CN114317518B - Application of SpRYn-CBE base editing system in base replacement in plant genomes - Google Patents

Abstract

The invention discloses application of a SpRYn-CBE base editing system in plant genome base substitution. The SpRYn-CBE base editing system comprises SpRYn, pmCDA1, UGI and sgRNA; the sgrnas target sequences. Experiments prove that: the SpRYn-CBE base editing system can edit the base C in a target sequence with the PAM sequence of NAN, NCN or NTN on a plant genome, realizes the replacement from the base C to the base T, and greatly expands the range of the editable C.

Description

Application of SpRYn-CBE base editing system in base substitution in plant genomes

Technical Field

The present invention relates to the field of biotechnology, and in particular to the application of the SpRYn-CBE base editing system in plant genome base replacement.

Background Art

CRISPR-Cas9 technology has become a powerful genome editing tool and has been widely used in many tissues and cells. The CRISPR/Cas9 protein-RNA complex is located on the target site through the guide RNA (guide RNA), cutting to produce double-stranded DNA breaks (dsDNA breaks, DSBs), and then the organism will instinctively initiate the DNA repair mechanism to repair DSBs. There are generally two types of repair mechanisms, one is non-homologous end joining (NHEJ) and the other is homologous recombination (homology-directed repair, HDR). Usually NHEJ accounts for the majority, so the random indels (insertions or deletions) generated by the repair are much higher than those of precise repair. For precise base replacement, the application of HDR to achieve precise base replacement is greatly limited because of the low efficiency of HDR and the need for a DNA template.

In 2016, David Liu and Akihiko Kondo's laboratories independently reported two different types of cytosine base editors (CBEs), using two different cytidine deaminases, rAPOBEC1 (rat APOBEC1) and PmCDA1 (activation-induced cytidine deaminase (AID) ortholog from sea lamprey), respectively. The principle of both is to use cytidine deaminase to directly edit a single cytosine (C) base, instead of generating DSB and initiating HDR repair, which greatly improves the base editing efficiency of replacing C with thymine (T). Specifically, dead Cas9 (dCas9) or the Cas9 nickase (Cas9n) is linked to rAPOBEC1 or PmCDA1 to locate the target through guide RNA. rAPOBEC1 or PmCDA1 catalyzes the deamination reaction of cytosine C on the unpaired single-stranded DNA to convert it into uracil (Uracil, U). Through DNA repair, U is paired with adenine (A), and through DNA replication, T is finally paired with A, thereby realizing the conversion from C to T. Among the editors tested, the SpCas9n(D10A)&rAPOBEC1/PmCDA1&UGI base editing system (which contains a uracil DNA glycosylase inhibitor (UGI)) has a higher average mutation rate for two reasons: first, UGI can inhibit the catalytic removal of U from DNA by uracil DNA glycosylase (UDG); second, SpCas9n(D10A) produces a cut on the non-edited strand, inducing the eukaryotic mismatch repair mechanism or the long-patch BER (base-excision repair) repair mechanism, prompting the U:G mismatch to be repaired more preferentially to U:A.

At present, the SpCas9n(D10A)&rAPOBEC1/PmCDA1&UGI base editing system has been widely used in rice to achieve the conversion from C to T, but the editing targets are mainly limited to sequences with PAM (Protospacer Adjacent Motif) as NGG, which greatly limits the range of editable C.

Summary of the invention

The first object of the present invention is to provide a method for mutating C to T in a target sequence of a plant genome.

The method provided by the present invention for mutating C in a plant genome target sequence to T is as follows 1) or 2) or 3) or 4):

The 1) comprises the following steps: introducing SpRYn, cytosine deaminase, sgRNA and UGI into the plant body to achieve mutation of C in the target sequence of the plant genome to T;

The 2) comprises the following steps: introducing SpRYn, cytosine deaminase and sgRNA into the plant body to achieve mutation of C in the target sequence of the plant genome to T;

The 3) comprises the following steps: introducing the coding gene of SpRYn, the coding gene of cytosine deaminase, the DNA molecule for transcribing sgRNA and the coding gene of UGI into the plant body, so that the SpRYn, the cytosine deaminase, the sgRNA and the UGI are all expressed, so as to achieve the mutation of C in the target sequence of the plant genome to T;

The method 4) comprises the following steps: introducing a gene encoding SpRYn, a gene encoding cytosine deaminase and a DNA molecule for transcribing sgRNA into a plant body, so that the SpRYn, the cytosine deaminase and the sgRNA are all expressed, thereby achieving a mutation of C in the target sequence of the plant genome to T;

The sgRNA targets a target sequence;

The PAM sequence of the target sequence is NAN or NCN or NTN; N is A, T, C or G.

In the above method for mutating C in a plant genome target sequence to T, the sgRNA is tRNA-esgRNA;

The tRNA-esgRNA is as shown in Formula I: tRNA-RNA transcribed from the target sequence-esgRNA backbone (Formula I);

The tRNA is m1) or m2) or m3):

m1) RNA molecule obtained by replacing T in positions 597-673 of sequence 1 with U;

m2) an RNA molecule having the same function as the RNA molecule shown in m1) after one or more nucleotides are replaced and/or deleted and/or added;

m3) an RNA molecule having 75% or more identity with the nucleotide sequence defined in m1) or m2) and having the same function;

The esgRNA backbone is n1) or n2) or n3):

n1) RNA molecule obtained by replacing T in positions 694-779 of sequence 1 with U;

n2) an RNA molecule having the same function as the RNA molecule shown in n1) after one or more nucleotides are substituted and/or deleted and/or added;

n3) An RNA molecule that has 75% or more identity with the nucleotide sequence defined in n1) or n2) and has the same function.

In the above method for mutating C in a plant genome target sequence to T, the SpRYn is A1) or A2) or A3):

A1) The amino acid sequence is the protein shown in SEQ ID NO: 2;

A2) a protein having the same function as the amino acid sequence shown in Sequence 2 in the sequence list, wherein one or more amino acid residues are substituted and/or deleted and/or added;

A3) A fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of A1) or A2).

The cytosine deaminase may be a protein such as human APOBEC3A, human AID, PmCDA1 or rAPOBEC1. In a specific embodiment of the present invention, the cytosine deaminase is PmCDA1.

The PmCDA1 is C1) or C2) or C3):

C1) The amino acid sequence is the protein shown in SEQ ID NO: 3;

C2) a protein having the same function as the amino acid sequence shown in Sequence 3 in the sequence list, wherein one or more amino acid residues are substituted and/or deleted and/or added;

C3) A fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of C1) or C2).

The UGI is E1) or E2) or E3):

E1) The amino acid sequence is the protein shown in SEQ ID NO:4;

E2) a protein having the same function as the amino acid sequence shown in Sequence 4 in the sequence list, wherein one or more amino acid residues are substituted and/or deleted and/or added;

E3) A fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of E1) or E2).

In order to facilitate the purification of the protein in A1), C1) or E1), a tag as shown in the following table may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in Sequence 2, Sequence 3 or Sequence 4 in the sequence listing.

Table, sequence of labels

Label Residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHQ c-myc 10 EQKLISEEDL

The protein in A2), C2) or E2) is a protein having 75% or more identity with the amino acid sequence of the protein shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 and having the same function. The 75% or more identity is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

The proteins in A2), C2) or E2) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

The coding gene of the protein in the above A2), C2) or E2) can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in positions 3167-7267, 7553-8176 or 8210-8458 of Sequence 1, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in the above table to its 5' end and/or 3' end. Positions 3167-7267, 7553-8176 and 8210-8458 of Sequence 1 encode the proteins shown in Sequence 2, Sequence 3 and Sequence 4, respectively.

The coding gene of the SpRYn is b1) or b2) or b3):

b1) the cDNA molecule or DNA molecule shown in positions 3167-7267 of Sequence 1 in the sequence listing;

b2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) and encodes the above-mentioned SpRYn;

b3) a cDNA molecule or a DNA molecule that hybridizes with the nucleotide sequence defined in b1) or b2) under stringent conditions and encodes the above-mentioned SpRYn;

The coding gene of PmCDA1 is d1) or d2) or d3):

d1) the cDNA molecule or DNA molecule shown in positions 7553-8176 of Sequence 1 in the sequence listing;

d2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in d1) and encodes the above-mentioned PmCDA1;

d3) a cDNA molecule or a DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the above-mentioned PmCDA1;

The coding gene of the UGI is f1) or f2) or f3):

f1) cDNA molecules or DNA molecules shown in positions 8210-8458 of Sequence 1 in the sequence listing;

f2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in f1) and encodes the above-mentioned UGI;

f3) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined in f1) or f2) under stringent conditions and encodes the above-mentioned UGI.

A person skilled in the art can easily mutate the nucleotide sequence encoding the SpRYn, the PmCDA1 or the UGI of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the SpRYn, the PmCDA1 or the UGI of the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the SpRYn, the PmCDA1 or the UGI and have the same function.

The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of a protein consisting of the amino acid sequence shown in the coding sequence 2, 3 or 4 of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

The stringent conditions are hybridization and washing the membrane twice at 68°C in a 2×SSC, 0.1% SDS solution for 5 min each time, and hybridization and washing the membrane twice at 68°C in a 0.5×SSC, 0.1% SDS solution for 15 min each time; or hybridization and washing the membrane at 65°C in a 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution.

The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

In the above method for mutating C in the target sequence of the plant genome to T, the tRNA-esgRNA obtained after the transcription of the DNA molecule that transcribes the tRNA-esgRNA is an immature RNA precursor, and the tRNA in the RNA precursor will be cut off by two enzymes (RNase P and RNase Z) to obtain a mature RNA. As many independent mature RNAs are obtained as there are targets in a recombinant expression vector, each mature RNA is composed of the RNA transcribed from the target sequence and the esgRNA skeleton, or is composed of the individual bases remaining in the tRNA, the RNA transcribed from the target sequence, and the esgRNA skeleton.

In the above method for mutating C in a plant genome target sequence to T, in 1) and 3), the number of the UGIs may be one or two or more. In a specific embodiment of the present invention, the number of the UGIs is specifically two.

