Methods and compositions for DNA base editing

Abstract

The present invention relates to methods and compositions for modifying a target site in the genome of a cell. Fusion proteins are provided that include one or more DNA binding domains and one or more heterologous domains, such as DNA modification domains, linked by an improved linker sequence. In some embodiments, codon optimized polynucleotides are provided that encode fusion proteins comprising one or more DNA binding domains and one or more heterologous domains linked by an improved linker sequence.

Description

[0001] This application is a divisional application of the invention patent application with application date of September 18, 2020, application number 202080081866.3, and invention name “Methods and compositions for DNA base editing”. Technical Field

[0002] The present invention relates to methods and compositions for targeted nucleotide base editing in the genome of a cell.

[0003] Statement Concerning Electronic Submission of Sequence Listings

[0004] Attached hereto and archived is a Sequence Listing in ASCII text format, filed pursuant to 37 C.FR §1.821, entitled “81945_ST25,” created on September 18, 2020, approximately 702 kilobytes, and incorporated herein by reference. Background Art

[0005] The ability to edit plant genomes is highly desirable in agriculture in order to create favorable alleles. It is possible to increase yields or prevent diseases. Genome editing is a new field, in which progress in plants lags behind. In addition, in addition to the expected changes, changes in the genome are a problem that limits the desired changes. CRISPR-CAS9 works by double-stranded cutting of DNA. Since this break is repaired by non-homologous end joining or homology-dependent repair, DNA base insertion or deletion may occur. A strategy called base editing can change DNA without cutting and producing insertions and deletions. In one version, an enzyme called cytidine deaminase targets specific bases through CAS9 (Shimatani et al., 2017. Nat. Biotechnol. [Natural Biotechnology] 35, 441-443) or CAS12a (Li et al., 2018. Nat. Biotechnol. [Natural Biotechnology] 36, 324-327) enzymes, and CAS9 or CAS12a enzymes are modified so that they cannot cut DNA. Cytidine deaminase and nuclease-deficient CAS9 or CAS12a are fused together by connecting an amino acid linker. Improvements in linker connection can improve the functionality of the fusion protein, such as improving the accuracy of cutting by reducing changes in target bases. Summary of the invention

[0006] In order to meet this need for improvement, we provide optimized and improved Cas12a enzymes and constructs. In particular, we provide a fusion protein comprising a heterologous domain, a first linker sequence, and a V-type CRISPR-Cas enzyme. The first linker sequence comprises a repeated GGGGS sequence. The heterologous domain can be a deaminase, a polymerase, a nuclease, a relaxase, an alkyltransferase, a methyltransferase, adenosine deaminase, a cytidine deaminase, an oxidase, a thymine alkyltransferase, adenine oxidase, adenosine methyltransferase, a glycosylase, or a nuclear localization signal. For base editing, the heterologous domain is a deaminase domain-such as a cytidine deaminase or adenine deaminase. The cytidine deaminase domain can be an activation-induced cytidine deaminase ("AID") or apolipoprotein B mRNA-editing complex ("APOBEC") domain, such as the APOBEC1 family from a deaminase. In some contexts, the APOBEC domain comprises a sequence having at least 70% identity to SEQ ID NO:1. When an adenine deaminase is desired, the adenine deaminase may be a TadA domain comprising an amino acid sequence having at least 70% identity to SEQ ID NO:92.

[0007] When the V-type CRISPR-Cas enzyme is a V-type-A ("Cas12a") enzyme, Cas12a is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48. The Cas12a domain can be catalytically inactive, but still binds to the target DNA and allows the heterologous domain to function. When Cas12a is inactive, its sequence is SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 22.

[0008] The first linker sequence between the heterologous domain and the Cas12a enzyme can include GGGGS repeated at least three times. In other uses, the first linker sequence can include GGGGS repeated at least six times.

[0009] The fusion protein may include SEQ ID NO: 11, 12, 13 or 44, and it may also include a uracil DNA glycosylase inhibitor ("UGI") domain (as represented by SEQ ID NO: 8). The UGI domain may be connected to the Cas12a enzyme via a second linker comprising the sequence SGGS. The fusion protein may include SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 or SEQ ID NO: 89. When contacted with DNA, these fusion proteins produce on-target editing at an increased frequency compared to the prior art fusion proteins lacking the first linker sequence of the repeated GGGGS sequence, and produce off-target editing at a reduced frequency.

[0010] We also provide a method for editing plant genomic DNA, the method comprising contacting the plant genomic DNA with: (a) a fusion protein as described in one of the above aspects, and the fusion protein optionally comprises a UGI domain; and (b) a guide RNA ("gRNA") that targets the fusion protein of step (a) to a target DNA sequence of the plant genomic DNA; wherein the edited plant genomic DNA comprises reduced off-target editing compared to plant genomic DNA edited by a fusion protein having a first linker other than a repeated GGGGS sequence.

[0011] We also provide a method for editing plant genomic DNA with reduced off-target editing, the method by contacting plant genomic DNA with: (a) a fusion protein as described in one of the above aspects, and the fusion protein optionally comprises a UGI domain; and (b) a guide RNA ("gRNA") that targets the fusion protein of step (a) to a target DNA sequence of plant genomic DNA; wherein the edited plant genomic DNA comprises reduced off-target editing compared to plant genomic DNA edited by a fusion protein having a first linker other than a repeated GGGGS sequence. In one aspect, the fusion protein comprises SEQ ID NO: 24.

[0012] We also provide a method for obtaining an edited plant population with reduced off-target editing by: (a) obtaining a population of plant cells comprising genomic DNA to be edited; (b) obtaining a nucleotide sequence encoding a fusion protein as described in one of the above aspects, and optionally a UGI domain; (c) transforming a population of plant cells with the nucleotide sequence of step (b), thereby expressing the fusion protein encoded by the nucleic acid sequence within the population of plant cells; (d) growing the transformed plant cell population into plants, wherein at least one plant is edited; and (e) selecting at least one edited plant from the product of step (d), thereby obtaining an edited plant population; wherein the edited plant population contains reduced off-target editing compared to plants edited by a fusion protein having a first linker other than a repeated GGGGS sequence. In one aspect, the nucleotide sequence encoding the fusion protein comprises SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87 or SEQ ID NO:89. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic representation of the DNA constructs of three versions of Cas12aBE are shown. (1) represents a promoter; (2) is a nuclear localization signal; (3) is a deaminase, such as an APOBEC deaminase; (4) is an XTEN linker; (5) is LbCas12a; (6) is an SGGS linker; (7) is a uracil glycosylase inhibitor; (8) is a long linker, such as a (G4S)6 linker; (9) is Mb2Cas12a; and (10) is an element encoding a guide RNA. Figure 1 A shows the LbCas12aBE plus guide RNA construct in a 5' to 3' orientation, where the deaminase (3) is operably linked to LbCas12a (5) via an XTEN linker (4). Figure 1 B shows the LbCas12aBE plus guide RNA construct in a 5' to 3' orientation, where the deaminase (3) is operably linked to LbCas12a (5) via a (G4S)6 linker (8). Figure 1 C shows the Mb2Cas12aBE plus guide RNA construct in a 5' to 3' orientation, where the deaminase (3) is operably linked to the Mb2Cas12a (9) via a (G4S)6 linker (8).

[0014] Figure 2 A schematic diagram of a DNA construct in the 5' to 3' direction is shown, the DNA construct comprising Cas12aBE and multiple guide RNAs. (1) represents a promoter; (2) is a nuclear localization signal; (3) is a deaminase, for example, an APOBEC deaminase; (6) is an SGGS linker; (7) is a uracil glycosylase inhibitor; (8) is a long linker, for example, a (G4S)6 linker; (9) is Cas12a; (10) is an element encoding a first guide RNA; (11) is an element encoding a second guide RNA; and (12) is an element encoding a third guide RNA. Each element encoding a guide RNA comprises a crRNA segment and a target sequence segment capable of hybridizing with a genomic target DNA sequence.

[0015] Brief description of the sequences in the sequence listing

[0016] SEQ ID NO: 1 is the amino acid sequence of Apobec1.

[0017] SEQ ID NO: 2 is the nucleotide sequence of Apobec1.

[0018] SEQ ID NO: 3 is the amino acid sequence of catalytically inactive Mb2Cas12a.

[0019] SEQ ID NO: 4 is the nucleotide sequence of catalytically inactive Mb2Cas12a.

[0020] SEQ ID NO: 5 is the nucleotide sequence of cLbCas12aBE without catalytic activity.

[0021] SEQ ID NO: 6 is the amino acid sequence of cLbCas12aBE without catalytic activity.

[0022] SEQ ID NO: 7 is the nucleotide sequence of uracil DNA glycosylase inhibitor (UGI).

[0023] SEQ ID NO: 8 is the amino acid sequence of uracil DNA glycosylase inhibitor (UGI).

[0024] SEQ ID NO:9 is a nucleotide sequence, which contains the expression cassette prSoUbi4:SV40NLS:cLbCas12aBE:GS6 linker:SV40NLS:SGGS linker:UGI:SGGS linker:SV40NLS:tNOS.

[0025] SEQ ID NO: 10 is the nucleotide sequence of the optimized (G4S)x6 linker.

[0026] SEQ ID NO: 11 is the amino acid sequence of the optimized (G4S)x6 linker.

[0027] SEQ ID NO: 12 is the amino acid sequence of 18 aa linker-SX.

[0028] SEQ ID NO: 13 is the amino acid sequence of 15 aa linker-(G4S)X3.

[0029] SEQ ID NO: 14 is a nucleotide sequence comprising the fusion protein cLBCas12aBE-07 from construct 25057.

[0030] SEQ ID NO: 15 is an amino acid sequence comprising the fusion protein cLBCas12aBE-07 from construct 25057.

[0031] SEQ ID NO: 16 is a nucleotide sequence comprising the fusion protein cLBCas12aBE-08 from construct 25058.

[0032] SEQ ID NO: 17 is an amino acid sequence comprising the fusion protein cLBCas12aBE-08 from construct 25058.

[0033] SEQ ID NO: 18 is a nucleotide sequence comprising the fusion protein cLBCas12aBE-01 from construct 24524.

[0034] SEQ ID NO: 19 is an amino acid sequence comprising the fusion protein cLBCas12aBE-01 from construct 24524.

[0035] SEQ ID NO:20 is the nucleotide sequence of cCas9BE-02.

[0036] SEQ ID NO:21 is the amino acid sequence of cCas9BE-02.

[0037] SEQ ID NO: 22 is the amino acid sequence of catalytically inactive AsCas12a.

[0038] SEQ ID NO: 23 is a nucleotide sequence comprising the fusion protein cLBCas12aBE-06 from construct 24904.

[0039] SEQ ID NO: 24 is an amino acid sequence comprising the fusion protein cLBCas12aBE-06 from construct 24904.

[0040] SEQ ID NO:25 is a nucleotide sequence comprising the promoter prSoUbi4-02.

[0041] SEQ ID NO:26 is a nucleotide sequence comprising the Cas12a gRNA waxy1 target sequence.

[0042] SEQ ID NO: 27 is a nucleotide sequence comprising the Cas9 gRNA waxy1 target sequence.

[0043] SEQ ID NO: 28 is a nucleotide sequence comprising exon 4 of the ZmWaxy1 gene.

[0044] SEQ ID NO: 29 is the forward primer of ZmWaxy1.

[0045] SEQ ID NO: 30 is the reverse primer of ZmWaxy1.

[0046] SEQ ID NO: 31 is the sequencing primer of ZmWaxy1.

[0047] SEQ ID NO:32 is a nucleotide sequence comprising the fusion protein cLbCpf1-02 from construct 24523.

[0048] SEQ ID NO:33 is the amino acid sequence comprising the fusion protein cLbCpf1-02 from construct 24523.

[0049] SEQ ID NO: 34 is a nucleotide sequence comprising the fusion protein cLbCas12a-05 from construct 25181.

[0050] SEQ ID NO: 35 is an amino acid sequence comprising the fusion protein cLbCas12a-05 from construct 25181.

[0051] SEQ ID NO: 36 is a nucleotide sequence comprising the fusion protein cLbCas12a-02 from construct 25205.

[0052] SEQ ID NO: 37 is an amino acid sequence comprising the fusion protein cLbCas12a-02 from construct 25205.

