Evitt, N.H. et al. High-performance CRISPR- Cas12a genome editing for combinatorial genetic screening. Nat Commun 11, 3455 , 2020). The use of multiple NLS’s on each end or stacking suggests that little effort has been put into proper presentation of the NLS for efficient import. The focus of this study was the optimal presentation of NLS sequence to improve import and retain editing activity without the need for multiple copies of the NLS. Two approaches were taken to optimize candidates. The first approach was to test three NLS peptides (Table 1) that could be introduced to the termini of LbCas12a along with linker elements to facilitate optimal presentation of the NLS element. NLS1 belongs to Class one type of classic NLSs and NLS-HSFA is a truncated Class 6 type of classic NLS. Table1: NLS sequences to be tested with LbCas12a NLS name Plant optimized DNA SEQ Protein SEQ ID [0583] Direct fusion of the NLS peptide to the nuclease without a linker could lead to undesirable outcomes, including sub-optimal folding of the fusion protein, low protein accumulation, or reduced bioactivity. Therefore, the selection or rational design of a linker to join the NLS to the nuclease is important. Different linkers were modelled and positions for NLS signals were tested. NLS signals were placed on the N and C-terminal ends. In all cases, Alphafold was leveraged to model the sequences to determine how the sequences might fold and impact function. The version of alphafold was downloaded in February 2022 from: https://github.com/deepmind/alphafoldAll. The following command was used to run alphafold -- fasta_paths=/data/input/[seqname].fasta --max_template_date=2022-02-17 [0584] Public structures that had the nucleic acid in the Cas12a nuclease were leveraged to understand what impact the additions would have. Specifically, Protein Data Bank (PDB) structures with accession numbers PDB: 6I1L (FnCas12-crRNA-TS-only complex) and PDB :6NME (Structure of LbCas12a-crRNA) was used for this analysis ( see Swarts et. al., Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a. Mol. Cell. ,2019; and Zhange et al., Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins, Cell Host and Microbe, 25:6 pg 815-826, 2019 ). Additionally, eight Cas12a fusion protein designs comprising NLS, linkers and YFP (Yellow flourescent protein) were modelled and evaluated via alphafold. These designs were (1) NLS_Cas12a_eYFP; (2) eYFP_Cas12a_NLS; (3) NLS_linker_Cas12a_linker_eYFP; (4) eYFP_linker_Cas12a_linker_NLS; (5) NLS_HSFA1_linker_Cas12a_linker_eYFP; (6) eYFP_linker_Cas12a_HSFA1_NLS_HSFA1_NLS; (7) Class1_NLS_Cas12a_linker_eYFP; and (8) eYFP_linker_Cas12a_HSFA1_NLS. [0585] Based on the analysis described above, six linker designs with varying lengths and flexibility were subsequently chosen for testing (See Table 2) . Linker L1 was a flexible linker GGSG with small or hydrophilic amino acids. Linkers L2 to L4 were linkers with increasing length and rigidity and comprised the core amino acid sequence (EAAAK) (PROT SEQ ID NO: 18; DNA SEQ ID NO: 6) flanked by flexible linker peptides. Linker L5 comprised the core amino acid sequence YETKQ (PROT SEQ ID NO:19; DNA SEQ ID NO: 7) flanked by flexible linker peptides. Linker L6 comprised the core amino acid sequence PVTAT(PROT SEQ ID NO:20; DNA SEQ ID NO: 8) flanked by flexible linker peptides . Modelling studies suggested that many of these linkers could create an alpha helical structure, causing the NLS to be further from Cas12a/Cpf1. Table2: Linker sequences to be tested with LbCas12a Linker Linker structure Linker Linker DNA Protein SEQ [05 ons where Alphafold generated a less confident model for Cas12a were areas that were targeted for embedding the NLS. In addition, regions that were unresolved in the crystal structures were also targeted. The hypothesis was that those areas may already be flexible and unstructured so modifying them to include the NLS may not make a difference in the function of the protein. The LbCas12a protein sequence (SEQ ID NO: 33) was interrogated and four regions : positions 370..379 (SEQ ID NO: 74), positions 449 to 461 (SEQ ID NO: 53) and position 487 to 496 (SEQ ID NO: 54) and position 1076..1085(SEQ ID NO: 73) were identified as potential sites to modify and embed an NLS sequence. Table 3: Motifs in LbCas12a that were modified to embed NLS sequences. Class 1 NLS conserved motif sequence is underlined. Nuclease/Embedded AA SEQ at AA SEQ at position AA SEQ at AA SEQ at LbCas12a_E1_E2 Same as WT DADFGRKRKRGGN GEGKKRKRDE Same as WT (SEQ ID : 36) (SEQ ID :55) (SEQ ID :56) p g . e 3. In LbCas12a_E1 variant, the amino acid sequence at position 449..461 was modified to SEQ ID NO:55 to embed an NLS peptide. In LbCas12a_E2 variant, the amino acid sequence at position 487..496 was modified to SEQ ID NO:56 to embed an NLS peptide. In LbCas12a_E1_E2 variant, the