A PAM-unrestricted dual base editing system

Abstract

The application provides a high-efficiency PAM-free double-base editing system. The technology realizes high-efficiency PAM-free double-base editing in rice protoplast by introducing different point mutations into SpRY, and fusing the high-efficiency adenine deaminase and cytosine deaminase to construct a double-base editor. The application can realize high-efficiency and PAM-free double-base editing, and is a plant base editing technology with a wider targeting range, higher efficiency and a higher proportion of double-base editing products.

Description

TECHNICAL FIELD

[0001] The application relates to the field of plant base editing, and particularly relates to a PAM-unrestricted double-base editing system. BACKGROUND

[0002] The CRISPR / Cas system is a genome editing tool derived from a bacterial immune system, and has been widely applied to gene function research and precision breeding of mammals, plants and microorganisms due to its high efficiency, precision and relatively simple operation process. As one of the derived technologies, the base editing system realizes targeted base conversion by fusing a deaminase with a Cas protein without introducing a double-strand break, such as CBE, ABE, CGBE, AYBE, AXBE, gGBE, TSBE and the like, which can realize various efficient single-base substitutions, regulate gene expression in plants, perform epigenetic modification and the like.

[0003] Single-base editors can only catalyze conversion and transversion of a single type of base, which limits their wide application. Therefore, developing a new base editor that can simultaneously produce two different base mutations has important significance in gene therapy, species improvement and molecular evolution.

[0004] Existing double-base editors, such as Target-ACEmax, SPACE, CABE-RY, A&C-Bemax, STCBE-2, STEME1, MoBE and TadDE, are mostly constructed based on SpCas9, and have the problems of PAM restriction and low proportion of double-base editing products. In 2020, the Benjamin Kleinstiver team developed the SpCas9 variant SpRY, which almost eliminated the PAM restriction and realized editing at any PAM, but SpRY has low editing efficiency in plants and has PAM preference.

[0005] Therefore, developing a new plant double-base editing system based on SpRY can eliminate PAM restriction and realize double-base simultaneous editing, improve editing efficiency, and has important significance for widening the editing range of plant genomes. SUMMARY

[0006] To solve the problems in the prior art, the application develops a high-efficiency PAM-unrestricted double-base editing tool. Efficient adenine deaminase and cytosine deaminase are fused with an engineered SpRY protein variant to realize PAM-less double-base simultaneous editing and widen the targeting range of plant double-base editing.

[0007] The technical scheme of the application is as follows:

[0008] A first object of the present application is to provide a base editing system based on SpRY variants, comprising:

[0009] (1) an expression construct containing a nucleotide sequence of a base editing protein selected from SpRY variants; the structure of the nucleotide sequence expression construct comprises (a) and (b):

[0010] (a) N-terminal of cytosine deaminase-C-terminal of adenine deaminase;

[0011] (b) N-terminal of SpRY variants-C-terminal of SpRY variants;

[0012] (2) an expression construct containing a nucleotide sequence encoding the guide RNA.

[0013] As used herein, "base editing system" refers to the combination of components required for editing the genome in cells or organisms. Each component of the system, such as nuclease editing protein, guide RNA, can exist independently, or can exist in any combination as a composition.

[0014] Further, (b) the SpRY variants are selected from SpRY, SpRY-AK, SpRY-K1, SpRY-K2, SpRY-K3, SpRY-KK, SpRY-V or SpRY-KV including or not including (D10A).