[0053] SEQ ID NO: 38 is a nucleotide sequence comprising the fusion protein cLbCas12a-25 from construct 25513.

[0054] SEQ ID NO: 39 is an amino acid sequence comprising the fusion protein cLbCas12a-25 from construct 25513.

[0055] SEQ ID NO:40 is a nucleotide sequence comprising the fusion protein cMb2Cas12a-01 from construct 25220.

[0056] SEQ ID NO:41 is an amino acid sequence comprising the fusion protein cMb2Cas12a-01 from construct 25220.

[0057] SEQ ID NO:42 is a nucleotide sequence comprising the fusion protein cMb2Cas12a-02 from construct 25382.

[0058] SEQ ID NO: 43 is an amino acid sequence comprising the fusion protein cMb2Cas12a-02 from construct 25382.

[0059] SEQ ID NO:44 is the amino acid sequence of the optimized (G4SG)x6 linker.

[0060] SEQ ID NO: 45 is the amino acid sequence of active LbCas12a.

[0061] SEQ ID NO: 46 is the amino acid sequence of active Mb2Cas12a.

[0062] SEQ ID NO: 47 is the amino acid sequence of active AsCas12a.

[0063] SEQ ID NO: 48 is the amino acid sequence of active FnCas12a.

[0064] SEQ ID NO: 49 is a nucleotide sequence comprising the fusion protein cMb2Cas12a-BE-01 from construct 25457.

[0065] SEQ ID NO: 50 is an amino acid sequence comprising the fusion protein cMb2Cas12a-BE-01 from construct 25457.

[0066] SEQ ID NO: 51 is a nucleotide sequence comprising the fusion protein cLbCas12a-BE-08 from construct 25268.

[0067] SEQ ID NO: 52 is an amino acid sequence comprising the fusion protein cLbCas12a-BE-08 from construct 25268.

[0068] SEQ ID NO: 53 is a nucleotide sequence comprising the fusion protein cLbCas12a-05 from construct 25173.

[0069] SEQ ID NO: 54 is an amino acid sequence comprising the fusion protein cLbCas12a-05 from construct 25173.

[0070] SEQ ID NO: 55 is a nucleotide sequence comprising the fusion protein cLbCas12a-05 from construct 25175.

[0071] SEQ ID NO: 56 is an amino acid sequence comprising the fusion protein cLbCas12a-05 from construct 25175.

[0072] SEQ ID NO: 57 is the amino acid sequence of a catalytically inactive LbCas12a with an optimized (G4SG)6 linker.

[0073] SEQ ID NO: 58 is the amino acid sequence of active Mb2Cas12a with an optimized (G4S)6 linker.

[0074] SEQ ID NO: 59 is the amino acid sequence of catalytically inactive Mb2Cas12a with an XTEN linker.

[0075] SEQ ID NO: 60 is the amino acid sequence of active AsCas12a with an XTEN linker.

[0076] SEQ ID NO: 61 is the amino acid sequence of catalytically inactive AsCas12a with an XTEN linker.

[0077] SEQ ID NO: 62 is the amino acid sequence of active FnCas12a with an XTEN linker.

[0078] SEQ ID NO: 63 is the amino acid sequence of active AsCas12a with an optimized (G4S)6 linker.

[0079] SEQ ID NO: 64 is the amino acid sequence of a catalytically inactive AsCas12a with an optimized (G4S)6 linker.

[0080] SEQ ID NO: 65 is the amino acid sequence of active FnCas12a with an optimized (G4S)6 linker.

[0081] SEQ ID NO: 66 is the amino acid sequence of a catalytically inactive Mb2Cas12a with an optimized (G4SG)6 linker.

[0082] SEQ ID NO: 67 is the amino acid sequence of active AsCas12a with an optimized (G4SG)6 linker.

[0083] SEQ ID NO: 68 is the amino acid sequence of a catalytically inactive AsCas12a with an optimized (G4SG)6 linker.

[0084] SEQ ID NO: 69 is the amino acid sequence of active FnCas12a with an optimized (G4SG)6 linker.

[0085] SEQ ID NO: 70 is the amino acid sequence of the XTEN linker.

[0086] SEQ ID NO:71 is a nucleotide sequence comprising the Cas12a gRNA SBEII target sequence.

[0087] SEQ ID NO:72 is a nucleotide sequence comprising the Cas12a gRNA GL2 target sequence.

[0088] SEQ ID NO:73 is a nucleotide sequence comprising the Cas12a gRNA Fad2 target sequence.

[0089] SEQ ID NO:74 is a nucleotide sequence comprising a Cas12a crRNA sequence for use with waxy1, SBEII, and Fad2 target sequences.

[0090] SEQ ID NO:75 is a nucleotide sequence comprising a Cas12acrRNA sequence for use with a GL2 target sequence.

[0091] SEQ ID NO:76 is a nucleotide sequence comprising the fusion protein cCas9ABE-01 from construct 24785.

[0092] SEQ ID NO:77 is an amino acid sequence comprising the fusion protein cCas9ABE-01 from construct 24785.

[0093] SEQ ID NO:78 is a nucleotide sequence comprising the fusion protein cLbCas1aABE-01 from construct 25459.

[0094] SEQ ID NO: 79 is an amino acid sequence comprising the fusion protein cLbCas1aABE-01 from construct 25459.

[0095] SEQ ID NO: 80 is a nucleotide sequence comprising the fusion protein cLbCas12aABE-02 from construct 25504.

[0096] SEQ ID NO: 81 is an amino acid sequence comprising the fusion protein cLbCas12aABE-02 from construct 25504.

[0097] SEQ ID NO: 82 is a nucleotide sequence comprising the fusion protein cLbCas12aBE-09 from construct 25289.

[0098] SEQ ID NO: 83 is an amino acid sequence comprising the fusion protein cLbCas12aBE-09 from construct 25289.

[0099] SEQ ID NO: 84 is a nucleotide sequence comprising the fusion protein cdLbCas12a-ABE-CBE-01 from construct 25658.

[0100] SEQ ID NO: 85 is an amino acid sequence comprising the fusion protein cdLbCas12a-ABE-CBE-01 from construct 25658.

[0101] SEQ ID NO: 86 is a nucleotide sequence comprising the fusion protein cdLbCas12a-ABE-CBE-02 from construct 25701.

[0102] SEQ ID NO: 87 is an amino acid sequence comprising the fusion protein cdLbCas12a-ABE-CBE-02 from construct 25701.

[0103] SEQ ID NO: 88 is a nucleotide sequence comprising the fusion protein cdLbCas12a-ABE-CBE-03 from construct 25702.

[0104] SEQ ID NO: 89 is an amino acid sequence comprising the fusion protein cdLbCas12a-ABE-CBE-03 from construct 25702.

[0105] SEQ ID NO:90 is a nucleotide sequence comprising the Cas12a gRNA ADH1 target sequence.

[0106] SEQ ID NO:91 is a nucleotide sequence comprising a TadA dimer.

[0107] SEQ ID NO:92 is an amino acid sequence comprising a TadA dimer. DETAILED DESCRIPTION

[0108] This description is not intended to be an exhaustive list of all the different ways in which the present invention may be implemented, or of all the features that may be added to the present invention. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be deleted from that embodiment. In addition, in view of this disclosure, a variety of variations of the various embodiments suggested herein and additions will be apparent to those skilled in the art without departing from the present invention. Therefore, the following description is intended to set forth some specific embodiments of the present invention, and does not exhaustively describe all permutations, combinations, and variations thereof.

[0109] definition

[0110] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. The terms used in the description of the invention herein are for the purpose of describing specific embodiments only and are not intended to limit the invention. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety.

[0111] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise indicated, the terms used herein should be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of general terms in molecular biology can also be found in Rieger et al., 遗传学词汇表:经典与分子 Glossary of Genetics: Standard and Molecular, 5th ed., Springer-Verlag, New York, 1994.

[0112] As used herein, the term "long linker" refers to a polypeptide chain of at least 10 amino acids for connecting a heterologous domain to a protein of interest. By way of example and not limitation, a long linker may include a sequence GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 11), additionally represented as (G4S) 6 or (G4S) x6 or (G4S) * 6. A long linker may include GGGGSGGGGGSGGGGGSGGGGGSGGGGGSGGGGGSGGGGGSG (SEQ ID NO: 44), additionally represented as (G4SG) 6 or (G4SG) x6 or (G4SG) * 6. A heterologous domain connected to a protein by a long linker includes cytidine deaminase, guanine deaminase, uracil glycosylase inhibitor ("UGI"), nuclease, and any other protein domain that may be operably connected to a protein of interest in a heterologous manner. Such proteins of interest include, but are not limited to, site-directed nucleases (e.g., Cas9, Cas12a, Cas12b, Cas12i, Cas12j or other CRISPR nucleases), zinc fingers, homing endonucleases, transcription activator-like effector nucleases ("TALEN"), and the like.

[0113] As used in the description of embodiments of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0114] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0115] As used herein, the term "about" when referring to a measurable value such as an amount of a compound, dosage, time, temperature, etc., is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0116] The terms “comprise”, “comprises” and / or “comprising” when used in this specification specify the presence of listed features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0117] As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim will be interpreted as covering the specified materials or steps mentioned in the claim and those materials or steps that do not materially affect one or more basic and novel features of the claimed invention. Therefore, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising".

[0118] As used herein, the term "amplified" means using at least one nucleic acid molecule as a template to construct multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule. See, for example, Diagnostic Molecular Microbiology: Principles and Applications, DHPERSING et al., eds., American Society for Microbiology, Washington, DC, (1993). The amplified product is called an amplicon.

[0119] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA (such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA). In some embodiments, the RNA is subsequently translated in vivo to produce a protein.

[0120] As used herein, the term "event" of transgenic refers to a recombinant plant produced by transforming and regenerating a single plant cell with heterologous DNA (e.g., an expression cassette comprising one or more target genes (e.g., transgenic)). The term "event" refers to the original transformant comprising heterologous DNA and / or the progeny of the transformant. The term "event" also refers to the progeny produced by sexual outcrossing (outcross) between a transformant and another strain. Even after repeated backcrossing to the recurrent parent, the inserted DNA and flanking DNA from the transformed parent are present in the same chromosomal position of the hybrid progeny. Typically, the transformation of plant tissues produces multiple events, each of which represents a different position in the genome of a plant cell inserted by a DNA construct. Based on the expression of transgenic or other desired features, a specific event is selected. Thus, as used herein, "event MIR604," "MIR604," or "MIR604 event" refers to the original MIR604 transformant and / or progeny of a MIR604 transformant (U.S. Pat. Nos. 7,361,813; 7,897,748; 8,354,519 and 8,884,102, incorporated herein by reference).

[0121] As used herein, "expression cassette" means a nucleic acid molecule capable of directing the expression of a specific nucleotide sequence in an appropriate host cell, the nucleic acid molecule comprising a promoter operably connected to a target nucleotide sequence (typically a coding region), the nucleotide sequence being operably connected to a termination signal. It also typically comprises a sequence required for the appropriate translation of the nucleotide sequence. The coding region usually encodes the target protein, but can also encode a functional RNA of interest (e.g., antisense RNA or non-translated RNA) in a sense or antisense direction. The expression cassette may also be included in a sequence not required for directing the expression of the target nucleotide sequence, but it exists because of a convenient restriction site for removing the expression cassette from the expression vector. The expression cassette comprising the target nucleotide sequence may be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components. The expression cassette may also be a naturally occurring expression cassette that has been obtained in a recombinant form useful for heterologous expression. However, the expression cassette is usually heterologous relative to the host, i.e., the specific nucleic acid sequence of the expression cassette is not naturally occurring in the host cell, and must have been introduced into the host cell or the ancestor of the host cell by a transformation method known in the art. The expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some specific external stimulus. In the case of multicellular organisms (such as plants), the promoter can also be specific to a specific tissue, or organ, or developmental stage. When transformed into a plant, an expression cassette or a fragment thereof may also be referred to as an "inserted sequence" or "inserted sequence."