amino acid sequence at position 449..461 was modified to SEQ ID NO:55 and position 487..496 was modified to SEQ ID NO:56 such that the resulting protein comprised two embedded NLS sequences. In LbCas12a_E3 variant, the amino acid sequence at position 487..496 was modified to SEQ ID NO:14 to embed an HSFA-NLS peptide. In LbCas12a_E4 variant, the amino acid sequence at position 1076..1085 was modified to SEQ ID NO:75 to embed an NLS peptide. In LbCas12a_E5 variant, the amino acid sequence at position 370..379 was modified to SEQ ID NO:76 to embed an NLS peptide. Specifically, K374R, A375R amino acid substitutions were made in LbCas12a so as to create the LbCas12a-E5 variant. Example 2: Nuclear localization of select LbCas12a NLS designs. [0588] This example describes the cellular localization analysis of LbCas12a NLS fusions and variants fused to eYFP(enhanced Yellow Fluorescent Protein) (SEQ ID NO:63) .Twelve expression constructs were designed and described in Table 4. Each construct comprised an LbCas12a fusion sequence operably fused to an enhanced 35S Promoter regulatory element (SEQ ID NO:57) and an Agrobacterium NOS(Nopaline Synthase) terminator sequence (SEQ ID NO: 64). A six nucleotide ATGGCG sequence encoding Met-Ala was introduced at the start of the sequence encoding the fusion protein. Table 4: YFP-LbCas12a cassette designs. Construct Design LbCas12a cassette design LbCas12a LbCas12a D 3: 7: 3: 1: 3: 7: 3: 2: 3: 7 3: 3: 5: 1: 3: 7: 5: 2: 3: 7: pM290 6 NLS1:L4:LbCas12a: L1: YFP SEQ ID 3: SEQ SEQ ID 15: ID 11: SEQ ID SEQ ID 23: 3: 7: 3: 7: 3: 4: 3: 7: 3: 5: 3: 7: 7 3: 7: 8 3: 7: 4 2: 7: 5 [0589] Corn leaf protoplasts were transformed with one of the twelve constructs described above using standard polyethylene glycol (PEG) mediated transformation. Four replicates were performed for each sample. A construct comprising an expression cassette for an RNA Binding protein known to localize to the nucleus was transformed as a positive control . Cells treated with transformation media lacking any DNA served as the negative control. After overnight incubation, a portion of the transformed corn leaf protoplasts were transferred into 384 wells imaging plate and imaged in high throughput microplate imager Operetta. The rest of protoplasts were fixed and stained by nuclei marker DAPI and imaged by confocal microscope to confirm the nuclear localization of the Cas12a protein. The images captured from high throughput microplate imager Operetta were analyzed by Operetta high content analysis system. Nuclear fluorescence intensity as well as nuclear fluorescence positive cells were calculated and found via establishing proper analysis blocks in the system. The data analysis is summarized in Figure 4 (A and B) . All tested designs showed nuclear accumulation of the fusion protein albeit at different levels. C terminal NLS designs (Designs 1-3) show better nuclear accumulation than N-terminal NLSs (Designs 4- 6). Designs comprising Linkers L5 and L6 also showed good nuclear accumulation (Design 7 and 8). All the modified LbCas12a protein variants with embedded NLS showed some level of nuclear accumulation suggesting that the embedded NLS sequences were capable and sufficient for targeting the protein to the nucleus. Among the four embedded NLS designs tested, LbCas12a_E1( Design 11) and LbCas12a_E2 (Design 12) had greater number of cells showing nuclear localization and also showed higher levels of nuclear accumulation per cell. These two designs were therefore advanced for further testing as described in Examples 3 and 4. Example 3: LbCas12a cassette designs and vectors for protoplast assays [0590] This example describes corn protoplast experiments designed to test the targeted cleavage activity of LbCas12a fusion proteins/variants comprising the NLS sequences with and without the linkers described in Example 1. Ten Cas12a expressing constructs were designed (see Table 5) . Each construct comprised an LbCas12a nuclease cassette comprising the LbCas12a variant/fusion sequence operably linked to an enhanced 35S Promoter (SEQ ID NO:57) and an Agrobacterium NOS terminator sequence (SEQ ID NO:64) and a selectable marker cassette. Table 5: Cas12a constructs and cassette designs. Construct Design LbCas12a cassette design LbCas12a LbCas12a name (35SProm ::LbCas12a:: fusion/variant fusion/variant s D D Q D D D D D [0591] Three independent corn target sites (see Table 6) for LbCas12a gRNAs were selected and gRNA expressing constructs were generated. Two gRNA cassette designs were utilized and six gRNA constructs were designed. In the first design, the gRNA expression cassette comprised a synthetic Pol III promoter GSP2273 (SEQ ID NO: 61) operably linked to a transcribable sequence comprising, in order: a Cas12a-compatible Direct repeat (DR) sequence (SEQ ID NO: 40) and a spacer listed in Table 6 