In the above method for mutating C in the target sequence of the plant genome to T, in 3), the coding gene of SpRYn, the DNA molecule for transcribing sgRNA, the coding gene of cytosine deaminase and the coding gene of UGI can be introduced into the plant body through one or more recombinant expression vectors. In a specific embodiment of the present invention, the coding gene of SpRYn, the DNA molecule for transcribing tRNA-esgRNA, the coding gene of PmCDA1 and the coding gene of UGI are introduced into the plant body through one recombinant expression vector.

Furthermore, the recombinant vector also includes a gene encoding a selection agent resistance protein.

Furthermore, the recombinant vector includes an expression cassette containing a DNA molecule for transcribing tRNA-esgRNA and an expression cassette containing, in sequence, the coding gene of the SpRYn, the coding gene of the PmCDA1, the coding gene of the UGI, the coding gene of the UGI, the coding gene of the self-cleaving oligopeptide and the coding gene of the selection agent resistance protein.

The number of expression cassettes containing the DNA molecule for transcribing tRNA-esgRNA may be one, two or more, and may be one, two or three.

The self-cleaving oligopeptide can be a 2A self-cleaving oligopeptide derived from a viral genome, such as a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis virus (ERAV) (E2A) peptide, a Thosea asigna virus (T2A) peptide, a porcine Teschovirus-1 (PTV-1) (P2A) peptide, a Theiler virus 2A peptide, and an encephalomyocarditis virus 2A peptide. Specifically, it can be a P2A peptide.

The selection agent resistance protein may specifically be hygromycin phosphotransferase.

In a specific embodiment of the present invention, the recombinant expression vector is specifically a SpRYn-CBE-9 recombinant expression vector, a SpRYn-CBE-10 recombinant expression vector, a SpRYn-CBE-11 recombinant expression vector, a SpRYn-CBE-12 recombinant expression vector, a SpRYn-CBE-13 recombinant expression vector, a SpRYn-CBE-14 recombinant expression vector, and a SpRYn-CBE-15 recombinant expression vector.

Another object of the present invention is to provide a new use of the above method for mutating C to T in a target sequence of a plant genome.

The present invention provides application of the method for mutating C in a plant genome target sequence to T in plant genome base replacement.

The present invention further provides application of the above-mentioned method of mutating C in a plant genome target sequence to T in plant genome base editing.

The present invention also provides application of the above method for mutating C in a target sequence of a plant genome to T in preparing plant mutants.

Another object of the present invention is to provide a new use of a set of reagents; the set of reagents is R1) or R2):

The R1) includes the SpRYn, the cytosine deaminase and the sgRNA;

The R2) includes the SpRYn, the cytosine deaminase, the sgRNA and the UGI;

The present invention provides a set of reagents for use in any of the following T1)-T7):

T1) Mutate C to T in the target sequence of the plant genome;

T2) preparing a product in which C in the target sequence of the plant genome is mutated to T;

T3) Plant genome base substitution;

T4) preparing products of plant genome base substitution;

T5) Plant genome base editing;

T6) preparing plant genome base editing products;

T7) preparing plant mutants;

The PAM sequence of the target sequence is NAN or NCN or NTN; N is A, T, C or G.

Furthermore, the kit also includes the self-cleaving oligopeptide and the screening agent resistance protein.

Furthermore, the set of reagents consists of the above-mentioned SpRYn, the above-mentioned cytosine deaminase, the above-mentioned sgRNA, the above-mentioned UGI, the above-mentioned self-cleaving oligopeptide and the above-mentioned screening agent resistance protein.

In any of the above methods or applications, the PAM sequence is a DNA sequence connected to the 3' end of the target sequence. The first N of the PAM sequence from the 5' end is connected to the 3' end of the target sequence. The size of the target sequence can be 15-25 bp, further 18-22 bp, and further 20 bp.

Furthermore, the NAN may be NAG or NAC.

The NCN may be NCA, NCG, NCC or NCT.

The NTN may be NTA, NTC or NTT.

Furthermore, the NAG may be AAG.

The NAC may be GAC or AAC.

The NCA may be GCA.

The NCG may be ACG.

The NCC may be GCC.

The NCT may be TCT.

The NTA may be a GTA.

The NTC may be a TTC.

The NTT may be a CTT.

In any of the above methods or applications, the target sequence may be one, two or more.

In any of the above methods or applications, the base editing or base replacement is to mutate C in the target sequence of the plant genome to T. The C can be a base C located at any position in the target sequence.

In any of the above methods or applications, the plant is S1) or S2) or S3):

S1) monocots or dicots;

S2) Gramineae;

S3) Rice (such as Nipponbare).

The present invention provides an application of a SpRYn-CBE base editing system in base replacement in a plant genome. The SpRYn-CBE base editing system of the present invention comprises SpRYn, PmCDA1, UGI and tRNA-esgRNA. Experiments have shown that the SpRYn-CBE base editing system of the present invention can edit the base C in the target sequence whose PAM sequence is NAN, NCN or NTN on the plant genome, thereby achieving the replacement of the base C to the base T, greatly expanding the range of editable C.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the structure of each element of the SpRYn-CBE base editing system vector. Among them, n is the number of targets, which can be 1, 2 or 3, and OsU6 can be OsU6a, OsU6b or OsU6c. OsU6a is used for one target, OsU6a and OsU6b are used for two targets, and OsU6a, OsU6b and OsU6c are used for three targets.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, instruments, etc. used in the following examples are all commercially available unless otherwise specified. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

The primer pair NAA-C1 consists of primer NAA-C1-F: 5’-CGCACGGCGGGAGGTACGTGC-3’ and primer NAA-C1-R: 5’-ATCAATAGCTGCAGTGTACTCTG-3’, which is used to amplify the target site NAA-C1.

The primer pair NAA-C2 consists of primer NAA-C2-F: 5’-TACCGCGCGCCGGAGCTGCT-3’ and primer NAA-C2-R: 5’-GCGCCTCCTCAACTGCATGTCA-3’, which is used to amplify the target site NAA-C2.

The primer pair NAA-C3 consists of primer NAA-C3-F: 5’-TATTCAGATCAGCATTTGGTGATAC-3’ and primer NAA-C3-R: 5’-AAGAAGATACAGTTAAGCTCCTG-3’, which is used to amplify the target site NAA-C3.

The primer pair NAA-C4 consists of primer NAA-C4-F: 5’-GATCATCGCATTGGATGGA-3’ and primer NAA-C4-R: 5’-ATGGGTTGTTGTTGAGGTTTAG-3’, which is used to amplify the target site NAA-C4.

The primer pair NAA-C5 consists of primer NAA-C5-F: 5’-CGCACGGCGGGAGGTACGTGC-3’ and primer NAA-C5-R: 5’-ATCAATAGCTGCAGTGTACTCTG-3’, which is used to amplify the target site NAA-C5.

The primer pair NAA-C6 consists of primer NAA-C6-F: 5’-CGCACGGCGGGAGGTACGTGC-3’ and primer NAA-C6-R: 5’-ATCAATAGCTGCAGTGTACTCTG-3’, which is used to amplify the target site NAA-C6.

The primer pair NAA-C7 consists of primer NAA-C7-F: 5’-GCACTGCCAGGTGAGTGAACT-3’ and primer NAA-C7-R: 5’-GCGCCTCCTCAACTGCATGTCA-3’, which is used to amplify the target site NAA-C7.

The primer pair NAA-C8 consists of primer NAA-C8-F: 5’-GGGCGAGCGCGGAGTGCGT-3’ and primer NAA-C8-R: 5’-TCAATGCGTGGCCCACATG-3’, which is used to amplify the target site NAA-C8.

The primer pair NAT-C1, consisting of primer NAT-C1-F: 5’-CGCACGGCGGGAGGTACGTGC-3’ and primer NAT-C1-R: 5’-ATCAATAGCTGCAGTGTACTCTG-3’, was used to amplify the target NAT-C1.

The primer pair NAT-C2, consisting of primer NAT-C2-F: 5’-AGAAGAGGAGGAGGGATTAGG-3’ and primer NAT-C2-R: 5’-GGGATCACATCCCTGATGCC-3’, was used to amplify the target NAT-C2.

The primer pair NAT-C3, consisting of primer NAT-C3-F: 5’-AGAAGAGGAGGAGGGATTAGG-3’ and primer NAT-C3-R: 5’-GGGATCACATCCCTGATGCC-3’, was used to amplify the target NAT-C3.

The primer pair NAT-C4, consisting of primer NAT-C4-F: 5’-TATTCAGATCAGCATTTGGTGATAC-3’ and primer NAT-C4-R: 5’-AAGAAGATACAGTTAAGCTCCTG-3’, was used to amplify the target NAT-C4.

The primer pair NAT-C5 consists of primer NAT-C5-F: 5’-TACCGCGCGCCGGAGCTGCT-3’ and primer NAT-C5-R: 5’-GCGCCTCCTCAACTGCATGTCA-3’, which is used to amplify the target NAT-C5.

The primer pair NAT-C6, consisting of primer NAT-C6-F: 5’-GGGCGAGCGCGGAGTGCGT-3’ and primer NAT-C6-R: 5’-TCAATGCGTGGCCCACATG-3’, was used to amplify the target NAT-C6.

The primer pair NAT-C7, consisting of primer NAT-C7-F: 5’-CCTAGCAAGGACAAGTACATCA-3’ and primer NAT-C7-R: 5’-GCCATGATGAGATGAGCAAGC-3’, was used to amplify the target NAT-C7.

The primer pair NAT-C8, consisting of primer NAT-C8-F: 5’-TACCGCGCGCCGGAGCTGCT-3’ and primer NAT-C8-R: 5’-GCGCCTCCTCAACTGCATGTCA-3’, was used to amplify the target site NAT-C8.

The primer pair NAC-C1, consisting of primer NAC-C1-F: 5’-TGATGTCACCTGATGATCTG-3’ and primer NAC-C1-R: 5’-GTGAGGCCGTGCGGGTTGG-3’, was used to amplify the target site NAC-C1.

The primer pair NAC-C2, consisting of primer NAC-C2-F: 5’-ACACAGCAAGGAGTGCCGG-3’ and primer NAC-C2-R: 5’-GCGTCGCATGTGATATTTGTCA-3’, was used to amplify the target site NAC-C2.