[0122] "Gene" is a limited region located in the genome, and in addition to the aforementioned coding nucleic acid sequence, it also includes other major regulatory nucleic acid sequences responsible for controlling the expression (i.e., transcription and translation) of the coding portion. A gene can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions). A gene typically expresses mRNA, functional RNA, or specific protein, including regulatory sequences. A gene may or may not be used to produce functional proteins. In certain embodiments, a gene refers only to a coding region. The term "natural gene" refers to a gene as found in nature. The term "chimeric gene" refers to any gene comprising the following: 1) a DNA sequence, including regulatory sequences and coding sequences that are not found together in nature, or 2) a sequence encoding a portion of an unnaturally adjacent protein, or 3) a portion of an unnaturally adjacent promoter. Therefore, a chimeric gene can include regulatory sequences and coding sequences obtained from different sources, or regulatory sequences and coding sequences obtained from the same source but arranged in a different manner from that found in nature. A gene can be "isolated," meaning a nucleic acid molecule that is substantially or essentially free of components normally found associated with the nucleic acid molecule in its natural state. Such components include other cellular materials, culture medium from recombinant production, and / or various chemicals used in chemically synthesizing nucleic acid molecules.

[0123] The terms "express" or "expression" with reference to a polynucleotide coding sequence mean that the sequence is transcribed, and optionally translated.

[0124] "Target gene" or "target nucleotide sequence" refers to any gene that, when transferred to a plant, confers a desired characteristic on the plant (such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other harmful organisms, herbicide tolerance, improved nutritional value, improved performance of industrial processes, or altered breeding capacity). "Target gene" can also be a gene transferred to a plant for the production of a commercially valuable enzyme or metabolite in the plant.

[0125] As used herein, "heterologous" refers to a nucleic acid molecule or nucleotide sequence that is naturally unrelated to the host cell into which it is introduced, the sequence being derived from another species or from the same species or organism, but being modified from its original form or the form primarily expressed in the cell, including multiple copies of non-naturally occurring nucleic acid sequences. Therefore, a nucleotide sequence derived from an organism or species different from the organism or species to which the cell into which it is introduced belongs is heterologous relative to that cell or the progeny of the cell. In addition, a heterologous nucleotide sequence includes a nucleotide sequence that is derived from and inserted into the same natural original cell type, but exists in a non-natural state, for example, exists in different copy numbers, and / or is under the control of regulatory sequences different from those found in the natural state of the nucleic acid molecule. The nucleic acid sequence can also be heterologous to other nucleic acid sequences associated therewith, for example in a nucleic acid construct, such as, for example, an expression vector. As a non-limiting example, a promoter can be present in a nucleic acid construct in combination with one or more regulatory elements and / or coding sequences that are not naturally present in relation to that particular promoter, i.e., they are heterologous to the promoter.

[0126] A "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with the host cell into which it is introduced. Homologous nucleic acid sequences can also be nucleic acid sequences naturally associated with other nucleic acid sequences, which can, for example, be present in a nucleic acid construct. As a non-limiting example, a promoter can be present in a nucleic acid construct in combination with one or more regulatory elements and / or encoding sequences, which naturally exist in association with that particular promoter, i.e., they are homologous to the promoter.

[0127] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid sequence so that the function of one affects the function of another. For example, when a promoter can affect the expression of a coding sequence or a functional RNA (i.e., the coding sequence or the functional RNA is under the transcriptional control of the promoter), the promoter is operably linked to the coding sequence or the functional RNA. A coding sequence in the sense direction or the antisense direction can be operably linked to a regulatory sequence. Therefore, a regulatory or control sequence (e.g., a promoter) that is operably associated with a nucleotide sequence can affect the expression of the nucleotide sequence. For example, a promoter that is operably linked to a nucleotide sequence encoding GFP will be able to affect the expression of the GFP nucleotide sequence.

[0128] Control sequences need not be adjacent to the nucleotide sequence of interest, as long as they function to direct its expression. Thus, for example, intervening untranslated, transcribed sequences may be present between a promoter and a coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

[0129] As used herein, "primers" are isolated nucleic acids that are annealed to complementary target DNA strands by nucleic acid hybridization to form hybridization between the primer and the target DNA strand, and then extended along the target DNA strand by a polymerase (such as a DNA polymerase). Primer pairs or primer sets can be used for amplification of nucleic acid molecules, such as by polymerase chain reaction (PCR) or other nucleic acid amplification methods.

[0130] "Probe" is a nucleic acid molecule that is complementary to the separation of a part of a target nucleic acid molecule, and is typically used to detect and / or quantify a target nucleic acid molecule. Therefore, in some embodiments, the probe can be a nucleic acid molecule that can detect a part or a reporter gene to which the separation is attached, such as a radioisotope, a ligand, a chemiluminescent agent, a fluorescent agent or an enzyme. Probe according to the present invention can not only include deoxyribonucleic acid or ribonucleic acid, but also include polyamides and other probe materials that are specifically combined with the target nucleic acid sequence and can be used to detect the presence of the target nucleic acid sequence or the amount of the quantitative target nucleic acid sequence.

[0131] The TaqMan probe is designed so that it anneals in the DNA region amplified by a specific primer set. Since the Taq polymerase extends the primer and synthesizes the nascent chain from the 3' to 5' single-stranded template of the complementary chain, the 5' to 3' exonuclease of the polymerase extends the nascent chain through the probe and thus degrades the probe that has been annealed to the template. The degradation of the probe releases the fluorophore therefrom and breaks the close contact with the quencher, thereby alleviating the quenching effect and allowing the fluorescence of the fluorophore. Therefore, the fluorescence detected in the quantitative PCR thermal cycler is proportional to the amount of the released fluorophore and the DNA template present in the PCR.

[0132] The length of primer and probe is generally between 5 and 100 nucleotide or more nucleotide.In certain embodiments, the length of primer and probe can be at least 20 nucleotide or more, or at least 25 nucleotide or more, or length at least 30 nucleotide or more.These primers and probes are hybridized with target sequence specificity under optimal hybridization conditions known in the art.Primer and probe according to the present invention can have the complete sequence complementary to the target sequence, although different from the target sequence and retain the probe with the ability of target sequence hybridization can be designed according to ordinary methods of the present invention.

[0133] Methods for making and using probes and primers are described, e.g., 分子克隆: 实验指南[Molecular Cloning: A Laboratory Manual], 2nd edition, Volumes 1-3, Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. PCR primer pairs can be derived from known sequences, for example by using a computer program designed for this purpose.

[0134] Polymerase chain reaction (PCR) is a technique for "amplifying" a specific DNA fragment. In order to perform PCR, at least a portion of the nucleotide sequence of the DNA molecule to be replicated must be known. Typically, primers or short oligonucleotides complementary to the nucleotide sequence (known sequence) at the 3' end of each strand of the DNA to be amplified (e.g., substantially complementary or completely complementary) are used. The DNA sample is heated to separate its chains and mixed with these primers. These primers hybridize with complementary sequences in their DNA samples. Synthesis (5' to 3' direction) is started using the original DNA chain as a template. The reaction mixture must contain all four deoxynucleotide triphosphates (dATP, dCTP, dGTP and dTTP) and DNA polymerase. Polymerization continues until each newly synthesized chain has proceeded far enough to contain a sequence recognized by another primer. Once this occurs, two DNA molecules identical to the original molecule are produced. The two molecules are heated to separate their chains, and the process is repeated. Each cycle doubles the number of DNA molecules. Using automated equipment, replication of each cycle can be completed in less than 5 minutes. After 30 cycles, the amplification starting from a single DNA molecule has exceeded 1 billion copies (2 30 =1.02x 10 9 ).

[0135] The oligonucleotides of the oligonucleotide primer pair are complementary to the DNA sequences on the relative DNA strand and flanking the region to be amplified. The annealed primers hybridize with the newly synthesized DNA strands. The first amplification cycle will result in two new DNA strands, whose 5' ends are fixed by the positions of the oligonucleotide primers, but whose 3' ends are variable ('irregular' 3' ends). The two new strands can successively serve as templates for the synthesis of complementary strands of the desired length (the 5' end is defined by the primer and the 3' end is fixed because the synthesis cannot exceed the end of the opposite primer). After several cycles, the desired fixed-length product begins to dominate.

[0136] Quantitative polymerase chain reaction (qPCR) (also referred to as real-time polymerase chain reaction) monitors the accumulation of DNA products from PCR reactions in real time. qPCR is a molecular biology laboratory technique based on polymerase chain reaction (PCR) for amplification and simultaneous quantitative target DNA molecules. Even one copy of a specific sequence can be amplified and detected in PCR. The PCR reaction generates copies of DNA templates in an exponential manner. This results in a quantitative relationship between the amount of the starting target sequence and the amount of PCR products accumulated under any particular cycle. Due to inhibitors of polymerase reaction found together with template, reagent limitation or accumulation of pyrophosphate molecules, the PCR reaction eventually stops generating templates (i.e., plateau phase) at an exponential rate, making the endpoint quantification of PCR products unreliable. Therefore, repeated reactions can generate variable amounts of PCR products. Only during the exponential phase of the PCR reaction is it possible to push back in order to determine the starting amount of the template sequence. Measurement during the accumulation of PCR products (i.e., real-time quantitative PCR) allows for quantification during the exponential phase of the reaction, and therefore eliminates the variability associated with conventional PCR. In real-time PCR assays, positive reactions are detected by accumulation of fluorescent signals. For one or more specific sequences in a DNA sample, quantitative PCR can perform both detection and quantification. The quantity can be the absolute number of copies or the relative amount when normalized to DNA input or an additional normalizing gene. Since the first record of real-time PCR, it has been used for an increasing number of applications, including mRNA expression studies, DNA copy number measurements in genomes or viral DNA, allele discrimination assays, expression analysis of specific splice variants of genes, and gene expression in paraffin-embedded tissues and laser-captured microdissected cells.

[0137] As used herein, the phrase "Ct value" refers to a "cycle threshold value", which is defined as "the fractional cycle number at which the amount of amplified target reaches a fixed threshold value". In some embodiments, it represents the intersection between the amplification curve and the threshold line. The amplification curve is typically in an "S" shape, which represents the change in relative fluorescence of each reaction (Y axis) at a given cycle (X axis), which is recorded during PCR by a real-time PCR instrument in some embodiments. In some embodiments, the threshold line is the detection level at which the reaction reaches a fluorescence intensity higher than the background. See Livak and Schmittgen (2001) 25 Methods [Methods] 402-408. It is a relative measure of the target concentration in PCR. Typically, in some embodiments, for a given reference gene, a good Ct value for quantitative determination such as qPCR is in the range of 10-40. The Ct level is inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct level, the higher the amount of detectable target nucleic acid in the sample). In addition, a good Ct value for quantitative determination such as qPCR shows a linear response range in the case of proportional dilution of the target gDNA.

[0138] In some embodiments, qPCR is performed under conditions where Ct values ​​can be collected in real time for quantitative analysis. For example, in a typical qPCR experiment, DNA amplification is monitored at each cycle of PCR during the extension phase. When DNA is in the log-linear phase of amplification, the amount of fluorescence typically increases above background. In some embodiments, Ct values ​​are collected at this time point.

[0139] As used herein, the term "cell" refers to any living cell. The cell can be a prokaryotic cell or a eukaryotic cell. The cell can be isolated. The cell may or may not be able to regenerate into an organism. The cell can be in the context of a tissue, callus, culture, organ, or part. In certain embodiments, the cell can be a plant cell. The plant cell of the present invention can be in the form of an isolated single cell, or can be a cultured cell, or can be a part of a higher organizational unit (such as, for example, a plant tissue or a plant organ). The plant cell can be derived from angiosperms or gymnosperms or a part thereof. In other embodiments, the plant cell can be a monocotyledonous plant cell, a dicotyledonous plant cell. The monocotyledonous plant cell can be, for example, corn, rice, sorghum, sugarcane, barley, wheat, oats, turf grass, or an ornamental grass cell. The dicotyledonous plant cell can be, for example, a tobacco, pepper, eggplant, sunflower, cruciferous plant, flax, potato, cotton, soybean, beet, or rapeseed cell.

[0140] As used herein, the term "plant part" includes, but is not limited to, embryos, pollen, ovules, seeds, leaves, stems, buds, flowers, branches, fruits, kernels, spikes, cobs, husks, stalks, roots, root tips, anthers, plant cells (including intact plant cells in plants and / or parts of plants), plant protoplasts, plant tissues, plant cell tissue cultures, plant callus, plant masses, etc. As used herein, "buds" refer to the above-ground parts including leaves and stems. In addition, as used herein, "plant cells" refer to the structural and physiological units of plants, including cell walls and may also refer to protoplasts.