followed by a polyT termination sequence. The second design was similar except that the spacer sequence was followed by a second Direct repeat (DR) sequence and the polyT terminator sequence. Table 6: Cas12a targets sites and cognate gRNA spacers for protoplast assays Target SEQ ID Spacer Table 7a: gRNA constructs for protoplast assays. All gRNAs were operably linked to a synthetic PolIII promoter and had a poly T terminator sequence. Construct name Target gRNA design gRNA SEQ ID NOs 0: 0: [0592] The nuclease and gRNA expression vectors disclosed in Table 5 and Table 7a were co- delivered into corn protoplasts using standard polyethylene glycol (PEG) mediated transformation. Four replicates were performed for each sample. For quantifying transformation frequency, a vector comprising a luciferase expression cassette was also co-delivered. Genomic DNA was isolated from the protoplast cells after transfection and incubation and target regions were amplified by PCR. The amplicons were sequenced by Next Generation Sequencing (NGS), using standard methods known in the art to identify modified sequences comprising insertions or deletions (InDels) around the three target sites that are indicative of editing. The editing rates were calculated by the number of reads containing an InDel compared to the total number of mappable reads. The editing rates of the ten Cas12a fusion proteins or variants at the three target sites are summarized in Figures 2-4. All LbCas12a designs comprising terminal NLSs with and without linkers (designs 1- 7) showed detectable editing rates at all tested target sites though the rates were variable. One of the modified LbCas12a proteins with an embedded NLS at positions 487..496 (LbCas12a_E2; design 9) showed considerable editing activity at all three target sites while the other two variants LbCas12a_E1(design 7) and LbCas12a_E1_E2 (design 10) did not show any edits suggesting that these modifications may led to significant loss of activity. Interestingly, as shown in Figure 3 , the LbCas12a-E2 variant showed the highest editing activity at ZmTS3 among all tested designs. The largest contributions to improving editing was gRNA design two which was used for subsequent in planta testing. Example 4: LbCas12a cassette designs and vectors for protoplast assays [0593] This example describes corn in planta experiments designed to test the targeted cleavage activity of LbCas12a fusion proteins or variants comprising the NLS sequences with and without the linkers described in Example 1. Twelve agrobacterium T-DNA constructs were generated. Each construct comprised a Cas12a nuclease cassette described in Table 7b, a gRNA array cassette targeting four unique corn genomic sites and a selectable marker cassette. Table 7b: Cas12a constructs and cassette designs. Construc Desig LbCas12a cassette design LbCas12a LbCas12a n Q 4: 3: 3: 7: SEQ ID 1: SEQ ID 14: SEQ ID 2 SEQ ID 14 3: 4: 7: 3: 4: 7: O: O: 5: 1: 5: 4: 5: 3: 3: 5: 3: 1: pM873 12 LbCas12a: L6: NLS1 SEQ ID 26: SEQ ID 33: SEQ ID 13: SEQ ID 25: [0594] Each vector had a functional cassette for the expression of Cas12a.The Cas12a expression cassette comprised the fusion Cas12a gene described in Table 7b operably linked to the 3’ end of a constitutive maize Ubiquitin promoter P-Zm.UbqM1 (SEQ ID NO: 77) and operably linked to the 5’ end of a transcription terminator sequence from a rice Lipid transfer protein (LTP) gene (SEQ ID NO: 78). Each construct comprised a gRNA array cassette targeting three unique corn target sites (see Table 8). The gRNA expression cassette comprised a synthetic Pol III promoter GSP2262 (SEQ ID NO: 83) operably linked to a transcribable sequence comprising, in order: a Cas12a-compatible Direct repeat (DR) sequence (SEQ ID NO: 40) : spacer SP4 (SEQ ID NO 46): a DR: spacer SP5 (SEQ ID NO: 47) a DR:: spacer SP6 (SEQ ID NO: 44) and a DR. The transcribable portion of the transcript is a pre-crRNA precursor RNA that can be processed by Cas12a into three copies of mature SP4, SP5, and SP6 guide RNAs. The T-DNA vector also comprised an expression cassette for the selectable marker CP4 conferring resistance to the herbicide glyphosate. Table 8: Cas12a targets sites and cognate gRNA spacers for protoplast assays [0595] Corn 01DKD2 cultivar embryos were transformed with the vectors described in Table 9 by agrobacterium-mediated transformation and R0 plants were regenerated from the transformed corn cells. DNA was extracted from leaf samples from 84 R0 seedlings per construct. Primers flanking Target sites TS4 to TS6 were amplified by PCR. The amplicons were sequenced by Next Generation Sequencing (NGS), using standard methods known in the art to identify modified sequences comprising insertions or deletions (InDels) around the four target sites that are indicative of editing. If a plant had a target site mutation