The primer pair NAC-C3, consisting of primer NAC-C3-F: 5’-GCCGCGACGGCCAAGACC-3’ and primer NAC-C3-R: 5’-AAGCCTCAATTTTCCCTGTC-3’, was used to amplify the target site NAC-C3.

The primer pair NAC-C4, consisting of primer NAC-C4-F: 5’-TGATGTCACCTGATGATCTG-3’ and primer NAC-C4-R: 5’-GTGAGGCCGTGCGGGTTGG-3’, was used to amplify the target site NAC-C4.

The primer pair NAG-C1 consisted of primer NAG-C1-F: 5’-GTGTCGCATCACGATTGCGA-3’ and primer NAG-C1-R: 5’-AAAACCAAAACTTCCATGGTTG-3’ and was used to amplify the target site NAG-C1.

The primer pair NAG-C2 consists of primer NAG-C2-F: 5’-TTTTGGTCGTTGCAGGGATGT-3’ and primer NAG-C2-R: 5’-GAACAACAAGATTAACCTAAGGCT-3’, which is used to amplify the target site NAG-C2.

The primer pair NCA-C1, consisting of primer NCA-C1-F: 5’-GGAGCTGGATGAGGTGCT-3’ and primer NCA-C1-R: 5’-GGAAGAAGAAAAGTAGGGAGA-3’, was used to amplify the target site NCA-C1.

The primer pair NCT-C1, consisting of primer NCT-C1-F: 5’-TTATTAACAGTGCATTTAGCA-3’ and primer NCT-C1-R: 5’-TGTGGATGCAGAAAGCAACCTG-3’, was used to amplify the target site NCT-C1.

The primer pair NCC-C1, consisting of primer NCC-C1-F: 5’-TAGTTGCCTCAAACAATAAAGACA-3’ and primer NCC-C1-R: 5’-CGGCGTCGGGACAGAGCTCCA-3’, was used to amplify the target site NCC-C1.

The primer pair NCG-C1, consisting of primer NCG-C1-F: 5’-CAATCCAAATTGTAATAAACTTCA-3’ and primer NCG-C1-R: 5’-CTGGTATCCCAAGCGTCCT-3’, was used to amplify the target site NCG-C1.

The primer pair NCG-C2, consisting of primer NCG-C2-F: 5’-ACGACTGGCACACTGGCCCA-3’ and primer NCG-C2-R: 5’-GATTGCCTGAAATTTGTCACTC-3’, was used to amplify the target site NCG-C2.

The primer pair NTA-C1 consists of primer NTA-C1-F: 5’-GCAGCAGCGGTCGGTGCAGCG-3’ and primer NTA-C1-R: 5’-TGTGGATGCAGAAAGCAACCTG-3’, which is used to amplify the target site NTA-C1.

The primer pair NTT-C1 consists of primer NTT-C1-F: 5’-GGCTCAATCATGTTAGACA-3’ and primer NTT-C1-R: 5’-TTCTGGCTTTTGTACTTCACCG-3’, which is used to amplify the target site NTT-C1.

The primer pair NTC-C1 consists of primer NTC-C1-F: 5’-ATTCCGTTGATGTTGCAAGCTT-3’ and primer NTC-C1-R: 5’-AGTCTCTAACAACAGTTATTACTT-3’, which is used to amplify the target site NTC-C1.

The primer pair NTG-C1, consisting of primer NTG-C1-F: 5’-GGCTCAATCATGTTAGACA-3’ and primer NTG-C1-R: 5’-TTCTGGCTTTTGTACTTCACCG-3’, was used to amplify the target site NTG-C1.

The primer pair NTG-C2 consists of primer NTG-C2-F: 5’-GCCGCGACGGCCAAGACC-3’ and primer NTG-C2-R: 5’-AAGCCTCAATTTTCCCTGTC-3’, which is used to amplify the target site NTG-C2.

The primer pair NTG-C3, consisting of primer NTG-C3-F: 5’-GAAGCAGGTGGTGCGGATGCT-3’ and primer NTG-C3-R: 5’-CGACGTACATACACGACGCG-3’, was used to amplify the target site NTG-C3.

In the following examples, C·T base substitution refers to the mutation of C to T at any position in the target sequence.

C·T base substitution efficiency = number of positive T0 seedlings with C·T base substitution/total number of positive T0 seedlings analyzed × 100%.

Nipponbare rice: References: Liang Weihong, Wang Gaohua, Du Jingyao, et al. Effects of sodium nitroprusside and its photolysis products on the growth of Nipponbare rice seedlings and the expression of five hormone marker genes [J]. Journal of Henan Normal University (Nature Edition), 2017(2):48-52.; Available to the public from Beijing Academy of Agricultural and Forestry Sciences.

Recovery medium: N6 solid medium containing 200 mg/L Timentin.

Screening medium: N6 solid medium containing 50 mg/L hygromycin.

Differentiation medium: N6 solid medium containing 2 mg/L KT, 0.2 mg/L NAA, 0.5 g/L glutamic acid, and 0.5 g/L proline.

Rooting medium: N6 solid medium containing 0.2 mg/L NAA, 0.5 g/L glutamic acid, and 0.5 g/L proline.

Example 1. Construction of the SpRYn-CBE base editing system vector and its application in base replacement of target sites with PAM sequences of NAN, NCN or NTN in the rice genome

1. Construction of the SpRYn-CBE base editing system vector

The following recombinant expression vectors were artificially synthesized, and each vector is a circular plasmid: SpRYn-CBE-1 recombinant expression vector, SpRYn-CBE-2 recombinant expression vector, SpRYn-CBE-3 recombinant expression vector, SpRYn-CBE-4 recombinant expression vector, SpRYn-CBE-5 recombinant expression vector, SpRYn-CBE-6 recombinant expression vector, SpRYn-CBE-7 recombinant expression vector, SpRYn-CBE-8 recombinant expression vector, SpRYn-CBE-9 recombinant expression vector, SpRYn-CBE-10 recombinant expression vector, SpRYn-CBE-11 recombinant expression vector, SpRYn-CBE-12 recombinant expression vector, SpRYn-CBE-13 recombinant expression vector, SpRYn-CBE-14 recombinant expression vector, SpRYn-CBE-15 recombinant expression vector and SpRYn-CBE-16 recombinant expression vector. The schematic diagram of the structure of each element of all recombinant expression vectors is shown in Figure 1. The specific structural descriptions are as follows:

The sequence of the SpRYn-CBE-1 recombinant expression vector is Sequence 1 in the sequence table. Sequence 1 has nucleotide sequences of OsU6a promoter from 131 to 596, tRNA from 597 to 673, target NAA-C1 from 674 to 693, esgRNA backbone from 694 to 779, and PolyT sequences from 780 to 786; Sequence 1 has nucleotide sequences of OsU6b promoter from 787 to 1119, tRNA from 1126 to 1202, esgRNA backbone from 1203 to 1222, target NAA-C3 from 1223 to 1308, and PolyT sequences from 1309 to 1320; Sequence 1 has nucleotide sequences of OsUbq3 promoter from 1327 to 3040 The nucleotide sequence of the ...

The sequence of the SpRYn-CBE-2 recombinant expression vector is obtained by replacing the NAA-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NAA-C2 target sequence, replacing the NAA-C3 target sequence with the NAA-C4 target sequence, and keeping the other sequences unchanged. The NAA-C2 target sequence and the NAA-C4 target sequence are shown in Table 1.

The sequence of the SpRYn-CBE-3 recombinant expression vector is obtained by replacing the NAA-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NAA-C5 target sequence, replacing the NAA-C3 target sequence with the NAA-C6 target sequence, and keeping other sequences unchanged. The NAA-C5 target sequence and the NAA-C6 target sequence are shown in Table 1.

The sequence of the SpRYn-CBE-4 recombinant expression vector is obtained by replacing the NAA-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NAA-C7 target sequence, replacing the NAA-C3 target sequence with the NAA-C8 target sequence, and keeping the other sequences unchanged. The NAA-C7 target sequence and the NAA-C8 target sequence are shown in Table 1.

The sequence of the SpRYn-CBE-5 recombinant expression vector is obtained by replacing the NAA-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NAT-C1 target sequence, replacing the NAA-C3 target sequence with the NAT-C4 target sequence, and keeping the other sequences unchanged. The NAT-C1 target sequence and the NAT-C4 target sequence are shown in Table 1.

The sequence of the SpRYn-CBE-6 recombinant expression vector is obtained by replacing the NAA-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NAT-C2 target sequence, replacing the NAA-C3 target sequence with the NAT-C3 target sequence, and keeping the other sequences unchanged. The NAT-C2 target sequence and the NAT-C3 target sequence are shown in Table 1.

The sequence of the SpRYn-CBE-7 recombinant expression vector is obtained by replacing the NAA-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NAT-C5 target sequence, replacing the NAA-C3 target sequence with the NAT-C8 target sequence, and keeping the other sequences unchanged. The NAT-C5 target sequence and the NAT-C8 target sequence are shown in Table 1.

The sequence of the SpRYn-CBE-8 recombinant expression vector is obtained by replacing the NAA-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NAT-C6 target sequence, replacing the NAA-C3 target sequence with the NAT-C7 target sequence, and keeping the other sequences unchanged. The NAT-C6 target sequence and the NAT-C7 target sequence are shown in Table 1.

The sequence of the SpRYn-CBE-9 recombinant expression vector is obtained by replacing the NAA-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NAC-C1 target sequence, replacing the NAA-C3 target sequence with the NAC-C2 target sequence, and keeping the other sequences unchanged. The NAC-C1 target sequence and the NAC-C2 target sequence are shown in Table 1.

The sequence of the SpRYn-CBE-10 recombinant expression vector is obtained by replacing the NAA-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NAC-C3 target sequence, and replacing the NAA-C3 target sequence with the NAC-C4 target sequence, while keeping other sequences unchanged. The NAC-C3 target sequence and the NAC-C4 target sequence are shown in Table 1.

The sequence of the SpRYn-CBE-11 recombinant expression vector is obtained by replacing the NAA-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NAG-C1 target sequence, replacing the NAA-C3 target sequence with the NAG-C2 target sequence, and keeping other sequences unchanged. The NAG-C1 target sequence and the NAG-C2 target sequence are shown in Table 1.