[0141] In the context of cells, prokaryotic cells, bacterial cells, eukaryotic cells, plant cells, plants and / or plant parts, the term "introducing" or "introduce" means contacting a nucleic acid molecule with a cell, eukaryotic cell, plant, plant part and / or plant cell in such a way that the nucleic acid molecule is able to enter the interior of the cell, eukaryotic cell, plant cell and / or plant and / or plant part. When more than one nucleic acid molecule is introduced, these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or assembled into separate polynucleotides or nucleic acid constructs and can be located on the same or different nucleic acid constructs. Thus, these polynucleotides can be introduced into plant cells in a single transformation event, in separate transformation events, or, for example, as part of a breeding program.

[0142] As used herein, the terms "transformed" and "transgenic" refer to any cell, prokaryotic cell, eukaryotic cell, plant, plant cell, callus, plant tissue, or plant part that contains all or part of at least one recombinant (e.g., heterologous) polynucleotide. In some embodiments, all or part of the recombinant polynucleotide is stably integrated into a chromosome or a stable extrachromosomal element so that it is passed on to successive generations. For purposes of the present invention, the term "recombinant polynucleotide" refers to a polynucleotide that has been altered, rearranged, or modified by genetic engineering. Examples include any cloned polynucleotide, or a polynucleotide that is linked or joined to a heterologous sequence. The term "recombinant" does not refer to polynucleotide changes that occur due to naturally occurring events (such as spontaneous mutations) or due to non-spontaneous mutagenesis followed by selective breeding.

[0143] As used herein, the term "conversion" refers to the introduction of heterologous nucleic acid into a cell. The transformation of a cell can be stable or transient. Therefore, transgenic cells, plant cells, plants and / or plant parts of the present invention can be stably transformed or transiently transformed. The term "conversion" can refer to the transfer of nucleic acid molecules into the genome of a host cell, resulting in genetically stable inheritance. In certain embodiments, the introduction of plants, plant parts and / or plant cells is via bacterial-mediated transformation, particle bombardment transformation, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, liposome-mediated transformation, nanoparticle-mediated transformation, polymer-mediated transformation, virus-mediated nucleic acid delivery, whisker-mediated nucleic acid delivery, microinjection, ultrasonic treatment, infiltration, polyethylene glycol-mediated transformation, protoplast transformation or any other electrical, chemical, physical and / or biological mechanism causing the introduction of nucleic acid into plants, plant parts and / or their cells, or any combination thereof.

[0144] Procedures for transforming plants are well known and routine in the art and are generally described in the literature. Non-limiting examples of methods for plant transformation include transformation via bacterial-mediated nucleic acid delivery (e.g., via bacteria from the genus Agrobacterium), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid absorption, and any other electrical, chemical, physical (mechanical) and / or biological mechanism that allows nucleic acid to be introduced into plant cells, including any combination thereof. General guides to the various plant transformation methods known in the art include Miki et al. ("Procedures for Introducing Foreign DNA into Plants," in Plant Molecular Biology and Biotechnology, eds. Glick, BR and Thompson, JE (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0145] Agrobacterium-mediated transformation is a common method for transforming plants because of its high transformation efficiency and because of its wide applicability with many different species. Agrobacterium-mediated transformation typically involves transferring a binary vector carrying the exogenous DNA of interest to an appropriate Agrobacterium strain, which may depend on the complement of the vir genes carried by the host Agrobacterium strain on a co-existing Ti plasmid or chromosomally (Uknes et al., 1993, Plant Cell [Plant Cell] 5: 159-169). The transfer of the recombinant binary vector to Agrobacterium can be achieved using an Escherichia coli strain carrying a recombinant binary vector, and an auxiliary Escherichia coli strain (the auxiliary Escherichia coli strain carries a plasmid capable of moving the recombinant binary vector into the target Agrobacterium strain) through a three-parent mating procedure. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by nucleic acid transformation ( and Willmitzer, 1988, Nucleic Acids Res. 16:9877).

[0146] Plant transformation by recombinant Agrobacterium usually involves co-cultivation of Agrobacterium with explants from plants and follows methods well known in the art. Transformed tissue is typically regenerated on selection media carrying antibiotic or herbicide resistance markers located between these binary plasmid T-DNA borders.

[0147] Another method for transforming plants, plant parts and plant cells involves advancing inert or biologically active particles on plant tissues and cells. See, for example, U.S. Patent Nos. 4,945,050; 5,036,006 and 5,100,792. Typically, this method involves advancing inert or biologically active particles at plant cells under conditions effective to penetrate the outer surface of the cell and provide for incorporation into its interior. When inert particles are used, the vector can be introduced into the cell by coating these particles with a vector containing the target nucleic acid. Alternatively, one or more cells can be surrounded by the vector so that the vector is brought into the cell by the excitation of the particle. Biologically active particles (e.g., dried yeast cells, dried bacteria or bacteriophages, each containing one or more nucleic acids intended to be introduced) can also be advanced into plant tissues.

[0148] "Transient transformation" in the context of a polynucleotide means that the polynucleotide is introduced into a cell and is not integrated into the genome of the cell.

[0149] As used herein, in the context of a polynucleotide introduced into a cell, "stably introducing (stably introducing)", "stable transformation (stable transformation or stably transformed)" means that the introduced polynucleotide is stably integrated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide. Therefore, the integrated polynucleotide can be inherited by its progeny, more specifically, by multiple successive generations of progeny. "Genome" as used herein includes nuclear and / or plastid genomes, and therefore includes the integration of polynucleotides into, for example, chloroplast genomes. Stable transformation as used herein can also refer to a polynucleotide that is maintained outside the chromosome, for example, as a minichromosome.

[0150] Transient transformation can be detected by, for example, enzyme-linked immunosorbent assay (ELISA) or Western blotting, both of which can detect the presence of peptides or polypeptides encoded by one or more nucleic acid molecules introduced into an organism. Stable transformation of cells can be detected by, for example, Southern blot hybridization assays of cellular genomic DNA and nucleic acid sequences (these sequences specifically hybridize with the nucleotide sequences of nucleic acid molecules introduced into an organism (e.g., a plant). Stable transformation of cells can be detected by, for example, Northern blot hybridization assays of cellular RNA and nucleic acid sequences (these sequences specifically hybridize with the nucleotide sequences of nucleic acid molecules introduced into a plant or other organism). Stable transformation of cells can also be detected by, for example, polymerase chain reaction (PCR) or other amplification reactions well known in the art, which employ specific primer sequences that hybridize with one or more target sequences of nucleic acid molecules, resulting in amplification of one or more target sequences, which can be detected according to standard methods. Conversion can also be detected by direct sequencing and / or hybridization schemes well known in the art.

[0151] Thus, in specific embodiments of the invention, plant cells can be transformed by any method known in the art and as described herein and whole plants can be regenerated from these transformed cells using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue culture and / or cultured protoplasts is described, for example, in Evans et al. ( 植物细胞培养手册 [Manual of Plant Cell Culture], Vol. 1, MacMilan Publishing Co., New York (1983)); and Vasil IR (ed.) ( 细胞培养与植物 体细胞遗传学 [Cell Culture and Somatic Cell Genetics of Plants], Academic Press, Orlando, Volume I (Acad. Press, Orlando) (1984) and Volume II (1986)). Methods for selecting transformed transgenic plants, plant cells and / or plant tissue cultures are routine in the art and can be used in the methods of the invention provided herein.

[0152] "Transformation and regeneration process" refers to the process of stably introducing a transgene into a plant cell and regenerating a plant from the transgenic plant cell. As used herein, transformation and regeneration include a selection process, wherein the transgene comprises a selectable marker, and the transformed cell has incorporated and expresses the transgene, such that the transformed cell will survive and thrive in the presence of the selection agent. "Regeneration" refers to the growth of a whole plant from a plant cell, group of plant cells, or plant piece (e.g., from a protoplast, callus, or tissue portion).

[0153] The terms "nucleotide sequence", "nucleic acid", "nucleic acid sequence", "nucleic acid molecule", "oligonucleotide" and "polynucleotide" are used interchangeably herein to refer to heteropolymers of nucleotides and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and chimeras of RNA and DNA. The term nucleic acid molecule refers to a nucleotide chain, regardless of the length of the chain. These nucleotides contain a sugar, a phosphate and a base, which is a purine or a pyrimidine. Nucleic acid molecules can be double-stranded or single-stranded. When single-stranded, nucleic acid molecules can be sense strands or antisense strands. Nucleic acid molecules can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can, for example, be used to prepare nucleic acid molecules with altered base pairing ability or enhanced resistance to nucleases. Nucleic acid sequences provided herein are represented herein from left to right in a 5' to 3' orientation and are represented using the standard code for representing nucleotide characters as set forth in the U.S. Sequence Rules, 37 CFR §§ 1.821-1.825 and World Intellectual Property Organization (WIPO) Standard ST.25.

[0154] A "nucleic acid fragment" is a portion of a given nucleic acid molecule. An "RNA fragment" is a portion of a given RNA molecule. A "DNA fragment" is a portion of a given DNA molecule. A "nucleic acid segment" is a portion of a given nucleic acid molecule and is not isolated from the molecule. An "RNA segment" is a portion of a given RNA molecule and is not isolated from the molecule. A "DNA segment" is a portion of a given DNA molecule and is not isolated from the molecule. A segment of a polynucleotide can be any length, such as at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, or 500 or more nucleotides in length. A segment or portion of a guide sequence can be about 50%, 40%, 30%, 20%, 10% of a guide sequence, such as one-third of a guide sequence or less, such as 7, 6, 5, 4, 3, or 2 nucleotides in length.

[0155] In the context of a molecule, the term "derived from" refers to a molecule that is isolated or made using a parent molecule or information from that parent molecule. For example, the Cas9 single mutant nickase and the Cas9 double mutant null nuclease are derived from the wild-type Cas9 protein.

[0156] In higher plants, deoxyribonucleic acid (DNA) is the genetic material, while ribonucleic acid (RNA) involves the transfer of information contained in DNA to proteins. "Genome" is the entirety of the genetic material contained in each cell of an organism. Unless otherwise indicated, the specific nucleic acid sequence of the present invention also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as sequences explicitly specified. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. [Nucleic Acids Research] 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. [Journal of Biological Chemistry] 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes [Molecular and Cellular Probes] 8: 91-98 (1994)). The term nucleic acid molecule is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0157] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or peptide sequences are invariant over the entire comparison window of components (e.g., nucleotides or amino acids). "Identity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, eds.) Humana Press, New Jersey (1994); 分子生物学中的序列分析 生物学 [Sequence Analysis in Molecular Biology] (von Heinje, G. ed.) Academic Press (1987); and 序列 分析引物 [Primers for sequence analysis] (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0158] As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, "percent identity" may refer to the percentage of identical amino acids in an amino acid sequence.

[0159] As used herein, in the context of two nucleic acid molecules, nucleotide sequences or protein sequences, the phrase "substantially identical" refers to two or more sequences or subsequences with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments of the present invention, there is substantial identity in a sequence region of at least about 50 residues to about 150 residues in length. Therefore, in some embodiments of the present invention, there is substantial consistency in a sequence region of at least about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, or more residues in length. In some specific embodiments, these sequences are substantially identical in at least about 150 residues. In other embodiments, the sequences are substantially identical over the entire length of the coding region. In addition, in representative embodiments, substantially identical nucleotide sequences or protein sequences perform substantially the same function (e.g., directing endonuclease cleavage to a specific genomic target table, a specific genomic target site).

[0160] For sequence comparison, typically, one sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer (if necessary, subsequence coordinates are specified), and the parameters of the sequence algorithm program are specified. The sequence comparison algorithm then calculates the sequence identity percentage of this or these test sequences relative to the reference sequence based on the specified program parameters.

[0161] Optimal alignment of sequences for comparison windows is well known to those skilled in the art and can be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and optionally by computerized implementations of these algorithms, such as Wisconsin (Accelrys Inc., San Diego, California). The "identity score" of the aligned segments of a test sequence and a reference sequence is the number of identical components common to the two aligned sequences divided by the total number of components in the reference sequence segment (i.e., a complete reference sequence or a smaller defined portion of a reference sequence). The percent sequence identity is expressed as the identity score multiplied by 100. The comparison of one or more polynucleotide sequences can be relative to a full-length polynucleotide sequence or a portion thereof, or relative to a longer polynucleotide sequence. For purposes of the present invention, "percent identity" can also be determined using BLASTX, version 2.0, for translated nucleotide sequences and BLASTN, version 2.0, for polynucleotide sequences.