and if greater than 10% of the amplicon reads covering the loci contained that mutation, it was identified as an edited plant. The transformations were done in three batches and for each batch, pM471 (Design 1) served as a control against which to measure edit rates. Table 9: Summary of edit rates at ZmTS4-ZmTS6 in R0 plants. Transformation batch 1 Construct ZmTS6 ZmTS4 ZmTS5 [0596] As shown in Table 9, several Cas12a designs were able to produce more edits in multiple loci as compared to the control Design 1. For instance , designs with two copies of the NLS-HSFA at the C terminus of Cas12a with a short flexible linker(Design 2 and Design 3) showed better editing efficiency at all three target sites than Design 1 which had an N and C terminal NLS- HSFA. Design with two C terminal copies of the NLS-HSFA( Design 2) also performed better than similar design 10 with two C terminal copies of NLS1. Design 12 comprising the L6 linker and a single copy of NLS1 and Design 11 comprising the L2 linker and a single copy of NLS1 also showed higher editing frequency at all three target sites compared to designs with multiple NLSs. [0597] The construct pM875 (design 5) comprising the LbCas12a_E2 embedded NLS , showed higher editing at ZmTS6 than the control. Combining the LbCas12a_E2 with a C terminal L2 linker and NLS1 (design 8), led to further improvement in editing frequencies at 2 of the 3 tested sites as compared to the control. The construct pM874(design 6) comprising the LbCas12a_E3 embedded design showed weaker editing activity than LbCas12a_E2 and a complete loss of activity atZmTS5. The E2 and E3 designs comprised modifications at the same region in LbCas12a suggesting that embedding an NLS into Cas12a may have to be empirically determined based upon the amino acid sequence of the NLS. Example 5: Nuclear localization of LbCas12a with classical NLS. [0598] This example describes the use of classical NLSs (cNLS) spanning five different classes that could be used to engineer targeting of proteins like RNA guided nucleases to nuclei of plant cells. Table 10 discloses six synthetic NLS peptides that were introduced to the termini of LbCas12a along with linker elements to facilitate optimal presentation of the NLS element. The sequences of these six synthetic NLS peptides were designed based on the approaches described by Kosugi et. al. J Biol Chem 2009; 284:478-85, for example, the use of weight matrices (PWMs). Table 10: cNLS sequences cNLS NLS Class Plant optimized Protein SEQ ID [0599] Eight expression constructs were designed as described in Table 11. Each construct comprised a YFP-LbCas12a-cNLS fusion sequence operably fused to an enhanced 35S Promoter regulatory element (SEQ ID NO:57) and a transcription terminator sequence from a rice Lipid transfer protein (LTP) gene (SEQ ID NO: 78). A six nucleotide ATGGCG sequence encoding Met-Ala was introduced 5’ to the YFP sequence corresponding to the start of the fusion protein coding sequence. Table 11: YFP-LbCas12a-NLS cassette designs. Construct LbCas12a cassette design LbCas12a LbCas12a name (35S r m::YFP-Cas12a- fusion fusion Q 3: 7: 3: 5: 3: 7: 3: 5: 3: 7: 3: 5: 3: 7: 3: 5: 3: 7: SEQ ID 26: SEQ ID 33: SEQ ID 13: SEQ ID 25: 3: 7: 3: 5: 3: 7: 3: 5: 3: 7: 3: 5: cribed above using standard polyethylene glycol (PEG) mediated transformation. Four replicates were performed for each sample. After overnight incubation, a portion of the transformed corn leaf protoplasts were transferred into 384 wells imaging plate and imaged in high throughput microplate imager Operetta. The rest of protoplasts were fixed and stained by nuclei marker DAPI and imaged by confocal microscope to confirm the nuclear localization of the Cas12a protein. The images captured from high throughput microplate imager Operetta were analyzed by Operetta high content analysis system. Nuclear fluorescence intensity as well as nuclear fluorescence positive cells were calculated and found via establishing proper analysis blocks in the system. The data analysis is summarized in Figure 5 (A and B) . All tested designs showed nuclear accumulation of the fusion protein albeit at different levels. The data generated demonstrates functionality of putative NLS sequences confirming that they are capable of localizing proteins to the nucleus of plant cells. Example 6: LbCas12a-cNLS cassette designs and vectors for protoplast assays [0601] This example describes corn protoplast experiments designed to test the targeted cleavage activity of LbCas12a fusion proteins comprising the classic NLS sequences described in Example 5. Eight expression constructs were generated. Each construct comprised two cassettes: an LbCas12a nuclease cassette comprising the LbCas12a fusion sequence as described in Table 12 operably linked to an enhanced 35S Promoter (SEQ ID NO:57) and a transcription terminator