The sequence of the SpRYn-CBE-12 recombinant expression vector is obtained by replacing the NAA-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NCA-C1 target sequence, replacing the NAA-C3 target sequence with the NCC-C1 target sequence, and keeping other sequences unchanged. The NCA-C1 target sequence and the NCC-C1 target sequence are shown in Table 1.

The sequence of the SpRYn-CBE-13 recombinant expression vector is obtained by replacing the sequence 1 in the sequence list with the sequence 5 in the sequence list at positions 131-1320, and keeping the other sequences unchanged. Positions 1-466 of sequence 5 are the nucleotide sequence of the OsU6a promoter, positions 467-543 are the nucleotide sequence of tRNA, positions 544-563 are the nucleotide sequence of the target NCT-C1, positions 564-649 are the nucleotide sequence of the esgRNA backbone, and positions 650-656 are the PolyT sequence; positions 657-989 of sequence 5 are the nucleotide sequence of the OsU6b promoter, positions 996-1072 are the nucleotide sequence of tRNA, and positions 1073-1092 are the target NCG -C1, the nucleotide sequence of the esgRNA backbone at positions 1093-1178, and the PolyT sequence at positions 1179-1185; the nucleotide sequence of the OsU6c promoter at positions 1186-1927 of sequence 5, the nucleotide sequence of the tRNA at positions 1934-2010, the nucleotide sequence of the target NCG-C2 at positions 2011-2030, the nucleotide sequence of the esgRNA backbone at positions 2031-2116, and the PolyT sequence at positions 2117-2128. The target sequences of NCT-C1, NCG-C1, and NCG-C2 are shown in Table 1.

The sequence of the SpRYn-CBE-14 recombinant expression vector is obtained by replacing the NAA-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NTT-C1 target sequence, replacing the NAA-C3 target sequence with the NTC-C1 target sequence, and keeping the other sequences unchanged. The NTT-C1 target sequence and the NTC-C1 target sequence are shown in Table 1.

The sequence of the SpRYn-CBE-15 recombinant expression vector is obtained by replacing the sequence 131-1320 in the sequence list with the sequence 6 in the sequence list, and keeping the other sequences unchanged. The 1st to 466th positions of sequence 6 are the nucleotide sequence of the OsU6a promoter, the 467th to 543rd positions are the nucleotide sequence of tRNA, the 544th to 563rd positions are the nucleotide sequence of the target NTA-C1, the 564th to 649th positions are the nucleotide sequence of the esgRNA backbone, and the 650th to 661st positions are the PolyT sequence. The NTA-C1 target sequence is shown in Table 1.

The sequence of the SpRYn-CBE-16 recombinant expression vector is obtained by replacing the NCT-C1 target sequence in the SpRYn-CBE-13 recombinant expression vector sequence with the NTG-C1 target sequence, the NCG-C1 target sequence with the NTG-C2 target sequence, the NCG-C2 target sequence with the NTG-C3 target sequence, and keeping other sequences unchanged. The NTG-C1 target sequence, the NTG-C2 target sequence, and the NTG-C3 target sequence are shown in Table 1.

The target nucleotide sequences of esgRNAs of each vector and the corresponding PAM sequences are shown in Table 1.

Table 1. Target nucleotide sequences and corresponding PAM sequences of esgRNAs in each vector

2. Base editing of target sites in rice plants

The SpRYn-CBE-1 recombinant expression vector, SpRYn-CBE-2 recombinant expression vector, SpRYn-CBE-3 recombinant expression vector, SpRYn-CBE-4 recombinant expression vector, SpRYn-CBE-5 recombinant expression vector, SpRYn-CBE-6 recombinant expression vector, SpRYn-CBE-7 recombinant expression vector, SpRYn-CBE-8 recombinant expression vector, SpRYn-CBE-9 recombinant expression vector, SpRYn-CBE-10 recombinant expression vector, SpRYn-CBE-11 recombinant expression vector, SpRYn-CBE-12 recombinant expression vector, SpRYn-CBE-13 recombinant expression vector, SpRYn-CBE-14 recombinant expression vector, SpRYn-CBE-15 recombinant expression vector and SpRYn-CBE-16 recombinant expression vector obtained in step 1 are respectively operated according to the following steps 1-11:

1. Introduce the vector into Agrobacterium EHA105 (product of Shanghai Weidi Biotechnology Co., Ltd., CAT#: AC1010) to obtain recombinant Agrobacterium.

2. The recombinant Agrobacterium was cultured in a culture medium (YEP medium containing 50 μg/ml kanamycin and 25 μg/ml rifampicin) at 28°C and 150 rpm with shaking until the _OD600 was 1.0-2.0. The culture was centrifuged at 10,000 rpm for 1 min at room temperature. The cells were resuspended in infection solution (the sugar in the N6 liquid culture medium was replaced with glucose and sucrose, and the concentrations of glucose and sucrose in the infection solution were 10 g/L and 20 g/L, respectively) and diluted to an _OD600 of 0.2 to obtain the Agrobacterium infection solution.

3. Mature seeds of the rice variety Nipponbare were shelled and threshed, placed in a 100 mL conical flask, added with a 70% (v/v) ethanol aqueous solution and soaked for 30 seconds, then placed in a 25% (v/v) sodium hypochlorite aqueous solution, shaken and sterilized at 120 rpm for 30 minutes, rinsed with sterile water three times, and dried with filter paper. Then, the seeds were placed with the embryo facing downward on an N6 solid culture medium, and cultured in the dark at 28° C. for 4-6 weeks to obtain rice callus.

4. After completing step 3, soak the rice callus in Agrobacterium infection solution A (Agrobacterium infection solution A is a liquid obtained by adding acetosyringone to the Agrobacterium infection solution, and the amount of acetosyringone added satisfies the volume ratio of acetosyringone to the Agrobacterium infection solution of 25 μl:50 ml) for 10 minutes, then place it on a culture dish (containing about 200 ml of infection solution without Agrobacterium) covered with two layers of sterilized filter paper, and culture it in the dark at 21°C for 1 day.

5. Place the rice callus obtained in step 4 on a recovery medium and culture it in the dark at 25-28°C for 3 days.

6. Take the rice callus obtained in step 5, place it on the screening medium, and culture it in the dark at 28°C for 2 weeks.

7. Take the rice callus obtained in step 6, place it on the screening medium again, and culture it in the dark at 28° C. for 2 weeks to obtain rice resistant callus.

8. Take the rice resistant callus obtained in step 7 and place it on a differentiation medium, culture it under light at 25°C for about 1 month, transfer the differentiated seedlings to a rooting medium, culture them under light at 25°C for 2 weeks, and obtain rice T0 seedlings.

9. Extract the genomic DNA of rice T0 seedlings and use it as a template, and use a primer pair consisting of primer F (5'-ttattgccactagttcattctacttat-3') and primer R (5'-ggggtacttctcgtggtagg-3') to perform PCR amplification to obtain a PCR amplification product; subject the PCR amplification product to agarose gel electrophoresis, and then make the following judgment: if the PCR amplification product contains a DNA fragment of about 729 bp, the corresponding rice T0 seedling is a rice-positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 729 bp, the corresponding rice T0 seedling is not a rice-positive T0 seedling.

10. Each vector takes the genomic DNA of the rice positive T0 seedling obtained in step 9 as a template, and for the NAA-C1 target site, uses the primer pair NAA-C1 to perform PCR amplification to obtain a PCR amplification product; for the NAA-C2 target site, uses the primer pair NAA-C2 to perform PCR amplification to obtain a PCR amplification product; for the NAA-C3 target site, uses the primer pair NAA-C3 to perform PCR amplification to obtain a PCR amplification product; for the NAA-C4 target site, uses the primer pair NAA-C4 to perform PCR amplification to obtain a PCR amplification product; for the NAA-C5 target site, uses the primer pair NAA-C5 to perform PCR amplification to obtain a PCR amplification product; for the NAA-C6 target site, uses the primer pair NAA-C6 to perform PCR amplification to obtain a PCR amplification product; for the NAA-C7 target site, uses the primer pair NAA-C7 to perform PCR amplification to obtain a PCR amplification product; for the NAA-C8 target site, uses the primer pair NAA-C8 to perform PCR amplification to obtain a PCR amplification product. PCR amplification was performed to obtain a PCR amplification product; for the NAT-C1 target, PCR amplification was performed using primer pair NAT-C1 to obtain a PCR amplification product; for the NAT-C2 target, PCR amplification was performed using primer pair NAT-C2 to obtain a PCR amplification product; for the NAT-C3 target, PCR amplification was performed using primer pair NAT-C3 to obtain a PCR amplification product; for the NAT-C4 target, PCR amplification was performed using primer pair NAT-C4 to obtain a PCR amplification product; for the NAT-C5 target, PCR amplification was performed using primer pair NAT-C5 to obtain a PCR amplification product; for the NAT-C6 target, PCR amplification was performed using primer pair NAT-C6 to obtain a PCR amplification product; for the NAT-C7 target, PCR amplification was performed using primer pair NAT-C7 to obtain a PCR amplification product; for the NAT-C8 target, PCR amplification was performed using primer pair NAT-C8 to obtain a PCR amplification product; For the NAC-C1 target, PCR amplification was performed using primer pair NAC-C1 to obtain a PCR amplification product; for the NAC-C2 target, PCR amplification was performed using primer pair NAC-C2 to obtain a PCR amplification product; for the NAC-C3 target, PCR amplification was performed using primer pair NAC-C3 to obtain a PCR amplification product; for the NAC-C4 target, PCR amplification was performed using primer pair NAC-C4 to obtain a PCR amplification product; for the NAG-C1 target, PCR amplification was performed using primer pair NAG-C1 to obtain a PCR amplification product; for the NAG-C2 target, PCR amplification was performed using primer pair NAG-C2 to obtain a PCR amplification product; for the NCA-C1 target, PCR amplification was performed using primer pair NCA-C1 to obtain a PCR amplification product; for the NCT-C1 target, PCR amplification was performed using primer pair NCT-C1 to obtain a PCR amplification product; for the NCC-C1 target, PCR amplification was performed using primer pair NCC-C 1 is used for PCR amplification to obtain a PCR amplification product; for the NCG-C1 target, the primer pair NCG-C1 is used for PCR amplification to obtain a PCR amplification product; for the NCG-C2 target, the primer pair NCG-C2 is used for PCR amplification to obtain a PCR amplification product; for the NTA-C1 target, the primer pair NTA-C1 is used for PCR amplification to obtain a PCR amplification product; for the NTT-C1 target, the primer pair NTT-C1 is used for PCR amplification to obtain a PCR amplification product; for the NTC-C1 target, the primer pair NTC-C1 is used for PCR amplification to obtain a PCR amplification product; for the NTG-C1 target, the primer pair NTG-C1 is used for PCR amplification to obtain a PCR amplification product; for the NTG-C2 target, the primer pair NTG-C2 is used for PCR amplification to obtain a PCR amplification product; for the NTG-C3 target, the primer pair NTG-C3 is used for PCR amplification to obtain a PCR amplification product.