[0162] Software for performing BLAST analysis can be publicly obtained through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words with length W in the query sequence, which match or meet the score T of some positive thresholds when compared with words with the same length in the database sequence. T is called the neighboring word score threshold (Altschul et al., 1990). These initial neighboring word hits serve as seeds for initial searches to find longer HSPs containing them. Then, these word hits are extended along each sequence in both directions until the cumulative comparison score can be increased. For nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty for mismatched residues; always<0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score decreases by the amount X from its maximum achieved value; when the cumulative score approaches or falls below zero due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0163] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1 to less than about 0.001. Thus, in some embodiments of the invention, the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.001.

[0164] When two nucleotide sequences hybridize to each other under stringent conditions, the two nucleotide sequences may also be considered to be substantially identical. In some representative embodiments, two nucleotide sequences considered to be substantially identical hybridize to each other under high stringency conditions.

[0165] In the context of nucleic acid hybridization experiments (such as DNA hybridization and RNA hybridization), "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. A comprehensive guide to nucleic acid hybridization is found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acids, Chapter 2, Part I, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York (1993). In general, highly stringent hybridization and wash conditions are selected to be above the thermal melting point (T) for a specific sequence at a defined ionic strength and pH. m ) is about 5℃ lower.

[0166] T m It is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T for a specific probe. m. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences (which have more than 100 complementary residues on the filter in a Southern or Northern blot) is 50% formamide with 1 mg of heparin at 42°C, where hybridization is performed overnight. An example of high stringency washing conditions is 0.15M NaCl at 72°C for about 15 minutes. An example of stringent washing conditions is washing at 65°C with 0.2x SSC for 15 minutes (see Sambrook, below, for a description of SSC buffer). Typically, a low stringency wash is performed before a high stringency wash to remove background probe signals. An example of a medium stringency wash for a duplex of, for example, more than 100 nucleotides is at 45°C with 1x SSC for 15 minutes. An example of a low stringency wash for a duplex of, for example, more than 100 nucleotides is at 40°C with 4-6xSSC for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. In general, a signal-to-noise ratio of 2x (or higher) observed in a particular hybridization assay compared to an unrelated probe indicates that specific hybridization has been detected. If the proteins encoded by nucleotide sequences that do not hybridize to each other under stringent conditions are substantially identical, then the nucleotide sequences are still substantially identical. For example, this may occur when a copy of a nucleotide sequence is generated using the maximum codon degeneracy allowed by the genetic code.

[0167] The following are examples of hybridization / washing conditions that can be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present invention. In one embodiment, the reference nucleotide sequence is hybridized to the "test" nucleotide sequence at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and washed at 50°C in 2X SSC, 0.1% SDS. In another embodiment, the reference nucleotide sequence is hybridized to the "test" nucleotide sequence at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and washed at 50°C in 1XSSC, 0.1% SDS; or hybridized at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and washed at 50°C in 0.5X SSC, 0.1% SDS. In still other embodiments, the reference nucleotide sequence is hybridized to a "test" nucleotide sequence at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and washed at 50°C in 0.1X SSC, 0.1% SDS; or hybridized at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and washed at 65°C in 0.1X SSC, 0.1% SDS.

[0168] "Isolated" nucleic acid molecules or nucleotide sequences or "isolated" polypeptides are nucleic acid molecules, nucleotide sequences or polypeptides that exist by artificially detaching from their natural environment and / or have different, modified, regulated and / or changed functions when compared with their functions in their natural environment and are therefore not natural products.Isolated nucleic acid molecules or isolated polypeptides can exist in purified form or can exist in non-natural environments (such as, for example, recombinant host cells).Therefore, for example, with respect to polynucleotides, the term isolated means that polynucleotides are separated from chromosomes and / or cells in which they are naturally present.If a polynucleotide is separated from chromosomes and / or cells in which it is naturally present, and then inserted into a genetic background, chromosome, chromosomal position, and / or cell in which it is not naturally present, the polynucleotide is also isolated.Recombinant nucleic acid molecules and nucleotide sequences of the present invention can be considered as "isolated" as defined above.

[0169] Thus, an "isolated nucleic acid molecule" or "isolated nucleotide sequence" is a nucleic acid molecule or nucleotide sequence that is not adjacent to adjacent nucleotide sequences (sequences at the 5' end or sequences at the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately adjacent to the coding sequence. Thus, the term includes, for example, recombinant nucleic acids that are incorporated into a vector, into a self-replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or that exist as a separate molecule (e.g., a cDNA or a genomic DNA fragment obtained by PCR or restriction endonuclease treatment) independent of other sequences. It also includes recombinant nucleic acids that are part of a hybrid nucleic acid molecule encoding another polypeptide or peptide sequence. An "isolated nucleic acid molecule" or "isolated nucleotide sequence" may also include a nucleotide sequence that is derived from and inserted into the same natural original cell type, but is present in a non-natural state, for example, in a different copy number, and / or under the control of regulatory sequences that are different from those found in the natural state of the nucleic acid molecule.

[0170] The term "isolated" may further refer to a nucleic acid molecule, nucleotide sequence, polypeptide, peptide or fragment that is substantially free of cellular material, viral material, and / or culture medium (e.g., when produced by recombinant DNA techniques), or chemical precursors or other chemicals (e.g., when chemically synthesized). Additionally, an "isolated fragment" is a fragment of a nucleic acid molecule, nucleotide sequence or polypeptide that does not naturally exist as a fragment and would not so exist in nature. "Isolated" does not necessarily mean that the preparation is industrially pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form that can be used for the intended purpose.

[0171] In representative embodiments of the invention, "isolated" nucleic acid molecules, nucleotide sequences and / or polypeptides are at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% pure (w / w) or purer. In other embodiments, "isolated" nucleic acids, nucleotide sequences and / or polypeptides refer to at least about 5-fold, 10-fold, 25-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold or greater enrichment (w / w) of nucleic acids compared to the starting material.

[0172] A "wild-type" nucleotide sequence or amino acid sequence refers to a naturally occurring ("native") or endogenous nucleotide sequence or amino acid sequence. Thus, for example, a "wild-type mRNA" is an mRNA naturally present in or endogenous to an organism. A "homologous" nucleotide sequence is a nucleotide sequence naturally associated with the host cell into which it is introduced.

[0173] The terms "open reading frame" and "ORF" refer to the amino acid sequence encoded between the translation start and stop codons of a coding sequence. The terms "start codon" and "stop codon" refer to a unit of three adjacent nucleotides ('codons') in a coding sequence that specify the initiation and chain termination of protein synthesis (mRNA translation), respectively.

[0174] "Promoter" refers to a nucleotide sequence, usually upstream (5') of its coding sequence, which controls the expression of the coding sequence by providing recognition of the RNA polymerase and other factors required for appropriate transcription."Promoter regulatory sequence" consists of proximal and more distal upstream elements. The promoter regulatory sequence affects the transcription, RNA processing or stability, or translation of the relevant coding sequence. Regulatory sequences include enhancers, promoters, non-translated leader sequences, introns, and polyadenylation signal sequences. They include natural sequences and synthetic sequences, and sequences that may be combinations of synthetic sequences and natural sequences. "Enhancer" is a DNA sequence, which can stimulate the activity of the promoter and can be an intrinsic element of the promoter or an inserted heterologous element to enhance the level or tissue specificity of the promoter. It can operate in two directions (normal or flipped), and can even play a role when moved to the upstream or downstream of the promoter. The meaning of the term "promoter" includes "promoter regulatory sequence".

[0175] "Primary transformants" and "T0 generation" refer to transgenic plants of the same genetic generation as the tissue that was initially transformed (i.e., has not undergone meiosis and fertilization since transformation). "Secondary transformants" and "T1, T2, T3, etc. generations" refer to transgenic plants that are derived from primary transformants through one or more meiotic and fertilization cycles. They can be derived by self-fertilization of primary or secondary transformants or hybridization of primary or secondary transformants with other transformed or untransformed plants.

[0176] "Transgenic" refers to a nucleic acid molecule that has been introduced into the genome by transformation and is stably maintained. A transgenic may comprise at least one expression cassette, typically at least two expression cassettes, and may comprise ten or more expression cassettes. A transgenic may include, for example, genes that are heterologous or homologous to the genes of a particular plant to be transformed. In addition, a transgenic may comprise a natural gene that is inserted into a non-natural organism, or a chimeric gene. The term "endogenous gene" refers to a natural gene in its natural location in the genome of an organism. An "exogenous" gene refers to a gene that is not normally found in the host organism but is introduced into the organism by gene transfer.

[0177] "Intron" refers to an intervening segment of DNA that occurs almost exclusively in eukaryotic genes, but which is not translated into an amino acid sequence in the gene product. These introns are removed from immature mRNA by a process called splicing, which leaves the exons untouched, thereby forming the mRNA. For the purposes of the present invention, the definition of the term "intron" includes modifications to the nucleotide sequence derived from an intron of a target gene, provided that the modified intron does not significantly reduce the activity of its associated 5' regulatory sequence.

[0178] "Exon" refers to a segment of DNA that carries the coding sequence for a protein or a portion thereof. Exons are separated by intervening, non-coding sequences (introns). For the purposes of the present invention, the definition of the term "exon" includes modifications to the nucleotide sequence of an exon derived from a target gene, provided that the modified exon does not significantly reduce the activity of its associated 5' regulatory sequence.

[0179] The term "cleavage" or "cleaving" refers to the break of the covalent phosphodiester linkage in the ribosylphosphodiester backbone of a polynucleotide. The term "cleavage" or "cleaving" covers both single-strand breaks and double-strand breaks. As a result of two different single-strand cleavage events, double-strand cleavage can occur. Cutting can result in the production of flat ends or staggered ends. "Nuclease cleavage site" or "genomic nuclease cleavage site" is a nucleotide region including a nuclease cleavage sequence, which is recognized by a specific nuclease, and the nuclease is used to cut the nucleotide sequence of genomic DNA in one or two chains. This cutting by nuclease triggers a DNA repair mechanism in the cell, which establishes an environment where homologous recombination occurs.

[0180] The present invention provides a fusion protein having an improved linker between a deaminase domain and a site-directed DNA-binding domain, providing increased editing efficiency and reduced mutation frequency. In some embodiments of the present invention, the deaminase domain is a cytidine deaminase. In other embodiments of the present invention, the deaminase domain is an adenine deaminase. In some embodiments, the cytidine deaminase domain is an activation-induced cytidine deaminase ("AID"). In some embodiments of the present invention, the cytidine deaminase domain is an apolipoprotein B mRNA-editing complex ("APOBEC") domain. In some embodiments, the APOBEC domain is an APOBEC1 family deaminase.

[0181] "Cytidine deaminase" refers to an enzyme that catalyzes the irreversible hydrolytic deamination of cytidine and deoxycytidine to produce uridine and deoxyuridine, respectively. Cytidine deaminase maintains the cytosine pool. The family of cytidine deaminases is APOBEC ("Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like"). Members of this family are C to U editing enzymes. The N-terminal domain of APOBEC-like proteins is the catalytic domain, while the C-terminal domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain and is important for cytidine deamination. RNA editing of APOBEC1 requires homodimerization, and the complex interacts with RNA-binding proteins to form an editing body. Non-limiting examples of APOBEC proteins include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase. A variety of mutants of APOBEC proteins are also known, and these mutants bring different editing features of base editors. For example, for human APOBEC3A, some mutants (e.g., Y130F, Y132D, W104A, and D131Y) are even better than wild-type human APOBEC3A according to editing efficiency. Therefore, the term APOBEC and each of its family members also covers a certain level (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%) of sequence identity with the corresponding wild-type APOBEC protein, and variants and mutants that retain cytidine deamination activity. Variants and mutants can be obtained by amino acid addition, deletion, and / or substitution. In some embodiments, such substitutions are conservative substitutions.

[0182] A “cytosine base editor” (“CBE”) converts a C·G base pair into a T·A base pair.