sequence from a rice Lipid transfer protein (LTP) gene (SEQ ID NO: 78) and a second cassette encoding a selectable marker. Table 12: LbCas12a-cNLS constructs and cassette designs. Construct LbCas12a cassette design LbCas12a LbCas12a D D D D D D D D pM659 LbCas12a: L6: NLS6(11) SEQ ID 26: SEQ SEQ ID 33: SEQ ID ID 13: SEQ ID 88 25: SEQ ID 94 n vectors pM790 targeting the ZmTS1 target site or pM791 targeting the ZmTS2 target site as disclosed in Table 5 and Table 7a using standard polyethylene glycol (PEG) mediated transformation. Four replicates were performed for each sample. For quantifying transformation frequency, a vector comprising a luciferase expression cassette was also co-delivered. Genomic DNA was isolated from the protoplast cells after transfection and incubation and target regions were amplified by PCR. The amplicons were sequenced by Next Generation Sequencing (NGS), using standard methods known in the art to identify modified sequences comprising insertions or deletions (InDels) around the three target sites that are indicative of editing. The editing rates were calculated by the number of reads containing an InDel compared to the total number of mappable reads. The editing rates of the eight Cas12a-cNLS fusion proteins at the two target sites are summarized in Table 13. All LbCas12a designs comprising terminal cNLSs showed detectable editing rates at both tested target sites. This suggests that all cNLS peptides were capable of targeting the Cas12a protein to the nuclease so as to cleave the cognate target site. Table 13: Summary of edit rates at ZmTS1 and-ZmTS2 Construct LbCas12a-cNLS cassette ZmTS1 ZmTS2 pM657 LbCas12a: L6: NLS5 10.63% 20.97% Example 7: Nuclear localization with non-classic NLS. [0603] This example describes the identification and validation of non classic NLSs (ncNLS) that could be used to engineer targeting of proteins to the nuclei of plant cells. [0604] Unlike classic nuclear localization signals, non-classic pathway NLS peptides engage the beta-importin (also known as karyopherin β) instead of the alpha-importin pathways for nuclear import. Diversity and complexity of signals recognized by Kapβs have prevented prediction of new Kapβ substrates and subsequent identification of the NLSs in candidate import substrates. The few characterized ncNLSs which are mostly from mammalian proteins, are diverse and encompass both structural domains and linear epitopes (see Lu, J., et al. Cell Commun Signal 19, 60 (2021). One of the best characterized Kapβ substrate is the human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). hnRNP A1 ncNLS belongs to the “proline-tyrosine” category, named PY-NLS. It assumes a disordered structure consisting of N-terminal hydrophobic or basic motifs and C-terminal R/K/H(X)2-5PY motifs (where X2-5 is any sequence of 2–5 residues). Other Kapβ substrates include ribosomal proteins, transcription factors and splicing factors. Characterized ncNLSs are longer than classic NLS’s, ranging from >20 amino acids and up to ~70 amino acids in length compared to 7-17 average length for classic NLS’s. [0605] Five mammalian Kapβ substrate proteins comprising ncNLS were sequence analysed against the corn genome database to identify putative homologs in corn. Factors like conserved amino acid residues and modelling was used to narrow down regions within the protein that could function as NLSs. The putative corn ncNLS sequences were extracted from the corn homologs and are disclosed in Table 14. Table 14: Zea mays ncNLS sequences Human protein Putative corn Similarity ncNLS ncNLS ncNLS homolog (PROT to human name SEQ ID SEQ ID [0606] Five expression constructs were designed to test the cellular localization of fusion proteins comprising the ncNLSs . As described in Table 15, each construct comprised an ncNLS described in Table 14 fused 5’ to a sequence encoding an eYFP(enhanced Yellow Fluorescent Protein) (SEQ ID NO:63) followed by a sequence encoding for an optimized bacterial beta-glucuronidase (GUS protein) (SEQ ID NO: 106). A six bp nucleotide sequence ATGGCG encoding Met-Ala was introduced 5’ to the start of the sequence encoding the fusion protein. Each cassette also comprised linker elements to facilitate optimal folding of proteins and optimal presentation of the NLS element. The coding sequence for the fusion protein was operably fused to an enhanced 35S Promoter regulatory element (SEQ ID NO:57) and an Agrobacterium NOS terminator sequence (SEQ ID NO:64). Table 15: ncNLS-YFP-GUS cassette designs. Construct ncNLS fusion cassette design Fusion Fusion name (35S ::ncNLS-YFP-GUS rotein rotein Q 0: 7: 3: 7: 1: 7: 3: 7: 2: 7: 3: 7: 3: 7: 3: 7: pMON957 ncNLS5:L1:YFP:L1:GUS SEQ ID 99: SEQ ID 104: SEQ ID 5: SEQ ID 17: 3: 7: ribed above . plasts were subsequently fixed, stained by nuclei marker DAPI and imaged by confocal microscope to