11. The PCR amplification products obtained in step 10 were subjected to Sanger sequencing and analysis. The sequencing results were analyzed only for each target region. The number of positive T0 seedlings with C·T base substitution at each target was counted, and the C·T base substitution efficiency was calculated. The results are shown in Table 2.

The results showed that the SpRYn-CBE base editing system could effectively edit the targets with PAM sequences, except for the eight targets with PAM sequence NAA (NAA-C1, NAA-C2, NAA-C3, NAA-C4, NAA-C5, NAA-C6, NAA-C7 and NAA-C8), the eight targets with PAM sequence NAT (NAT-C1, NAT-C2, NAT-C3, NAT-C4, NAT-C5, NAT-C6, NAT-C7 and NAT-C8), and the three targets with PAM sequence NTG (NTG-C1, NTG-C2 and NTG-C3), to obtain T0 seedlings with C·T base substitution, with a base editing efficiency of 7.5%-70%. This shows that the SpRYn-CBE base editing system can base edit target sequences with PAM sequences of NAC, NAG, NCA, NCT, NCC, NCG, NTA, NTT and NTC in the rice genome, achieving C·T base replacement, greatly expanding the scope of base editing.

Table 2. C·T base substitution efficiency

The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Sequence Listing

<110> Beijing Academy of Agricultural and Forestry Sciences

<120> Application of SpRYn-CBE base editing system in plant genome base replacement