[0183] "Adenine deaminase" refers to an enzyme that catalyzes the hydrolytic deamination of adenosine to produce inosine. Inosine pairs with a C and is therefore read or copied as a G. An exemplary enzyme is TadA from E. coli, which functions as a homodimer.

[0184] An “adenine base editor” (“ABE”) converts an A·T base pair into a G·C base pair.

[0185] Lachnospiraceae bacterium Cpf1 (LbCpf1) is one of a large group of many Cpf1 proteins. The terms "Cpf1" and "Cas12a" are used interchangeably throughout the text. Cpf1 is a Cas protein. The term "Cas protein" or "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein" refers to RNA-guided DNA endonucleases Cas proteins associated with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-adaptive immune systems found in, for example, Streptococcus pyogenes and other bacteria, including Cas9, Cas12a, Cas12b, Cas12i, Cas12j, etc. In some embodiments of the present invention, the site-directed DNA binding domain is a catalytically inactive Cas12a from Lachnospiraceae bacteria ("dLbCas12a"). In other embodiments, the site-directed DNA binding domain is catalytically active and is from a bacterium of the genus Lachnospiraceae ("LbCas12a") or Moraxella bovoculi AAX08_00205 ("Mb2Cas12a"). In some embodiments of the invention, a Cas12a protein from a bacterium of the genus Lachnospiraceae, a species of Acidococcus, a species of Moraxella bovoculi, a species of Thiomicrospira sp., a species of Moraxella lacunata, Methanomethylophilus alvus, a species of Btyrivibrio sp., or a species of Bacteroidetesoral sp. is provided as a site-directed DNA-binding domain of a fusion protein.

[0186] The fusion protein may include other fragments such as uracil DNA glycosylase inhibitor (UGI) and nuclear localization sequence (NLS).

[0187] "Uracil glycosylase inhibitor" (UGI), which can be prepared from Bacillus subtilis phage PBS1, is a small protein (9.5 kDa) that inhibits Escherichia coli uracil-DNA glycosylase (UDG) as well as UDG from other species. Inhibition of UDG occurs by reversible protein binding with a 1:1 UGD:UGI stoichiometry. UGI is capable of dissociating UDG-DNA complexes. A non-limiting example of UGI is found in Bacillus phage AR9 (YP_009283008.1). In some embodiments, UGI comprises the amino acid sequence of SEQ ID NO:8 or has at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:8, and retains uracil glycosylase inhibitory activity.

[0188] In some embodiments, the UGI is located on the C-terminal side of the cytidine deaminase-Cpf1 portion. In some embodiments, the fusion protein comprises at least two UGIs.

[0189] In some embodiments, at least one nuclear localization signal ("NLS") is located at the C-terminus of the first segment and the second segment (the cytidine deaminase-Cpf1 portion), such as between the second segment (including Cpf1) and UGI. In some embodiments, at least two NLSs are located between the second segment and UGI. In some embodiments, at least three NLSs are located between the second segment and UGI. In some embodiments, at least one NLS is located at the N-terminus of the first segment and the second segment (the cytidine deaminase-Cpf1 portion).

[0190] Non-limiting example arrangements of components in the fusion protein include, from N-terminus to C-terminus, (a) NLS, cytidine deaminase, Cas12a, NLS, UGI, NLS, 2A, and UGI; (b) NLS, cytidine deaminase, Cas12a, NLS, NLS, UGI, NLS, 2A, and UGI; (c) NLS, cytidine deaminase, Cas12a, NLS, UGI, NLS, 2A, UGI, 2A, and UGI; (d) NLS, cytidine deaminase, Cas12a, NLS, UGI, NLS, 2A, UGI, 2A, UGI, 2A, and UGI.

[0191] In some embodiments, a peptide linker is optionally provided between each fragment in the fusion protein. In some embodiments, the peptide linker has 1 to 100 amino acid residues (or 3-20, 4-15, without limitation). In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the amino acid residues of the peptide linker are selected from the group consisting of: alanine, glycine, cysteine ​​and serine.

[0192] The present invention also provides nucleic acid molecules comprising nucleic acid sequences encoding guide RNAs of the present invention. Nucleic acid molecules can be DNA or RNA molecules. In some embodiments, nucleic acid molecules are cyclized. In other embodiments, nucleic acid molecules are linear. In some embodiments, nucleic acid molecules are single-stranded, partially double-stranded, or double-stranded. In some embodiments, nucleic acid molecules are compounded with at least one polypeptide. The polypeptide may have a nucleic acid recognition domain or a nucleic acid binding domain. In some embodiments, the polypeptide is used to mediate, for example, a shuttle for the delivery of a chimeric RNA of the present invention and optionally a nuclease. In some embodiments, the polypeptide is a Feldan shuttle (U.S. Patent Publication No. 20160298078, incorporated herein by reference).

[0193] "On-target editing" is the replacement of thymine by cytosine in the region after the PAM site targeted by the gRNA. The primary editing window is 8 to 13 bases after the PAM site. "Off-target editing" is an insertion or deletion (indel) or base change other than C to T in the gRNA target region, or a base change or insertion or deletion outside the gRNA target region.

[0194] A "site-directed modifying polypeptide" modifies a target DNA (e.g., cleavage or methylation of a target DNA) and / or a polypeptide associated with a target DNA (e.g., methylation or acetylation of a histone tail). A site-directed modifying polypeptide is also referred to herein as a "site-directed polypeptide" or "RNA-binding site-directed modifying polypeptide." Due to the association of a site-directed modifying polypeptide with a guide RNA, the site-directed modifying polypeptide interacts with the guide RNA (the guide RNA is a single RNA molecule or an RNA duplex of at least two RNA molecules) and is guided to a DNA sequence (e.g., a chromosomal sequence or an extrachromosomal sequence, such as an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.).

[0195] In some cases, the site-directed modifying polypeptide is a naturally occurring modified polypeptide. In other cases, the site-directed modifying polypeptide is not a naturally occurring modified polypeptide (e.g., a chimeric polypeptide or a modified (e.g., mutated, deleted, inserted) naturally occurring polypeptide). Exemplary naturally occurring site-directed modifying polypeptides are known in the art (see, e.g., Makarova et al., 2017, Cell 168:328-328.e1, and Shmakov et al., 2017, Nat Rev Microbiol 15(3):169-182, both of which are incorporated herein by reference). These naturally occurring polypeptides bind to the DNA targeting RNA and are thereby directed to a specific sequence within the target DNA and cut the target DNA, thereby producing a double-strand break.

[0196] The site-directed modifying polypeptide comprises two parts, namely an RNA binding part and an active part. In some embodiments, the site-directed modifying polypeptide comprises: (i) an RNA binding part that interacts with a DNA-targeting RNA, wherein the DNA-targeting RNA comprises a nucleotide sequence complementary to a sequence in the target DNA; and (ii) an active part that exhibits site-directed enzymatic activity (e.g., DNA methylation activity, DNA cleavage activity, histone acetylation activity, histone methylation activity, etc.), wherein the site of enzymatic activity is determined by the DNA-targeting RNA. In other embodiments, the site-directed modifying polypeptide comprises: (i) an RNA binding part that interacts with a DNA-targeting RNA, wherein the DNA-targeting RNA comprises a nucleotide sequence complementary to a sequence in the target DNA; and (ii) an active part that regulates transcription (e.g., increases or decreases transcription) in the target DNA, wherein the site of regulated transcription in the target DNA is determined by the DNA-targeting RNA.

[0197] In some cases, the site-directed modification polypeptide has an operably connected heterologous domain. The heterologous domain can be an enzyme or a signal peptide. In the aspect that the heterologous domain is an enzyme domain, the domain has an enzymatic activity (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, reverse transcriptase activity, dismutase activity, alkylation activity, methylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity) that modifies the target nucleic acid. In other cases, the site-directed modifying polypeptide has an operably linked enzyme domain whose enzymatic activity modifies a polypeptide (e.g., a histone) associated with the target DNA (e.g., a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, an ubiquitin ligase activity, a deubiquitinating activity, adenylating activity, deadenylating activity, SUMOylating activity, deSUMOylating activity, ribosylating activity, deribosylating activity, myristoylation activity, or demyristoylation activity). Exemplary enzyme domains include adenosine deaminase, oxidase, thymine alkyltransferase, adenine oxidase, adenosine methyltransferase, adenosine deaminase, glycosylase, either alone or in combination with other enzyme domains. In aspects where the heterologous domain is a signal peptide, the signal peptide can be a nuclear localization signal ("NLS"), such as an SV40 NLS.

[0198] In some cases, different site-directed modification polypeptides, such as different Cas9 proteins (i.e., Cas9 proteins from a variety of species) can be advantageously used in a variety of methods provided by the present invention to utilize a variety of enzyme features of different Cas9 proteins (e.g., for different PAM sequence preferences; for increased or decreased enzyme activity; for increased or decreased cytotoxicity levels; for changing the balance between NHEJ, homology-directed repair, single-strand breaks, double-strand breaks, etc.). Cas9 proteins from a variety of species (e.g., those disclosed in Shmakov et al., 2017, or polypeptides derived therefrom) may require different PAM sequences in the target DNA. Therefore, for the selection of a specific Cas9 enzyme, the PAM sequence requirement may be different from the 5'-NGG-3' sequence (wherein N is A, T, C, or G) known to be required for Cas9 activity. Many Cas9 orthologs from a variety of species have been identified herein, and these proteins share only a few identical amino acids. All identified Cas9 orthologs have the same domain architecture as the central HNH endonuclease domain and the separate RuvC / RNase H domain. Cas9 proteins share 4 key motifs with conserved structures; motifs 1, 2, and 4 are RuvC-like motifs, while motif 3 is an HNH motif. In contrast, Cas12a proteins from different species may have different PAM sequence requirements compared to the canonical PAM of LbCas12a of TTTV.

[0199] The site-directed modification polypeptide can also be a chimeric and modified CRISPR / Cas nuclease. For example, it can be a modified Cas9 "base editor". Base editing enables a target DNA base to be directly and irreversibly converted to another base in a programmable manner without the need for DNA cutting or donor DNA molecules. For example, Komor et al. (2016, Nature [Nature], 533: 420-424) teach Cas9-cytidine deaminase fusion, in which Cas9 has also been engineered to be inactive and does not induce double-stranded DNA breaks. In addition, Gaudelli et al. (2017, Nature [Nature], doi: 10.1038 / nature24644) teach catalytically impaired Cas9 fused to tRNA adenosine deaminase, which can mediate the conversion of A / T to G / C in the target DNA sequence. Another class of engineered Cas9 nucleases that can serve as site-directed modifying polypeptides in the methods and compositions of the present invention are variants that can recognize a wide range of PAM sequences, including NG, GAA, and GAT (Hu et al., 2018, Nature, doi: 10.1038 / nature26155).

[0200] Example

[0201] In one embodiment, we provide a fusion protein comprising a heterologous domain, a first linker sequence, and a V-type CRISPR-Cas enzyme in the direction of N-terminus to C-terminus, wherein the first linker sequence comprises a repeated GGGGS sequence. On the one hand, the heterologous domain is a deaminase, a polymerase, a nuclease, a relaxase, an alkyltransferase, a methyltransferase, adenosine deaminase, a cytidine deaminase, an oxidase, a thymine alkyltransferase, adenine oxidase, adenosine methyltransferase, a glycosylase, or a nuclear localization signal. On the other hand, the heterologous domain is a deaminase domain. On the other hand, the deaminase domain is a cytidine deaminase. On the other hand, the cytidine deaminase domain is an activation-induced cytidine deaminase ("AID"). On the other hand, the cytidine deaminase domain is an apolipoprotein B mRNA-editing complex ("APOBEC") domain. On the other hand, the APOBEC domain is an APOBEC1 family deaminase. In yet another aspect, the APOBEC domain comprises a sequence at least 70% identical to SEQ ID NO: 1. In another aspect, the deaminase domain is an adenine deaminase. In yet another aspect, the adenine deaminase is a TadA domain comprising a sequence at least 70% identical to SEQ ID NO:92.

[0202] On the one hand, the V-type CRISPR-Cas enzyme is a VA-type ("Cas12a") enzyme. On the other hand, the Cas12a domain is selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48. On the other hand, the Cas12a domain is catalytically inactive and is selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 22.