determine the cellular localization of the YFP-GUS fusion proteins. Nuclear localized YFP signals were observed for all five proteins indicating that all ncNLS sequences were capable of directing the nuclear targeting of the GUS-YFP fusion protein. [0608] The next set of experiments tested the ability the ncNLS peptides to target LbCas12a to the nuclei of plant cells. Five expression constructs were generated. Each construct comprised a YFP-LbCas12a-ncNLS fusion sequence operably fused to an enhanced 35S Promoter regulatory element (SEQ ID NO:57) and a transcription terminator sequence from a rice Lipid transfer protein (LTP) gene (SEQ ID NO: 78). A six nucleotide ATGGCG sequence encoding Met-Ala was introduced 5’ to the YFP sequence corresponding to the start of the fusion protein coding sequence. L1 and L6 linker elements was incorporated into the cassette design to facilitate optimal presentation of YFP and the NLS elements. Table 16: LbCas12a-ncNLS constructs and cassette designs. Construct LbCas12a cassette desi n LbCas12a LbCas12a D pM455 YFP:L1:LbCas12a: L6: ncNLS2 SEQ ID 62: SEQ SEQ ID 63: SEQ ID 5: SEQ ID 26: ID 17: SEQ ID 33: D 3: D D 3: D [0609] Corn leaf protoplasts were transformed with one of the five constructs described above using standard polyethylene glycol (PEG) mediated transformation. Four replicates were performed for each sample. A construct comprising an expression cassette for an RNA Binding protein known to localize to the nucleus was transformed as a positive control . A construct comprising an expression cassette for Green Fluorescent protein (GFP) served as the flourescent protein control. After overnight incubation, a portion of the transformed corn leaf protoplasts were transferred into 384 wells imaging plate and imaged in high throughput microplate imager Operetta. The rest of protoplasts were fixed and stained by nuclei marker DAPI and imaged by confocal microscope to confirm the nuclear localization of the YFP-LbCas12a protein. The images captured from high throughput microplate imager Operetta were analyzed by Operetta high content analysis system. The percentage of cells with fluorescent nuclei in transformed protoplasts was calculated and is summarized in Figure 6. All tested designs showed nuclear accumulation of the fusion protein. This shows that the novel ncNLS sequences disclosed in Table 14 can target heterologous proteins like GUS, YFP and Cas12a into the nuclei of plant cells. Example 8: LbCas12a-ncNLS cassette designs and vectors for protoplast assays [0610] This example describes corn protoplast experiments designed to test the targeted cleavage activity of LbCas12a fusion proteins comprising four non classic NLS sequences described in Example 7. Four expression constructs were generated. Each construct comprised two cassettes. The first was an LbCas12a nuclease cassette comprising the LbCas12a fusion sequence as described in Table 16 operably linked to an enhanced 35S Promoter (SEQ ID NO:57) and an Agrobacterium NOS terminator sequence (SEQ ID NO:64). An L6 linker element was incorporated into the cassette design to facilitate optimal presentation of the NLS element. The second cassette encoded a selectable marker. Table 17: LbCas12a-ncNLS constructs and cassette designs. Construct LbCas12a cassette design LbCas12a LbCas12a D 0 2 D D [0611] Each nuclease vector was co-delivered in corn protoplasts with a gRNA expression vector pM790 targeting the ZmTS1 target site previously disclosed in Table 7a using standard polyethylene glycol (PEG) mediated transformation. Simultaneous transformations were carried out with LbCas12a fusion proteins comprising classic NLSs NLS-HSFA(pM652) and NLS1 (pM771). These served as positive controls for detecting editing efficacy. Four replicates were performed for each sample. For quantifying transformation frequency, a vector comprising a luciferase expression cassette was also co-delivered. Genomic DNA was isolated from the protoplast cells after transfection and incubation and target regions were amplified by PCR. The amplicons were sequenced by Next Generation Sequencing (NGS), using standard methods known in the art to identify modified sequences comprising insertions or deletions (InDels) around the two target sites that are indicative of editing. The editing rates were calculated by the number of reads containing an InDel compared to the total number of mappable reads. The editing rates of the four Cas12a-ncNLS and two Cas12a-cNLS fusion proteins at the ZmTS1 target site are summarized in Table 17. All LbCas12a designs comprising terminal ncNLSs showed detectable editing rates suggesting that the novel ncNLS peptides were capable of targeting the Cas12a protein to the nucleus so as to cleave the cognate target site. Table 18: Summary of edit rates at ZmTS1. Construct LbCas12a-cNLS cassette ZmTS1