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 16842

<212> DNA

<213> Artificial Sequence

<400> 1

ggtggcagga tatattgtgg tgtaaacatg gcactagcct caccgtcttc gcagacgagg 60

ccgctaagtc gcagctacgc tctcaacggc actgactagg tagtttaaac gtgcacttaa 120

ttaaggtacc tggaatcggc agcaaaggat tttttcctgt agttttccca caaccatttt 180

ttaccatccg aatgatagga taggaaaaat atccaagtga acagtattcc tataaaattc 240

ccgtaaaaag cctgcaatcc gaatgagccc tgaagtctga actagccggt cacctgtaca 300

ggctatcgag atgccataca agagacggta gtaggaacta ggaagacgat ggttgattcg 360

tcaggcgaaa tcgtcgtcct gcagtcgcat ctatgggcct ggacggaata ggggaaaaag 420

ttggccggat aggagggaaa ggcccaggtg cttacgtgcg aggtaggcct gggctctcag 480

cacttcgatt cgttggcacc ggggtaggat gcaatagaga gcaacgttta gtaccacctc 540

gcttagctag agcaaactgg actgccttat atgcgcgggt gctggcttgg ctgccgaaca 600

aagcaccagt ggtctagtgg tagaatagta ccctgccacg gtacagaccc gggttcgatt 660

cccggctggt gcaccgtcct gaggggatgt tcagtttcag agctatgctg gaaacagcat 720

agcaagttga aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 780

tttttttgca agaacgaact aagccggaca aaaaaaaaag gagcacatat acaaaccggt 840

tttattcatg aatggtcacg atggatgatg gggctcagac ttgagctacg aggccgcagg 900

cgagagaagc ctagtgtgct ctctgcttgt ttgggccgta acggaggata cggccgacga 960

gcgtgtacta ccgcgcggga tgccgctggg cgctgcgggg gccgttggat ggggatcggt 1020

gggtcgcggg agcgttgagg ggagacaggt ttagtaccac ctcgcctacc gaacaatgaa 1080

gaacccacct tataaccccg cgcgctgccg cttgtgttgg gatccaacaa agcaccagtg 1140

gtctagtggt agaatagtac cctgccacgg tacagacccg ggttcgattc ccggctggtg 1200

cacccttcat gagatatatg atgtttcaga gctatgctgg aaacagcata gcaagttgaa 1260

ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttttttt 1320

aagcttacaa attcgggtca aggcggaagc cagcgcgcca ccccacgtca gcaaatacgg 1380

aggcgcgggg ttgacggcgt cacccggtcc taacggcgac caacaaacca gccagaagaa 1440

attacagtaa aaaaaaagta aattgcactt tgatccacct tttattacct aagtctcaat 1500

ttggatcacc cttaaaccta tcttttcaat ttgggccggg ttgtggtttg gactaccatg 1560

aacaactttt cgtcatgtct aacttccctt tcagcaaaca tatgaaccat atatagagga 1620

gatcggccgt atactagagc tgatgtgttt aaggtcgttg attgcacgag aaaaaaaaat 1680

ccaaatcgca acaatagcaa atttatctgg ttcaaagtga aaagatatgt ttaaaggtag 1740

tccaaagtaa aacttataga taataaaatg tggtccaaag cgtaattcac tcaaaaaaaa 1800

tcaacgagac gtgtaccaaa cggagacaaa cggcatcttc tcgaaatttc ccaaccgctc 1860

gctcgcccgc ctcgtcttcc cggaaaccgc ggtggtttca gcgtggcgga ttctccaagc 1920

agacggagac gtcacggcac gggactcctc ccaccaccca accgccataa ataccagccc 1980

cctcatctcc tctcctcgca tcagctccac ccccgaaaaa tttctcccca atctcgcgag 2040

gctctcgtcg tcgaatcgaa tcctctcgcg tcctcaaggt acgctgcttc tcctctcctc 2100

gcttcgtttc gattcgattt cggacgggtg aggttgtttt gttgctagat ccgattggtg 2160

gttagggttg tcgatgtgat tatcgtgaga tgtttagggg ttgtagatct gatggttgtg 2220

atttgggcac ggttggttcg ataggtggaa tcgtggttag gttttgggat tggatgttgg 2280

ttctgatgat tggggggaat ttttacggtt agatgaattg ttggatgatt cgattgggga 2340

aatcggtgta gatctgttgg ggaattgtgg aactagtcat gcctgagtga ttggtgcgat 2400

ttgtagcgtg ttccatcttg taggccttgt tgcgagcatg ttcagatcta ctgttccgct 2460

cttgattgag ttattggtgc catgggttgg tgcaaacaca ggctttaata tgttatatct 2520

gttttgtgtt tgatgtagat ctgtagggta gttcttctta gacatggttc aattatgtag 2580

cttgtgcgtt tcgatttgat ttcatatgtt cacagattag ataatgatga actcttttaa 2640

ttaattgtca atggtaaata ggaagtcttg tcgctatatc tgtcataatg atctcatgtt 2700

actatctgcc agtaatttat gctaagaact atattagaat atcatgttac aatctgtagt 2760

aatatcatgt tacaatctgt agttcatcta tataatctat tgtggtaatt tctttttatact 2820

atctgtgtga agattattgc cactagttca ttctacttat ttctgaagtt caggatacgt 2880

gtgctgttac tacctatctg aatacatgtg tgatgtgcct gttactatct ttttgaatac 2940

atgtatgttc tgttggaata tgtttgctgt ttgatccgtt gttgtgtcct taatcttgtg 3000

ctagttctta ccctatctgt ttggtgatta tttcttgcag tacgtaatgg actacaagga 3060

ccacgacggc gactacaagg atcatgacat cgactacaag gacgacgacg acaagatggc 3120

tcctaagaag aagcggaagg ttggtattca cggggtgcct gcggctgaca agaagtactc 3180

catcggcctc gccatcggca ccaacagcgt cggctgggcg gtgatcaccg acgagtacaa 3240

ggtcccgtcc aagaagttca aggtcctggg caacaccgac cgccactcca tcaagaagaa 3300

cctcatcggc gccctcctct tcgactccgg cgagacggcg gagcgcaccc gcctcaagcg 3360

caccgcccgc cgccgctaca cccgccgcaa gaaccgcatc tgctacctcc aggagatctt 3420

ctccaacgag atggcgaagg tcgacgactc cttcttccac cgcctcgagg agtccttcct 3480

cgtggaggag gacaagaagc acgagcgcca ccccatcttc ggcaacatcg tcgacgaggt 3540

cgcctaccac gagaagtacc ccactatcta ccaccttcgt aagaagcttg ttgactctac 3600

tgataaggct gatcttcgtc tcatctacct tgctctcgct cacatgatca agttccgtgg 3660

tcacttcctt atcgagggtg accttaaccc tgataactcc gacgtggaca agctcttcat 3720

ccagctcgtc cagacctaca accagctctt cgaggagaac cctatcaacg cttccggtgt 3780

cgacgctaag gcgatccttt ccgctaggct ctccaagtcc aggcgtctcg agaacctcat 3840

cgcccagctc cctggtgaga agaagaacgg tcttttcggt aacctcatcg ctctctccct 3900

cggtctgacc cctaacttca agtccaactt cgacctcgct gaggacgcta agcttcagct 3960

ctccaaggat acctacgacg atgatctcga caacctcctc gctcagattg gagatcagta 4020

cgctgatctc ttccttgctg ctaagaacct ctccgatgct atcctccttt cggatatcct 4080

tagggttaac actgagatca ctaaggctcc tctttctgct tccatgatca agcgctacga 4140

cgagcaccac caggacctca ccctcctcaa ggctcttgtt cgtcagcagc tccccgagaa 4200

gtacaaggag atcttcttcg accagtccaa gaacggctac gccggttaca ttgacggtgg 4260

agctagccag gaggagttct acaagttcat caagccaatc cttgagaaga tggatggtac 4320

tgaggagctt ctcgttaagc ttaaccgtga ggacctcctt aggaagcaga ggactttcga 4380

taacggctct atccctcacc agatccacct tggtgagctt cacgccatcc ttcgtaggca 4440

ggaggacttc taccctttcc tcaaggacaa ccgtgagaag atcgagaaga tccttacttt 4500

ccgtattcct tactacgttg gtcctcttgc tcgtggtaac tcccgtttcg cttggatgac 4560

taggaagtcc gaggagacta tcaccccttg gaacttcgag gaggttgttg acaagggtgc 4620

ttccgcccag tccttcatcg agcgcatgac caacttcgac aagaacctcc ccaacgagaa 4680

ggtcctcccc aagcactccc tcctctacga gtacttcacg gtctacaacg agctcaccaa 4740

ggtcaagtac gtcaccgagg gtatgcgcaa gcctgccttc ctctccggcg agcagaagaa 4800

ggctatcgtt gacctcctct tcaagaccaa ccgcaaggtc accgtcaagc agctcaagga 4860

ggactacttc aagaagatcg agtgcttcga ctccgtcgag atcagcggcg ttgaggaccg 4920

tttcaacgct tctctcggta cctaccacga tctcctcaag atcatcaagg acaaggactt 4980

cctcgacaac gaggagaacg aggacatcct cgaggacatc gtcctcactc ttactctctt 5040

cgaggatagg gagatgatcg aggagaggct caagacttac gctcatctct tcgatgacaa 5100

ggttatgaag cagctcaagc gtcgccgtta caccggttgg ggtaggctct cccgcaagct 5160

catcaacggt atcagggata agcagagcgg caagactatc ctcgacttcc tcaagtctga 5220

tggtttcgct aacaggaact tcatgcagct catccacgat gactctctta ccttcaagga 5280

ggatattcag aaggctcagg tgtccggtca gggcgactct ctccacgagc acattgctaa 5340

ccttgctggt tcccctgcta tcaagaaggg catccttcag actgttaagg ttgtcgatga 5400

gcttgtcaag gttatgggtc gtcacaagcc tgagaacatc gtcatcgaga tggctcgtga 5460

gaaccagact acccagaagg gtcagaagaa ctcgagggag cgcatgaaga ggattgagga 5520

gggtatcaag gagcttggtt ctcagatcct taaggagcac cctgtcgaga acacccagct 5580

ccagaacgag aagctctacc tctactacct ccagaacggt agggatatgt acgttgacca 5640

ggagctcgac atcaacaggc tttctgacta cgacgtcgac cacattgttc ctcagtcttt 5700

ccttaaggat gactccatcg acaacaaggt cctcacgagg tccgacaaga acaggggtaa 5760

gtcggacaac gtcccttccg aggaggttgt caagaagatg aagaactact ggaggcagct 5820

tctcaacgct aagctcatta cccagaggaa gttcgacaac ctcacgaagg ctgagagggg 5880

tggcctttcc gagcttgaca aggctggttt catcaagagg cagcttgttg agacgaggca 5940

gattaccaag cacgttgctc agatcctcga ttctaggatg aacaccaagt acgacgagaa 6000

cgacaagctc atccgcgagg tcaaggtgat caccctcaag tccaagctcg tctccgactt 6060

ccgcaaggac ttccagttct acaaggtccg cgagatcaac aactaccacc acgctcacga 6120

tgcttacctt aacgctgtcg ttggtaccgc tctttatcaag aagtacccta agcttgagtc 6180

cgagttcgtc tacggtgact acaaggtcta cgacgttcgt aagatgatcg ccaagtccga 6240

gcaggagatc ggcaaggcca ccgccaagta cttcttctac tccaacatca tgaacttctt 6300

caagaccgag atcaccctcg ccaacggcga gatccgcaag cgccctctta tcgagacgaa 6360

cggtgagact ggtgagatcg tttgggacaa gggtcgcgac ttcgctactg ttcgcaaggt 6420

cctttctatg cctcaggtta acatcgtcaa gaagaccgag gtccagaccg gtggcttctc 6480

caaggagtct atccgcccaa agagaaactc ggacaagctc atcgctagga agaaggattg 6540

ggaccctaag aagtacggtg gtttcctgtg gcctactgtc gcctactccg tcctcgtggt 6600

cgccaaggtg gagaagggta agtcgaagaa gctcaagtcc gtcaaggagc tcctcggcat 6660

caccatcatg gagcgctcct ccttcgagaa gaacccgatc gacttcctcg aggccaaggg 6720

ctacaaggag gtcaagaagg acctcatcat caagctcccc aagtactctc ttttcgagct 6780

cgagaacggt cgtaagagga tgctggcttc cgctaagcag ctccagaagg gtaacgagct 6840

tgctcttcct tccaagtacg tgaacttcct ctacctcgcc tcccactacg agaagctcaa 6900

gggttcccct gaggataacg agcagaagca gctcttcgtg gagcagcaca agcactacct 6960

cgacgagatc atcgagcaga tctccgagtt ctccaagcgc gtcatcctcg ctgacgctaa 7020

cctcgacaag gtcctctccg cctacaacaa gcaccgcgac aagcccatcc gcgagcaggc 7080

cgagaacatc atccacctct tcacgctcac gcgcctcggc gcccctcgcg ctttcaagta 7140

cttcgacacc accatcgacc ccaagcagta ccgctccacc aaggaggttc tcgacgctac 7200

tctcatccac cagtccatca ccggtcttta cgagactcgt atcgaccttt cccagcttgg 7260

tggtgatgga ggaggaggca cgggaggagg aggctccgcc gagtatgtgc gcgcgctctt 7320

cgacttcaac ggcaatgacg aggaggatct ccctttcaag aagggcgaca tcctccgcat 7380

ccgcgataag ccggaggagc agtggtggaa cgcagaggac tccgagggca agcggggcat 7440

gatcctggtg ccatacgtcg agaagtacag cggcgattac aaggaccacg atggcgacta 7500

caaggatcat gacatcgatt acaaggacga tgacgataag tccggcgtcg acatgacgga 7560

cgcggagtat gtgcgcatcc acgagaagct cgatatctac accttcaaga agcagttctt 7620

caacaataag aagtcggtgt cccatcggtg ctacgtcctc ttcgagctga agcgcagggg 7680

agagcgccgc gcctgcttct ggggctacgc ggtgaataag ccgcagtcag gcacagagcg 7740

cggcatccac gccgagatct tctcgatccg gaaggtcgag gagtacctcc gcgacaaccc 7800

aggccagttc acgatcaatt ggtactccag ctggtcccct tgcgcagatt gcgcagagaa 7860

gatcctcgag tggtacaacc aggagctgag gggcaatggc cataccctca agatctgggc 7920

ctgcaagctg tactacgaga agaacgcgag gaatcagatc ggcctctgga acctgcggga 7980

taatggcgtg ggcctcaacg tgatggtgtc cgagcactac cagtgctgcc gcaagatctt 8040

catccagtcc tcccacaatc agctgaacga gaataggtgg ctcgaaaaga ccctgaagcg 8100

cgccgagaag tggaggagcg agctgtctat catgatccag gtcaagatcc tgcacaccac 8160

aaagtcaccg gcggtgggcg gcggcggcag cgatgattcc ggcggcagca ccaacctctc 8220

cgacatcatc gagaaggaga caggcaagca gctcgtgatc caggagagca tcctcatgct 8280

cccggaggag gtggaggagg tcatcggcaa caagccggag tccgacatcc tcgtgcacac 8340

cgcctacgac gagtccaccg acgagaacgt gatgctcctc acctcagatg caccagagta 8400

caagccatgg gcactcgtga tccaggacag caacggcgag aacaagatca agatgctctc 8460

cggcggcagc accaacctct ccgacatcat cgagaaggag acaggcaagc agctcgtgat 8520

ccaggagagc atcctcatgc tcccggagga ggtggaggag gtcatcggca acaagccgga 8580

gtccgacatc ctcgtgcaca ccgcctacga cgagtccacc gacgagaacg tgatgctcct 8640

cacctcagat gcaccagagt acaagccatg ggcactcgtg atccaggaca gcaacggcga 8700