[0203] In one aspect, the first linker sequence comprises GGGGS repeated at least three times. In another aspect, the first linker sequence comprises GGGGS repeated at least six times.

[0204] On the one hand, the fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO: 11, 12, 13 and 44. On the other hand, the fusion protein further comprises a uracil DNA glycosylase inhibitor ("UGI") domain. On the other hand, the UGI domain comprises SEQ ID NO: 8. On the other hand, the UGI domain is connected to the Cas12a enzyme via a second linker comprising the sequence SGGS. On the other hand, the fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 and SEQ ID NO: 89. In another aspect, the fusion protein produces on-target edits at an increased frequency and off-target edits at a decreased frequency when contacted with DNA compared to a fusion protein having a first linker sequence in addition to a repeated GGGGS sequence.

[0205] In another embodiment, we provide a method for editing plant genomic DNA, the method comprising contacting the plant genomic DNA with: (a) the fusion protein of the above aspects, and the fusion protein optionally comprises a UGI domain; and (b) a guide RNA ("gRNA") that targets the fusion protein of step (a) to a target DNA sequence of the plant genomic DNA; wherein the edited plant genomic DNA comprises reduced off-target editing compared to plant genomic DNA edited by a fusion protein having a first linker other than a repeated GGGGS sequence.

[0206] In another embodiment, we provide a method for editing plant genomic DNA with reduced off-target editing, the method comprising contacting the plant genomic DNA with: (a) the fusion protein of the above aspect, and the fusion protein optionally comprises a UGI domain; and (b) a guide RNA ("gRNA") that targets the fusion protein of step (a) to a target DNA sequence of the plant genomic DNA; wherein the edited plant genomic DNA comprises reduced off-target editing compared to the plant genomic DNA edited by the fusion protein having a first linker other than a repeated GGGGS sequence. In one aspect, the fusion protein comprises SEQ ID NO: 24.

[0207] In another embodiment, we provide a method for obtaining an edited plant population with reduced off-target editing, the method comprising: (a) obtaining a population of plant cells comprising genomic DNA to be edited; (b) obtaining a nucleotide sequence encoding the fusion protein of the above aspects, and optionally a UGI domain; (c) transforming the population of plant cells with the nucleotide sequence of step (b), thereby expressing the fusion protein encoded by the nucleic acid sequence within the population of plant cells; (d) growing the transformed plant cell population into plants, wherein at least one plant is edited; and (e) selecting at least one edited plant from the product of step (d), thereby obtaining an edited plant population; wherein the edited plant population comprises reduced off-target editing compared to plants edited by a fusion protein having a first linker other than a repeated GGGGS sequence. In one aspect, the nucleotide sequence encoding the fusion protein comprises SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 89. In some embodiments, codon-optimized polynucleotides are provided that encode fusion proteins comprising one or more DNA binding domains and one or more DNA modifying domains connected by an improved linker sequence.

[0208] Examples

[0209] The following examples provide a number of illustrative embodiments. Based on this disclosure and the general level of skill in the art, it should be understood by those of ordinary skill that the following examples are intended to be illustrative only and that many changes, modifications, and variations may be employed without departing from the scope of the subject matter of this disclosure. Example 1. Vector construction and guide RNA expression of dLbCas12a-BE

[0210] We fused a catalytically inactive Lachnospiraceae bacterial Cas12a containing D832A / E925A / D1148A mutations (hereafter "dLbCas12a", formerly known as dLbCpf1), rat cytidine deaminase (APOBEC1), and uracil DNA glycosylase inhibitor (UGI) connected by an amino acid linker into a single protein to exploit the beneficial properties of base editing in plants. The fusion construct was codon-optimized for maize (Zea mays) and commercially synthesized (GenScript, Nanjing, China), and cloned under the promoter of the sugarcane ubiquitin-4 (SoUbi4) gene to constitutively produce dLbCas12a-BE.

[0211] In the dLbCas12a-BE of construct 24524, the nuclear localization signal (SV40-NLS) is in front of APOBEC1 connected to dLbCas12a via an XTEN protein linker, followed by SV40-NLS connected to UGI via an SGGS linker. SV40-NLS is also incorporated into the C-terminus of UGI via an SGGS linker to improve the targeting of the fusion protein to the nucleus. The synthetic sequence of dLbCas12a-BE made using maize optimized codons is listed in SEQ ID NO: 18.

[0212] In the dLbCas12a-BE of construct 24904, the SV40-NLS is in front of APOBEC1 connected to dLbCas12a by a 30 amino acid linker GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 11) with six GGGGS amino acid repeats (referred to as (G4S) x6), followed by the SV40NLS connected to UGI by a SGGS linker. SV40-NLS is also incorporated into the C-terminus of UGI by a SGGS linker to improve the targeting of the fusion protein to the nucleus. The synthetic sequence of dLbCas12a-BE made using maize optimized codons is listed in SEQ ID NO: 23.

[0213] In the dLbCas12a-BE of construct 25057, the SV40-NLS is in front of APOBEC1 connected to dLbCas12a by an XTEN protein linker, followed by the SV40-NLS connected to UGI by an 18 amino acid linker GGSTG GGSGG GSGGG SSG (SEQ ID NO: 12) (referred to as SX). The SV40-NLS is also incorporated into the C-terminus of UGI by a 15 amino acid linker GGGGS GGGGS (referred to as (G4S) x3) to improve the targeting of dLbCas12a-BE to the nucleus. The dLbCas12a-BE synthetic sequence made using maize optimized codons is listed in SEQ ID NO: 14.

[0214] In the dLbCas12a-BE of construct 25058, the SV40-NLS is in front of APOBEC1 connected to dLbCas12a by a 30 amino acid linker ((G4S)x6), followed by the SV40-NLS connected to UGI by a SX linker. SV40-NLS is also incorporated into the C-terminus of UGI by (G4S)x3 to improve the targeting of dLbCas12a-BE to the nucleus. The dLbCas12a-BE synthetic sequence made using corn optimized codons is listed in SEQ ID NO: 16.

[0215] In the dLbCas12a-BE construct, CRISPR / Cas12a guide RNA transcripts are expressed under the control of the SoUbi4 promoter, which targets the 4th exon region of maize Waxy1, following the PAM sequence in exon 4 to change C9, C10 or C22 to T. It also includes a direct repeat of LbCrRNA as a scaffold. The synthetic sequence of the guide RNA is listed in SEQ ID NO: 26.

[0216] In construct 24784, the nuclear localization signal (xSV40NLS-06) is in front of the cytidine deaminase (xAPOBEC1-01) connected to the maize optimized Cas9 gene (cCas9BE-02) through xXTEN-02, followed by the nuclear localization signal (xSV40NLS-04) connected to the uracil DNA glycosylase inhibitor xUGI-02 through xSGGS linker-02, which is connected to the nuclear localization signal xSV40NLS-07 through xSGGS linker-02. The fusion protein is under the control of the sugarcane ubiquitin-4 promoter (prSoUbi4-02) and then driven under the control of the NOS terminator (tNOS-05-01). The Cas9 protein is a nickase Cas9 mutant in which D10A is fused to rat APOBEC1 and uracil DNA glycosylase inhibitor (UGI). A nuclear localization signal was also incorporated into the C-terminus of Cas9 to improve its targeting to the nucleus. The synthetic sequence of cCas9BE-02 is listed in SEQ ID NO:20.

[0217] Example 2. Agrobacterium-mediated transformation of maize embryos

[0218] To generate potential events for editing in maize Wx1, the elite maize transformation variety NP2222 was selected for all experiments described (WO 16106121, incorporated herein by reference).

[0219] Maize variety NP2222 was used for maize transformation. Maize ears were harvested from GH when the immature embryos were about 1.2 mm, and then the ears were sterilized with 20% Clorox solution for 20 minutes and rinsed 3 times with sterile water.

[0220] The Agrobacterium tumefaciens strain LBA4404 17740RecA carrying the vector was electroporated - Streak on YP medium containing Gent (25 μg / ml) and Spec (100 μg / ml) antibiotics and grow at 28°C for 2 days. Before transformation, select a single colony and streak onto a fresh YP plate and grow at 28°C for 1 day. Resuspend the Agrobacterium using inoculation medium. Set the OD 660 Adjust to 0.25.

[0221] We removed the endosperm, then separated and collected the immature embryos with a sterile scalpel and injected them into the Agrobacterium suspension for two to three minutes. The infected immature embryos were transferred to co-cultivation medium at 22°C for two to four days.

[0222] After the co-cultivation period, the embryos were transferred to a medium containing the selection agent for four weeks at 28°C in the dark. The resistant embryonic callus was transferred to a regeneration medium and cultured at 28°C with a 16 / 8 photoperiod. After approximately three weeks, the regenerated plantlets were transferred to a growth container containing a rooting medium under the same culture temperature and light conditions. Example 3. Analysis of edited bases in the target region

[0223] We used Phire Plant Direct PCR Master Mix (Thermo Fisher, F160L) to directly amplify a DNA fragment of approximately 410 bp containing the target region from corn leaf samples. No DNA purification was required before PCR. The amplified DNA fragment was subjected to Sanger DNA sequencing to analyze mutations at the target site.

[0224] DNA extraction and PCR amplification were performed according to the manufacturer's recommendations. A young leaf (e.g., a punch of approximately 2 mm in diameter) was placed in 30 μL of dilution buffer. The leaf sample was crushed with a 100 μL pipette tip by briefly pressing the tube wall and adding 20 μL of dilution buffer. After crushing the leaf, the solution was green. The plant material was spun down in a centrifuge and 1 μL of the supernatant was used as a template for a 20 μL PCR reaction.

[0225] The PCR system consists of:

[0226]

[0227] PCR primers for ZmWaxy1:

[0228] Forward primer: 5'-AGATGGGAGACGGGTACGAGACGG-3' (SEQ ID NO: 29)

[0229] Reverse primer: 5'-GTATGGGTTGTTGTTGAGGCTCAGG-3' (SEQ ID NO: 30)

[0230] DNA sequencing primer: 5'-GACCACCCACTGTTCCTGGAGAGGG-3' (SEQ ID NO: 31)

[0231] PCR conditions:

[0232] 98°C for 5 minutes;

[0233] 35 cycles: 98°C for 5 seconds, followed by 60°C for 5 seconds;

[0234] 72°C for 20 seconds;

[0235] 72°C for 1 minute; and

[0236] Keep at 4 °C until ready for analysis.

[0237] Sequencing:

[0238] PCR products were separated by agarose gel electrophoresis and purified with specific primers before Sanger DNA sequencing. For heterozygous mutations, double peaks were observed at the target nucleotide position, while the only single peak different from the control was considered to be a homozygous mutation. Transgenic events of constructs 24524, 24904, and 24784 were used to amplify the ZmWxy1 exon 4 region for sequencing to evaluate base editing.

[0239] Table 1. CRISPR / Cas cytidine base editors (“CBE”) APOBECs containing an XTEN linker between a cytidine deaminase and a nickase Cas9 (“nCas9-CBE”)

[0240]

[0241] The edited nucleotides are shaded in gray. As shown above, this version of the Cas12a base editor, which contains an XTEN linker between the APOBEC domain and the site-directed nuclease, most efficiently edits cysteine ​​to thiamine at positions 5 and 6. However, there are instances of guanine to adenine editing at positions -2, 7, and 49. The position is determined by the number of nucleotides from the start of the PAM site.

[0242] Table 2. CRISPR / Cas cytidine base editors containing an XTEN linker between APOBEC deaminases and dLbCas12a

[0243]

[0244] The edited nucleotides are shaded in gray. In this version, the Cas12a base editor, which contains an XTEN linker between the APOBEC domain and the inactivated site-directed nuclease, edits cysteine ​​to thiamine at positions 9, 10, and 22, and edits guanine to adenine at positions 39, 44, 52, and especially 53. The editing of guanine to adenine indicates that the editing occurs on the complement strand.

[0245] Table 3. CRISPR / Cas cytidine base editors containing long linkers between deaminases and dLbCas12a

[0246]

[0247] The edited nucleotides are shaded in gray. In this version, a Cas12a base editor containing a long linker containing (G4S)6 between the APOBEC domain and the inactivated site-directed nuclease edits cysteine ​​to thiamine at positions 9 and 10, and guanine to adenine at positions 19 and 53. The editing of guanine to adenine indicates that the editing occurred on the complement strand.