Claims

CLAIMS 1. A ribonucleoprotein complex comprising: a) an RNA guided polypeptide comprising: i) an effector polypeptide which is or is derived from a Crispr/CAS protein; ii) one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a terminus of the effector polypeptide; b) at least one guide RNA; wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are connected to the effector polypeptide through a linker amino acid sequence comprising GGSG or GGGS.

2. The ribonucleoprotein complex according to claim 1, wherein the linker further comprises at least two or more repeats of the amino acid sequence EAAAK (SEQ ID NO: 18), or at least four or more repeats of the amino acid sequence EAAAK (SEQ ID NO: 18), or at least six repeats of the amino acid sequence EAAAK (SEQ ID NO: 18), or wherein the linker comprises an amino acid sequence selected from the group of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.

3. The ribonucleoprotein complex according to claim 1, wherein the linker comprises an amino acid sequence selected from SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 24 or SEQ ID NO: 25.

4. The ribonucleoprotein complex according to claim 1, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise a nuclear localization signal (NLS) selected from a Class 1 nuclear localization signal having the formula KR(K/R)R or K(K/R)RK, a Class 2 nuclear localization signal having the formula (P/R)XXKR(^DE)(K/R), a Class 3 nuclear localization signal having the formula KRX(W/F/Y)XXAF, a Class 4 nuclear localization signal having the formula (R/P)XXKR(K/R)(^DE), a Class 5 nuclear localization signal having the formula LGKR(K/R)(W/F/Y) or a Class 5 nuclear localization signal having the formula KRX_[10- 12]K(KR)(KR)

5. The ribonucleoprotein complex according to claim 1, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell comprise at least one copy of an NLS amino acid sequence from a tomato Heat-shock inducible protein HSFA1 (HsFA NLS), optionally comprising the amino acid sequence of SEQ ID No: 14, or wherein the NLS sequence comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 ,SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103,and SEQ ID NO: 104

6. The ribonucleoprotein complex according to claim 1, wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are connected to the effector polypeptide at the C-terminus.

7. The ribonucleoprotein complex according to of claim 1, wherein the effector polypeptide is or is derived from a Type II or Type V Crispr/Cas protein or wherein the effector polypeptide is or is derived from a Cas9 polypeptide or a Cas12a polypeptide.

8. The ribonucleoprotein complex according to claim 1, wherein the RNA guided polypeptide has an amino acid sequence having at least 90% or 95% or 99% or 100% sequence identity to an amino acid selected from the group consisting of a) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 17, SEQ ID NO:14and SEQ ID NO: 14; b) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:14, SEQ ID NO: 17, and SEQ ID NO:14; c) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:14, SEQ ID NO: 17:, SEQ ID NO:15; d) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 21 and SEQ ID NO: 15; e) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 22 and SEQ ID NO: 15; f) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 23 and SEQ ID NO: 15; g) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 24 and SEQ ID NO: 15; and h) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO: 25 and SEQ ID NO: 15. i) an amino acid sequence comprising in order SEQ ID NO: 35, SEQ ID NO:21, SEQ ID NO: 15; j) an amino acid sequence comprising in order SEQ ID NO: 35, SEQ ID NO:24, SEQ ID NO: 15; k) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:21, SEQ ID NO: 15; l) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 15; m) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 14; n)an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 15; o) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 89; p) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 90; q) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 91; r) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 92; s) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 93; t) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 94; u) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 100; v) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 101; w) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 102;. x) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 103; and y) an amino acid sequence comprising in order SEQ ID NO: 33, SEQ ID NO:25, SEQ ID NO: 104.

9. A ribonucleoprotein complex comprising: a) an RNA guided polypeptide comprising: i) an effector polypeptide derived from a Crispr/CAS protein; ii) one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell; b) at least one guide RNA; wherein the one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell are embedded within the effector polypeptide in an exposed loop of the Crispr/Cas protein.

10. The ribonucleoprotein complex according to claim 9, wherein the effector protein is or is derived from Cas12a and the exposed loop corresponds to the amino acid sequence from position 487-496 of the amino acid sequence of SEQ ID NO: 33.

11. The ribonucleoprotein complex according to claim 9, wherein the effector protein has an amino acid sequence having at least 90% or 95% or 99% or 100% sequence identity to a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid of SEQ ID NO: 34, the amino acid of SEQ ID NO: 35, the amino acid of SEQ ID NO: 36, the amino acid of SEQ ID NO: 37, the amino acid of SEQ ID NO: 67 and the amino acid of SEQ ID NO: 68.

12. The ribonucleoprotein complex according to claim 9, further comprising one or more heterologous polypeptides that facilitate uptake of the RNP complex into the nucleus of a eukaryotic cell located at or near or in proximity to a C-terminus of the effector polypeptide through a linker amino acid sequence comprising GGSG or wherein the linker comprises an amino acid sequence selected from the group of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.

13. A recombinant DNA molecule comprising the following operably linked DNA fragments: a) a promoter expressible in a eukaryotic cell; b) a DNA fragment encoding an RNA guided polypeptide according to any one of claims 1 to 12; c) optionally a transcription termination and/or polyadenylation signal.

14. The recombinant DNA molecule according to claim 13 wherein the promoter is a plant- expressible promoter selected from constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue-specific promoters or is selected from a meiotic promoter, an egg cell-preferred or embryo-tissue preferred promoter such as a DSUL1 promoter, an EA1 promoter, an ES4 promoter, a DMC1 promoter, a Mps1 promoter, an Adf1 promoter or an EAL promoter, or is a floral-tissue preferred or floral cell-preferred promoter.

15. A method for editing the genome of a eukaryotic cell at at least one target site in the eukaryotic cell comprising the step of providing the eukaryotic cell with, or introducing into the eukaryotic cell, one or more ribonucleoprotein complexes according to any one of claims 1 to 12.

16. A method for editing the genome of a eukaryotic cell at at least one target site in the eukaryotic cell comprising a. providing the eukaryotic cell with, or introducing into the cell, one or more recombinant DNA molecules according to claim 13 or claim 14; b. providing the eukaryotic cell with, or introducing into the eukaryotic cell at least one guide RNA or a nucleic acid encoding at least one guide RNA comprising a complementarity region to the nucleotide sequence of the at least one target site of the eukaryotic cell; c. optionally, further introducing or providing a donor template into the eukaryotic cell.