gaacaagatc aagatgctct ccggcggctc cccgaagaag aagaggaaag tgggatcagg 8760

agccaccaac ttctccctcc tcaagcaggc cggcgacgtg gaggagaacc cgggcccaat 8820

gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 8880

cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 8940

aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 9000

ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg 9060

ggagtttagc gagagcctga cctattgcat ctcccgccgt tcacagggtg tcacgttgca 9120

agacctgcct gaaaccgaac tgcccgctgt tctacaaccg gtcgcggagg ctatggatgc 9180

gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 9240

cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 9300

ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 9360

gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc 9420

caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 9480

gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg 9540

tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccacg 9600

actccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 9660

caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 9720

cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg 9780

tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagaa 9840

atagactagt tcagccagtt tggtggagct gccgatgtgc ctggtcgtcc cgagcctctg 9900

ttcgtcaagt atttgtggtg ctgatgtcta cttgtgtctg gtttaatgga ccatcgagtc 9960

cgtatgatat gttagtttta tgaaacagtt tcctgtggga cagcagtatg ctttatgaat 10020

aagttggatt tgaacctaaa tatgtgctca atttgctcat ttgcatctca ttcctgttga 10080

tgttttatct gagttgcaag tttgaaaatg ctgcatattc ttattaaatc gtcatttatact 10140

tttatcttaa tgagctttgc aatggcctat gggatataaa agagatcgtt caaacatttg 10200

gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt 10260

tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag 10320

atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat 10380

atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac tagatccctg 10440

caggacgcgt ttaattaagt gcacgcggcc gcctacttag tcaagagcct cgcacgcgac 10500

tgtcacgcgg ccaggatcgc ctcgtgagcc tcgcaatctg tacctagtgt ttaaactatc 10560

agtgtttgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt attagaataa 10620

cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg tgcatgccaa 10680

ccacagggtt cccctcggga tcaaagtact ttgatccaac ccctccgctg ctatagtgca 10740

gtcggcttct gacgttcagt gcagccgtct tctgaaaacg acatgtcgca caagtcctaa 10800

gttacgcgac aggctgccgc cctgcccttt tcctggcgtt ttcttgtcgc gtgttttagt 10860

cgcataaagt agaatacttg cgactagaac cggagacatt acgccatgaa caagagcgcc 10920

gccgctggcc tgctgggcta tgcccgcgtc agcaccgacg accaggactt gaccaaccaa 10980

cgggccgaac tgcacgcggc cggctgcacc aagctgtttt ccgagaagat caccggcacc 11040

aggcgcgacc gcccggagct ggccaggatg cttgaccacc tacgccctgg cgacgttgtg 11100

acagtgacca ggctagaccg cctggcccgc agcacccgcg acctactgga cattgccgag 11160

cgcatccagg aggccggcgc gggcctgcgt agcctggcag agccgtgggc cgacaccacc 11220

acgccggccg gccgcatggt gttgaccgtg ttcgccggca ttgccgagtt cgagcgttcc 11280

ctaatcatcg accgcacccg gagcgggcgc gaggccgcca aggcccgagg cgtgaagttt 11340

ggcccccgcc ctaccctcac cccggcacag atcgcgcacg cccgcgagct gatcgaccag 11400

gaaggccgca ccgtgaaaga ggcggctgca ctgcttggcg tgcatcgctc gaccctgtac 11460

cgcgcacttg agcgcagcga ggaagtgacg cccaccgagg ccaggcggcg cggtgccttc 11520

cgtgaggacg cattgaccga ggccgacgcc ctggcggccg ccgagaatga acgccaagag 11580

gaacaagcat gaaaccgcac caggacggcc aggacgaacc gtttttcatt accgaagaga 11640

tcgaggcgga gatgatcgcg gccgggtacg tgttcgagcc gcccgcgcac gtctcaaccg 11700

tgcggctgca tgaaatcctg gccggtttgt ctgatgccaa gctggcggcc tggccggcca 11760

gcttggccgc tgaagaaacc gagcgccgcc gtctaaaaag gtgatgtgta tttgagtaaa 11820

acagcttgcg tcatgcggtc gctgcgtata tgatgcgatg agtaaataaa caaatacgca 11880

aggggaacgc atgaaggtta tcgctgtact taaccagaaa ggcgggtcag gcaagacgac 11940

catcgcaacc catctagccc gcgccctgca actcgccggg gccgatgttc tgttagtcga 12000

ttccgatccc cagggcagtg cccgcgattg ggcggccgtg cgggaagatc aaccgctaac 12060

cgttgtcggc atcgaccgcc cgacgattga ccgcgacgtg aaggccatcg gccggcgcga 12120

cttcgtagtg atcgacggag cgccccaggc ggcggacttg gctgtgtccg cgatcaaggc 12180

agccgacttc gtgctgattc cggtgcagcc aagcccttac gacatatggg ccaccgccga 12240

cctggtggag ctggttaagc agcgcattga ggtcacggat ggaaggctac aagcggcctt 12300

tgtcgtgtcg cgggcgatca aaggcacgcg catcggcggt gaggttgccg aggcgctggc 12360

cgggtacgag ctgcccattc ttgagtcccg tatcacgcag cgcgtgagct acccaggcac 12420

tgccgccgcc ggcacaaccg ttcttgaatc agaacccgag ggcgacgctg cccgcgaggt 12480

ccaggcgctg gccgctgaaa ttaaatcaaa actcatttga gttaatgagg taaagagaaa 12540

atgagcaaaa gcacaaacac gctaagtgcc ggccgtccga gcgcacgcag cagcaaggct 12600

gcaacgttgg ccagcctggc agacacgcca gccatgaagc gggtcaactt tcagttgccg 12660

gcggaggatc acaccaagct gaagatgtac gcggtacgcc aaggcaagac cattaccgag 12720

ctgctatctg aatacatcgc gcagctacca gagtaaatga gcaaatgaat aaatgagtag 12780

atgaatttta gcggctaaag gaggcggcat ggaaaatcaa gaacaaccag gcaccgacgc 12840

cgtggaatgc cccatgtgtg gaggaacggg cggttggcca ggcgtaagcg gctgggttgt 12900

ctgccggccc tgcaatggca ctggaacccc caagcccgag gaatcggcgt gacggtcgca 12960

aaccatccgg cccggtacaa atcggcgcgg cgctgggtga tgacctggtg gagaagttga 13020

aggccgcgca ggccgcccag cggcaacgca tcgaggcaga agcacgcccc ggtgaatcgt 13080

ggcaagcggc cgctgatcga atccgcaaag aatcccggca accgccggca gccggtgcgc 13140

cgtcgattag gaagccgccc aagggcgacg agcaaccaga ttttttcgtt ccgatgctct 13200

atgacgtggg cacccgcgat agtcgcagca tcatggacgt ggccgttttc cgtctgtcga 13260

agcgtgaccg acgagctggc gaggtgatcc gctacgagct tccagacggg cacgtagagg 13320

tttccgcagg gccggccggc atggccagtg tgtgggatta cgacctggta ctgatggcgg 13380

tttcccatct aaccgaatcc atgaaccgat accgggaagg gaagggagac aagcccggcc 13440

gcgtgttccg tccacacgtt gcggacgtac tcaagttctg ccggcgagcc gatggcggaa 13500

agcagaaaga cgacctggta gaaacctgca ttcggttaaa caccacgcac gttgccatgc 13560

agcgtacgaa gaaggccaag aacggccgcc tggtgacggt atccgagggt gaagccttga 13620

ttagccgcta caagatcgta aagagcgaaa ccgggcggcc ggagtacatc gagatcgagc 13680

tagctgattg gatgtaccgc gagatcacag aaggcaagaa cccggacgtg ctgacggttc 13740

accccgatta ctttttgatc gatcccggca tcggccgttt tctctaccgc ctggcacgcc 13800

gcgccgcagg caaggcagaa gccagatggt tgttcaagac gatctacgaa cgcagtggca 13860

gcgccggaga gttcaagaag ttctgtttca ccgtgcgcaa gctgatcggg tcaaatgacc 13920

tgccggagta cgatttgaag gaggaggcgg ggcaggctgg cccgatccta gtcatgcgct 13980

accgcaacct gatcgagggc gaagcatccg ccggttccta atgtacggag cagatgctag 14040

ggcaaattgc cctagcaggg gaaaaaggtc gaaaaggtct ctttcctgtg gatagcacgt 14100

acattgggaa cccaaagccg tacattggga accggaaccc gtacattggg aacccaaagc 14160

cgtacattgg gaaccggtca cacatgtaag tgactgatat aaaagagaaa aaaggcgatt 14220

tttccgccta aaactcttta aaacttatta aaactcttaa aacccgcctg gcctgtgcat 14280

aactgtctgg ccagcgcaca gccgaagagc tgcaaaaagc gcctaccctt cggtcgctgc 14340

gctccctacg ccccgccgct tcgcgtcggc ctatcgcggc cgctggccgc tcaaaaatgg 14400

ctggcctacg gccaggcaat ctaccaggggc gcggacaagc cgcgccgtcg ccactcgacc 14460

gccggcgccc acatcaaggc accctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc 14520

tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 14580

caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag 14640

tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac 14700

tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 14760

tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 14820

gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 14880

caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 14940

tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 15000

gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 15060

ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 15120

cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 15180

tcgttcgctc caagctgggc tgtgtgcacg aacccccgt tcagcccgac cgctgcgcct 15240

tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 15300

cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 15360

agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 15420

agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 15480

gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 15540

aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 15600

ggattttggt catgcattct aggtactaaa acaattcatc cagtaaaata taatatttta 15660

ttttctccca atcaggcttg atccccagta agtcaaaaaa tagctcgaca tactgttctt 15720

ccccgatatc ctccctgatc gaccggacgc agaaggcaat gtcataccac ttgtccgccc 15780

tgccgcttct cccaagatca ataaagccac ttactttgcc atctttcaca aagatgttgc 15840

tgtctcccag gtcgccgtgg gaaaagacaa gttcctcttc gggcttttcc gtctttaaaa 15900

aatcatacag ctcgcgcgga tctttaaatg gagtgtcttc ttcccagttt tcgcaatcca 15960

catcggccag atcgttattc agtaagtaat ccaattcggc taagcggctg tctaagctat 16020

tcgtataggg acaatccgat atgtcgatgg agtgaaagag cctgatgcac tccgcataca 16080

gctcgataat cttttcaggg ctttgttcat cttcatactc ttccgagcaa aggacgccat 16140

cggcctcact catgagcaga ttgctccagc catcatgccg ttcaaagtgc aggacctttg 16200

gaacaggcag ctttccttcc agccatagca tcatgtcctt ttcccgttcc acatcatagg 16260

tggtcccttt ataccggctg tccgtcattt ttaaatatag gttttcattt tctcccacca 16320

gcttatatac cttagcagga gacattcctt ccgtatcttt tacgcagcgg tatttttcga 16380

tcagtttttt caattccggt gatattctca ttttagccat ttattatttc cttcctcttt 16440

tctacagtat ttaaagatac cccaagaagc taattataac aagacgaact ccaattcact 16500

gttccttgca ttctaaaacc ttaaatacca gaaaacagct ttttcaaagt tgttttcaaa 16560

gttggcgtat aacatagtat cgacggagcc gattttgaaa ccgcggtgat cacaggcagc 16620

aacgctctgt catcgttaca atcaacatgc taccctccgc gagatcatcc gtgtttcaaa 16680

cccggcagct tagttgccgt tcttccgaat agcatcggta acatgagcaa agtctgccgc 16740

cttacaacgg ctctcccgct gacgccgtcc cggactgatg ggctgcctgt atcgagtggt 16800

gattttgtgc cgagctgccg gtcggggagc tgttggctgg ct 16842

<210> 2

<211> 1368

<212> PRT

<213> Artificial Sequence

<400> 2

Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Arg Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1010 1015 1020

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

1025 1030 1035

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1040 1045 1050

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1055 1060 1065

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1085 1090 1095

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg Pro Lys

1100 1105 1110

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Leu Trp Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1145 1150 1155

Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1175 1180 1185

Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Lys

1205 1210 1215

Gln Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Arg Leu Gly Ala Pro Arg Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Pro Lys Gln Tyr Arg Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