[0248] Example 4. Measuring editing efficiency.

[0249] Table 4. Base editing efficiency of maize Wxy1 by the dLbCas12a-CBE system.

[0250]

[0251]

[0252] Table 4 shows how the base editing efficiency of Cas12a with a long linker is comparable to that of Cas9. Without optimization, Cas12aBE has a poor editing efficiency of about 5%, far lower than the editing efficiency of Cas9 (87%). However, by adding a long linker to operably connect the deaminase to the catalytically inactive Cas12a, the editing efficiency is increased by 12 times.

[0253] Table 5. Editing efficiency of SBEIIb by LbCas12a with long linkers.

[0254]

[0255] Table 5 shows a direct comparison between the editing efficiencies of LbCas12a base editors when operably linked to an XTEN linker or a long linker. When the deaminase was operably linked to the site-directed nuclease via a long linker (e.g., (G4S)6), the editing efficiency of difficult targets was increased by nearly 5-fold.

[0256] Table 6. Editing efficiency of Waxy1 by LbCas12a with long linkers.

[0257]

[0258] Table 7. Multiple editing of SBEIIb, Waxy1 and Glossy2 by LbCas12a with long linkers.

[0259]

[0260] High editing efficiencies were achieved using multiple simultaneous editing of several guide RNA molecules within the same construct ("multiplexing" or "multiplex editing") and long linkers between the nuclear localization signal and active Cas12a. Even challenging targets, such as SBEIIb, achieved acceptable editing efficiencies when used as part of a multiplex editing experimental design.

[0261] Example 5. Improved editing in soybean.

[0262] Using a combination of long linkers and Cas12a also greatly improved soybean editing. Standard Cas12a and long linker-Cas12a improved GmFAD2 editing by nearly 7-fold.

[0263] Table 8. GmFAD2 editing

[0264]

[0265] Example 6. Long linkers improve Mb2Cas12a editing in maize.

[0266] Long linkers also improve the editing efficiency of alternative Cas12 enzymes, such as Mb2Cas12a.

[0267] Table 9. Editing by Mb2Cas12a with long linkers.

[0268]

[0269]

[0270] Without the long linker, Mb2Cas12a did not edit the target sequence. However, with the long linker, the editing efficiency was significantly improved.

[0271] Example 7. Other heterologous domains are operably linked to Cas12a via long linkers.

[0272] It is also within the scope of the present invention that a heterologous domain (except only APOBEC deaminase) is connected to Cas12a by a long linker. Such heterologous domains include, but are not limited to, deaminases, polymerases, nucleases, relaxases, alkyltransferases, methyltransferases, adenosine deaminases, cytidine deaminases, oxidases, thymine alkyltransferases, adenine oxidases, adenosine methyltransferases, glycosylases or nuclear localization signals.

[0273] We operably linked adenine deaminase to Cas12a to create a Cas12a adenine base editor ("Cas12a-ABE"). We fused catalytically inactive LbCas12a (containing D832A, E925A, and D1148A mutations) to E. coli wild-type adenine deaminase (engineered "TadA" containing W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N amino acid substitutions) operably linked via an amino acid linker. The fusion construct was codon-optimized for maize (Zea mays) and commercially synthesized (GenScript, Nanjing, China) and cloned under the promoter of the Sugarcane ubiquitin-4 (SoUbi4) gene to constitutively produce dLbCal2a-ABE.

[0274] In the dLbCas12a-ABE of construct 25459, a 189 bp potato intron was inserted into the TadA coding sequence, which was preceded by a TadA variant connected by an XTEN protein linker to produce a TadA dimer. It was fused to dLbCas12a, and SV40-NLS was incorporated into the C-terminus of dLbCas12a via a GS linker to improve the targeting of the fusion protein to the nucleus. The dLbCas12a-ABE synthetic sequence made using corn optimized codons is listed in SEQ ID NO: 79.

[0275] In the dLbCas12a-ABE of construct 25504, a 189bp potato intron is inserted into the TadA coding sequence, which is preceded by a TadA variant to produce a TadA dimer. It is connected to dLbCas12a by 30 amino acid linkers (G4S) x6 protein linkers, and SV40-NLS is incorporated into the C-terminus of dLbCas12a by a GS linker to improve the targeting of the fusion protein to the nucleus. The dLbCas12a-ABE synthetic sequence made using corn optimized codons is listed in SEQ ID NO:81.

[0276] In the dLbCas12a-ABE construct, CRISPR / Cas12a guide RNA transcripts are expressed under the control of the SoUbi4 promoter, which targets the corn Waxy1 gene. It also includes a direct repeat of the LbCrRNA as a scaffold. The synthetic sequence of the guide RNA is listed in SEQ ID NO: 74.

[0277] In experiments with construct 25459, in which adenine deaminase was linked to dLbCas12a via an XTEN linker, no detectable editing occurred when used in corn plants. In experiments with construct 25504, in which adenine deaminase was linked to dLbCas12a via a (G4S)*6 long linker, an editing efficiency of 7% was achieved, about half that of the Cas9ABE control (construct 24785). See Table 10.

[0278] Table 10.dLbCas12aABE

[0279]

[0280] We believe this represents the first time that Cas12aABE has been demonstrated to be effective in plants. It is believed that the use of a long linker to operably link adenine deaminase to Cas12a is the reason for the success of this technology.

[0281] Example 8. Dual base editor in corn

[0282] Dual base editor (fused to the cytidine deaminase domain and adenine deaminase domain of the Cas enzyme). In this concept, targeted saturation mutagenesis of crop genes can be applied to generate genetic variants with improved agronomic performance, for example, C:G>T:A and A:T>G:C substitutions in the same target region. We multiplexed four guide RNAs: one targeting the ZmWaxy1 gene, and three different guide RNAs targeting the ZmADH gene.

[0283] Table 11. Dual CBE-ABE Cas12a editing frequency in maize.

[0284]

[0285] Table 12. Editing by dual CBE-ABE Cas12a in maize.

[0286]

[0287] In total, dLbCas12a-based CBE-ABEs had 1% C to T and A to G mutations. Adding introns increases vector stability but may reduce enzyme activity due to inefficient splicing. This is believed to be the first example of dual CBE-ABE editing in plants using Cas12a.

[0288] Summary table

[0289]

[0290]

[0291] In the above table, most Cas12aBE constructs follow the pattern of heterologous enzyme domain-linker-Cas enzyme. The exceptions to this pattern are: 25702 [TadA dimer-linker-PmCDA-linker-Cas enzyme], 25701 [PmCDA-linker-TadA dimer-linker-Cas enzyme], and 25658 [TadA dimer-linker-Cas enzyme-PmCDA]. Additional nuclear localization sequences, uracil glycosylase inhibitors, and other components may be present, but are not shown in this table. Such details appear in the sequences provided in the attached sequence table.

[0292] These examples and embodiments provided herein are non-limiting illustrations of the claims and should not be construed as the only available examples. Additional variations may be made by those skilled in the art.

[0293] Some embodiments of the present invention are as follows:

[0294] 1. A fusion protein comprising a heterologous domain, a first linker sequence, and a V-type CRISPR-Cas enzyme in an N-terminal to C-terminal direction, wherein the first linker sequence comprises a repeated GGGGS sequence.

[0295] 2. The fusion protein of embodiment 1, wherein the heterologous domain is a deaminase, a polymerase, a nuclease, a relaxase, an alkyltransferase, a methyltransferase, an adenosine deaminase, a cytidine deaminase, an oxidase, a thymine alkyltransferase, adenine oxidase, an adenosine methyltransferase, a glycosylase, or a nuclear localization signal.

[0296] 3. The fusion protein of embodiment 2, wherein the heterologous domain is a deaminase domain.

[0297] 4. The fusion protein of embodiment 3, wherein the deaminase domain is cytidine deaminase.

[0298] 5. The fusion protein of embodiment 4, wherein the cytidine deaminase domain is activation-induced cytidine deaminase ("AID").

[0299] 6. The fusion protein of embodiment 4, wherein the cytidine deaminase domain is an apolipoprotein B mRNA editing complex ("APOBEC") domain.

[0300] 7. The fusion protein of embodiment 6, wherein the APOBEC domain is an APOBEC1 family deaminase.

[0301] 8. The fusion protein of embodiment 7, wherein the APOBEC domain comprises a sequence that is at least 70% identical to SEQ ID NO: 1.

[0302] 9. The fusion protein of embodiment 3, wherein the deaminase domain is adenine deaminase.

[0303] 10. The fusion protein of embodiment 9, wherein the adenine deaminase is a TadA domain.

[0304] 11. The fusion protein of embodiment 10, wherein the TadA domain comprises a sequence that is at least 70% identical to SEQ ID NO:92.

[0305] 12. The fusion protein of embodiment 1, wherein the type V CRISPR-Cas enzyme is a type VA (Cas12a) enzyme.

[0306] 13. A fusion protein as described in embodiment 12, wherein the Cas12a domain is selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48.

[0307] 14. The fusion protein of embodiment 13, wherein the Cas12a domain is catalytically inactive and is selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 22.

[0308] 15. The fusion protein of embodiment 1, wherein the first linker sequence comprises GGGGS repeated at least three times.

[0309] 16. The fusion protein of embodiment 15, wherein the first linker sequence comprises GGGGS repeated at least six times.

[0310] 17. The fusion protein of embodiments 1-16, wherein the fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO: 11, 12, 13 and 44.

[0311] 18. The fusion protein of embodiments 1-17, further comprising a uracil DNA glycosylase inhibitor ("UGI") domain.

[0312] 19. The fusion protein of embodiment 18, wherein the UGI domain comprises SEQ ID NO:8.

[0313] 20. A fusion protein as described in embodiment 19, wherein the UGI domain is connected to the Cas12a enzyme via a second linker comprising the sequence SGGS.

[0314] 21. The fusion protein of embodiment 1, wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 89.

[0315] 22. The fusion protein of embodiment 1, wherein the fusion protein produces on-target editing at an increased frequency and off-target editing at a reduced frequency when contacted with DNA compared to a fusion protein having a first linker sequence in addition to a repeated GGGGS sequence.

[0316] 23. A method for editing plant genomic DNA, the method comprising contacting the plant genomic DNA with:

[0317] (a) a fusion protein as described in embodiments 1-17, and the fusion protein optionally comprises a UGI domain; and

[0318] (b) a guide RNA ("gRNA") that targets the fusion protein of step (a) to a target DNA sequence of the plant genomic DNA;

[0319] Wherein the edited plant genomic DNA comprises reduced off-target editing compared to plant genomic DNA edited by a fusion protein having a first linker other than a repeated GGGGS sequence.

[0320] 24. A method of editing plant genomic DNA with reduced off-target editing, the method comprising contacting the plant genomic DNA with:

[0321] (a) a fusion protein as described in embodiments 1-17, and the fusion protein optionally comprises a UGI domain; and

[0322] (b) a guide RNA ("gRNA") that targets the fusion protein of step (a) to a target DNA sequence of the plant genomic DNA;

[0323] Wherein the edited plant genomic DNA comprises reduced off-target editing compared to plant genomic DNA edited by a fusion protein having a first linker other than a repeated GGGGS sequence.

[0324] 25. A method as described in embodiment 24, wherein the fusion protein comprises SEQ ID NO:24.

[0325] 26. A method of obtaining an edited plant population with reduced off-target editing, the method comprising:

[0326] (a) obtaining a population of plant cells comprising genomic DNA to be edited;

[0327] (b) obtaining a nucleotide sequence encoding a fusion protein as described in embodiments 1-16, and optionally a UGI domain;

[0328] (c) transforming the population of plant cells with the nucleotide sequence of step (b), thereby expressing the fusion protein encoded by the nucleic acid sequence in the population of plant cells;

[0329] (d) growing the population of transformed plant cells into plants, wherein at least one plant cell is edited; and

[0330] (e) selecting the at least one edited plant from the product of step (d), thereby obtaining an edited plant population;

[0331] wherein the edited plant population comprises reduced off-target edits compared to plants edited by a fusion protein having a first linker other than a repeated GGGGS sequence.

[0332] 27. A method as described in embodiment 26, wherein the nucleotide sequence encodes a fusion protein selected from the group consisting of: SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87 and SEQ ID NO:89.

Claims

1. A fusion protein, wherein the fusion protein consists of SEQ ID NO: 17.

Images