17. The method according to claim 16, wherein the eukaryotic cell is selected from an in vitro eukaryotic cell, an animal cell, a fungal cell or a plant cell, optionally further comprising the step of regenerating a plant from the plant cell.

18. A eukaryotic cell comprising one or more ribonucleoprotein complexes according to claim 1 to 12 or one or more recombinant DNA molecules according to claim 13 or claim 14.

19. The eukaryotic cell according to claim 18, wherein the eukaryotic cell is selected from an in vitro eukaryotic cell, an animal cell, a fungal cell or a plant cell.

20. A plant comprising a plant cell or consisting essentially of a plant cells of claim 19.

21. A fusion protein comprising at least two polypeptide domains linked by a linker polypeptide comprising the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20 or SEQ ID NO: 24 or SEQ ID NO: 25.

22. A method for inserting a heterologous polypeptide sequence in a Cas12a protein comprising a) identifying an exposed loop in said Cas12a protein b) inserting a DNA sequence encoding said heterologous polypeptide into a DNA sequence encoding the exposed loop of said Cas12a protein.

23. The method according to claim 22, wherein said exposed loop corresponds to the amino acid sequence from position 449 to 461 or the amino acid sequence from position 487 to496 of the amino acid sequence of SEQ ID NO: 33.

24. A modified Cas12a protein comprising a heterologous polypeptide in an exposed loop, obtainable by the method of claim 22.

25. A modified Cas12a protein comprising a heterologous polypeptide at the amino acid sequence corresponding to the amino acid sequence from position 449 to 461 or the amino acid sequence from position 487 to 496 of the amino acid sequence of SEQ ID NO: 33.

26. A heterologous polypeptide that facilitates uptake of a protein into the nucleus of a eukaryotic cell, such as a plant cell, comprising an amino acid sequence having at least 95% or at least 96% or at least 97% or at least 98% or at least 99 % or 100% sequence identity to an amino acid selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 90; SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 94.

27. A heterologous polypeptide that facilitates uptake of a protein into the nucleus of a eukaryotic cell, such as a plant cell, comprising an amino acid sequence having at least 95% or at least 96% or at least 97% or at least 98% or at least 99 % or 100% sequence identity to an amino acid selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 101; SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104.

28. A nucleic acid encoding the heterologous polypeptide of claim 26.

29. The nucleic acid of claim 28 comprising a nucleotide sequence having at least 95% or at least 96% or at least 97% or at least 98% or at least 99 % or 100% sequence identity to a nucleic acid selected from the group consisting of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO: 88.

30. A nucleic acid encoding the heterologous polypeptide of claim 27.

31. The nucleic acid of claim 30 comprising a nucleotide sequence having at least 95% or at least 96% or at least 97% or at least 98% or at least 99 % or 100% sequence identity to a nucleic acid selected from the group consisting of SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99.

32. A fusion protein comprising a polypeptide that facilitates uptake of a protein into the nucleus of a eukaryotic cell according to any one of claim 26 or claim 27, operably linked to a heterologous polypeptide of interest.

33. A recombinant DNA molecule comprising: a) a promoter operably in a eukaryotic cell, such as a plant cell; b) a nucleic acid according to any one of claims 28 to 31; c) a nucleic acid encoding a heterologous polypeptide of interest; and d) optionally a transcription termination and/or polyadenylation signal.

34. A method of facilitating uptake of a heterologous protein of interest into the nucleus of a eukaryotic cell, such as a plant cell, comprising providing to said eukaryotic cell a fusion protein according to claim 32 or a recombinant DNA molecule according to claim 33.

35. A eukaryotic cell comprising a fusion protein according to claim 32 or comprising a recombinant DNA molecule according to claim 33.

36. The eukaryotic cell according to claim 35, which is an in vitro cell.

37. The eukaryotic cell according to any one of claims 35 or 36, which is an animal cell.

38. The eukaryotic cell according to claim 37, which is a non-human animal cell.

39. The eukaryotic cell according to claim 35 or 36, which is a fungal cell.

40. The eukaryotic cell according to any one of claims 35 or 36, which is a plant cell.

41. The eukaryotic cell according to claim 40, wherein the plant cell is from a plant selected from a monocotyledonous species, a dicotyledonous species, an angiosperm species or a gymnosperm species.

42. The eukaryotic cell according to claim 40 or claim 41, wherein the plant cell is from a plant selected from a corn plant, a rice plant, a sorghum plant, a wheat plant, an alfalfa plant, a barley plant, a millet plant, a rye plant, a sugarcane plant, a cotton plant, a soybean plant, a canola plant, a tomato plant, an onion plant, a cucumber plant, an Arabidopsis plant, or a potato plant.

43. A plant comprising a cell or consisting essentially of cells according to any one of claims 40 to 42.