<210> 3

<211> 208

<212> PRT

<213> Artificial Sequence

<400> 3

Met Thr Asp Ala Glu Tyr Val Arg Ile His Glu Lys Leu Asp Ile Tyr

1 5 10 15

Thr Phe Lys Lys Gln Phe Phe Asn Asn Lys Lys Ser Val Ser His Arg

20 25 30

Cys Tyr Val Leu Phe Glu Leu Lys Arg Arg Gly Glu Arg Arg Ala Cys

35 40 45

Phe Trp Gly Tyr Ala Val Asn Lys Pro Gln Ser Gly Thr Glu Arg Gly

50 55 60

Ile His Ala Glu Ile Phe Ser Ile Arg Lys Val Glu Glu Tyr Leu Arg

65 70 75 80

Asp Asn Pro Gly Gln Phe Thr Ile Asn Trp Tyr Ser Ser Trp Ser Pro

85 90 95

Cys Ala Asp Cys Ala Glu Lys Ile Leu Glu Trp Tyr Asn Gln Glu Leu

100 105 110

Arg Gly Asn Gly His Thr Leu Lys Ile Trp Ala Cys Lys Leu Tyr Tyr

115 120 125

Glu Lys Asn Ala Arg Asn Gln Ile Gly Leu Trp Asn Leu Arg Asp Asn

130 135 140

Gly Val Gly Leu Asn Val Met Val Ser Glu His Tyr Gln Cys Cys Arg

145 150 155 160

Lys Ile Phe Ile Gln Ser Ser His Asn Gln Leu Asn Glu Asn Arg Trp

165 170 175

Leu Glu Lys Thr Leu Lys Arg Ala Glu Lys Trp Arg Ser Glu Leu Ser

180 185 190

Ile Met Ile Gln Val Lys Ile Leu His Thr Thr Lys Ser Pro Ala Val

195 200 205

<210> 4

<211> 83

<212> PRT

<213> Artificial Sequence

<400> 4

Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val

1 5 10 15

Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile

20 25 30

Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu

35 40 45

Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr

50 55 60

Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile

65 70 75 80

Lys Met Leu

<210> 5

<211> 2128

<212> DNA

<213> Artificial Sequence

<400> 5

tggaatcggc agcaaaggat tttttcctgt agttttccca caaccatttt ttaccatccg 60

aatgatagga taggaaaaat atccaagtga acagtattcc tataaaattc ccgtaaaaag 120

cctgcaatcc gaatgagccc tgaagtctga actagccggt cacctgtaca ggctatcgag 180

atgccataca agagacggta gtaggaacta ggaagacgat ggttgattcg tcaggcgaaa 240

tcgtcgtcct gcagtcgcat ctatgggcct ggacggaata ggggaaaaag ttggccggat 300

aggagggaaa ggcccaggtg cttacgtgcg aggtaggcct gggctctcag cacttcgatt 360

cgttggcacc ggggtaggat gcaatagaga gcaacgttta gtaccacctc gcttagctag 420

agcaaactgg actgccttat atgcgcgggt gctggcttgg ctgccgaaca aagcaccagt 480

ggtctagtgg tagaatagta ccctgccacg gtacagaccc gggttcgatt cccggctggt 540

gcaatctcaa gatcttataa cttgtttcag agctatgctg gaaacagcat agcaagttga 600

aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttttgca 660

agaacgaact aagccggaca aaaaaaaaag gagcacatat acaaaccggttttattcatg 720

aatggtcacg atggatgatg gggctcagac ttgagctacg aggccgcagg cgagagaagc 780

ctagtgtgct ctctgcttgt ttgggccgta acggaggata cggccgacga gcgtgtacta 840

ccgcgcggga tgccgctggg cgctgcgggg gccgttggat ggggatcggt gggtcgcggg 900

agcgttgagg ggagacaggt ttagtaccac ctcgcctacc gaacaatgaa gaacccacct 960

tataaccccg cgcgctgccg cttgtgttgg gatccaacaa agcaccagtg gtctagtggt 1020

agaatagtac cctgccacgg tacagacccg ggttcgattc ccggctggtg caccaccatg 1080

tcggctctca ccgtttcaga gctatgctgg aaacagcata gcaagttgaa ataaggctag 1140

tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttctcat tagcggtatg 1200

catgttggta gaagtcggag atgtaaataa ttttcattat ataaaaaagg tacttcgaga 1260

aaaataaatg catacgaatt aattcttttt atgtttttta aaccaagtat atagaattta 1320

ttgatggtta aaatttcaaa aatatgacga gagaaaggtt aaacgtacgg catatacttc 1380

tgaacagaga gggaatatgg ggtttttgtt gctcccaaca attcttaagc acgtaaagga 1440

aaaaagcaca ttatccacat tgtacttcca gagatatgta cagcattacg taggtacgtt 1500

ttctttttct tcccggagag atgatacaat aatcatgtaa acccagaatt taaaaaatat 1560

tctttactat aaaaatttta attagggaac gtattatttt ttacatgaca ccttttgaga 1620

aagagggact tgtaatatgg gacaaatgaa caatttctaa gaaatgggca tatgactctc 1680

agtacaatgg accaaattcc ctccagtcgg cccagcaata caaagggaaa gaaatgaggg 1740

ggcccacagg ccacggccca cttttctccg tggtggggag atccagctag aggtccggcc 1800

cacaagtggc ccttgccccg tgggacggtg ggattgcaga gcgcgtgggc ggaaacaaca 1860

gtttagtacc acctcgctca cgcaacgacg cgaccacttg cttataagct gctgcgctga 1920

ggctcaggga tccaacaaag caccagtggt ctagtggtag aatagtaccc tgccacggta 1980

cagacccggg ttcgattccc ggctggtgca acgacgcaac cacggtaaga gtttcagagc 2040

tatgctggaa acagcatagc aagttgaaat aaggctagtc cgttatcaac ttgaaaaagt 2100

ggcaccgagt cggtgctttttttttttt 2128

<210> 6

<211> 661

<212> DNA

<213> Artificial Sequence

<400> 6

tggaatcggc agcaaaggat tttttcctgt agttttccca caaccatttt ttaccatccg 60

aatgatagga taggaaaaat atccaagtga acagtattcc tataaaattc ccgtaaaaag 120

cctgcaatcc gaatgagccc tgaagtctga actagccggt cacctgtaca ggctatcgag 180

atgccataca agagacggta gtaggaacta ggaagacgat ggttgattcg tcaggcgaaa 240

tcgtcgtcct gcagtcgcat ctatgggcct ggacggaata ggggaaaaag ttggccggat 300

aggagggaaa ggcccaggtg cttacgtgcg aggtaggcct gggctctcag cacttcgatt 360

cgttggcacc ggggtaggat gcaatagaga gcaacgttta gtaccacctc gcttagctag 420

agcaaactgg actgccttat atgcgcgggt gctggcttgg ctgccgaaca aagcaccagt 480

ggtctagtgg tagaatagta ccctgccacg gtacagaccc gggttcgatt cccggctggt 540

gcactacctc agccacaacg ctggtttcag agctatgctg gaaacagcat agcaagttga 600

aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttttttt 660

t 661

Claims (9)

1. A method of mutating C to T in a plant genome target sequence, as follows 1) or 2): Described 1) includes the following steps: introducing SpRYn, cytosine deaminase, sgRNA and UGI into plants to mutate C to T in the plant genome target sequence; Described 2) includes the following steps: introducing the coding gene of SpRYn, the coding gene of cytosine deaminase, the DNA molecule for transcribing sgRNA and the coding gene of UGI into the plant, so that the SpRYn, the cytosine deaminase, Both the sgRNA and the UGI are expressed, thereby mutating the C in the plant genome target sequence to T; The SpRYn is a protein whose amino acid sequence is shown in sequence 2; The cytosine deaminase is PmCDA1; the PmCDA1 is a protein whose amino acid sequence is shown in sequence 3; The UGI is a protein whose amino acid sequence is shown in sequence 4; The sgRNA is tRNA-esgRNA; the tRNA is an RNA molecule obtained by replacing T in positions 597-673 of sequence 1 with U; the esgRNA skeleton is obtained by replacing T with U in positions 694-779 of sequence 1 The resulting RNA molecule; The sgRNA targeting target sequence; The PAM sequence of the target sequence is NAN or NCN or NTN; N is A, T, C or G; The plant is rice. 2. The method according to claim 1, characterized in that the coding gene of SpRYn is the DNA molecule shown at positions 3167-7267 of Sequence 1. 3. The method according to claim 1, characterized in that: the coding gene of PmCDA1 is the DNA molecule shown at positions 7553-8176 of Sequence 1. 4. The method according to claim 1, characterized in that: the encoding gene of UGI is the DNA molecule shown at positions 8210-8458 of Sequence 1. 5. Application of the method of any one of claims 1 to 4 in base editing of plant genomes; the plant is rice. 6. Application of the method of any one of claims 1 to 4 in preparing plant mutants; the plant is rice. 7. Application of complete set of reagents in mutating C to T in plant genome target sequence or preparing products to mutate C in plant genome target sequence to T; The set of reagents includes the SpRYn described in any one of claims 1-4, the cytosine deaminase described in any one of claims 1-4, the sgRNA described in any one of claims 1-4, and the sgRNA described in any one of claims 1-4. UGI as described in any one of 1-4; The PAM sequence of the target sequence is NAN or NCN or NTN; N is A, T, C or G; The plant is rice. 8. Application of complete sets of reagents in plant genome base editing or preparation of plant genome base editing products; The set of reagents includes the SpRYn described in any one of claims 1-4, the cytosine deaminase described in any one of claims 1-4, the sgRNA described in any one of claims 1-4, and the sgRNA described in any one of claims 1-4. UGI as described in any one of 1-4; The PAM sequence of the target sequence is NAN or NCN or NTN; N is A, T, C or G; The plant is rice. 9. Application of complete set of reagents in preparing plant mutants; The set of reagents includes the SpRYn described in any one of claims 1-4, the cytosine deaminase described in any one of claims 1-4, the sgRNA described in any one of claims 1-4, and the sgRNA described in any one of claims 1-4. UGI as described in any one of 1-4; The PAM sequence of the target sequence is NAN or NCN or NTN; N is A, T, C or G; The plant is rice.