[0015] Further, SpRY variants not including (D10A) are selected from SpRY-AK, SpRY-K1, SpRY-K2, SpRY-K3, SpRY-KK, SpRY-V or SpRY-KV;

[0016] SpRY-AK is SpRY with R61A and G1104K mutations, SpRY-K1 is SpRY with G1104K mutation, SpRY-K2 is SpRY with R221K mutation, SpRY-K3 is SpRY with N394K mutation, SpRY-KK is SpRY with R221K and N394K mutations, SpRY-V is SpRY with L1245V mutation, and SpRY-KV is SpRY with N394K and L1245V mutations;

[0017] The nucleotide sequence of SpRY-AK is shown as SEQ ID NO. 1, the nucleotide sequence of SpRY-K1 is shown as SEQ ID NO. 2, the nucleotide sequence of SpRY-K2 is shown as SEQ ID NO. 3, the nucleotide sequence of SpRY-K3 is shown as SEQ ID NO. 4, the nucleotide sequence of SpRY-KK is shown as SEQ ID NO. 5, the nucleotide sequence of SpRY-V is shown as SEQ ID NO. 6, and the nucleotide sequence of SpRY-KV is shown as SEQ ID NO. 7.

[0018] Further, the SpRY variant comprising (D10A) is selected from SpRY-K1(D10A), SpRY-K2(D10A), SpRY-K3(D10A), or SpRY-KK(D10A); the nucleotide sequence of SpRY-K1(D10A) is shown as SEQ ID NO. 16, the nucleotide sequence of SpRY-K2(D10A) is shown as SEQ ID NO. 17, the nucleotide sequence of SpRY-K3(D10A) is shown as SEQ ID NO. 18, and the nucleotide sequence of SpRY-KK(D10A) is shown as SEQ ID NO. 19.

[0019] SpRY(D10A) is used as a control, and the nucleotide sequence is shown as SEQ ID NO. 15.

[0020] Further, (b) further comprises a nucleoplasmin NLS, and (b) is a SpRY variant-nucleoplasmin NLS connected in sequence; the nucleotide sequence of (b) is shown as SEQ ID NO. 8 to SEQ ID NO. 14.

[0021] Wherein:

[0022] SEQ ID NO. 8 is a SpRY-AK-nucleoplasmin NLS sequence;

[0023] SEQ ID NO. 9 is a SpRY-K1-nucleoplasmin NLS sequence;

[0024] SEQ ID NO. 10 is a SpRY-K2-nucleoplasmin NLS sequence;

[0025] SEQ ID NO. 11 is a SpRY-K3-nucleoplasmin NLS sequence;

[0026] SEQ ID NO. 12 is a SpRY-KK-nucleoplasmin NLS sequence;

[0027] SEQ ID NO. 13 is a SpRY-V-nucleoplasmin NLS sequence;

[0028] SEQ ID NO. 14 is a SpRY-KV-nucleoplasmin NLS sequence.

[0029] Further, (a) the cytosine deaminase is APOBEC3A, and the purine deaminase is TadA8e.

[0030] Further, (1) the nucleotide sequence expression construct is connected with (a) and (b) by a 32aa Linker, and one or more UGI and BpNLS are connected at the C-terminus.

[0031] Further, (1) the nucleotide sequence expression construct is connected with (a) and (b) by a 32aa Linker, and one or more UGI and BpNLS are connected at the C-terminus.

[0032] The 32aa Linker, nucleoplasmin NLS, and BpNLS elements are elements for constructing base editing proteins, and BpNLs and Nucleoplasmin NLS are different nuclear localization signals responsible for localizing proteins to the nucleus; the 32aa Linker is a linker responsible for connecting the deaminase and SpRY(D10A); wherein the types and positions of the nuclear localization signal (NLS) and the linker (Linker) are limited.

[0033] Further, when the SpRY variant is SpRY(D10A), the nucleotide sequence of (1) is as shown in SEQ ID NO. 20; when the SpRY variant is SpRY-K1(D10A), the nucleotide sequence of (1) is as shown in SEQ ID NO. 21; when the SpRY variant is SpRY-K2(D10A), the nucleotide sequence of (1) is as shown in SEQ ID NO. 22; when the SpRY variant is SpRY-K3(D10A), the nucleotide sequence of (1) is as shown in SEQ ID NO. 23; and when the SpRY variant is SpRY-KK(D10A), the nucleotide sequence of (1) is as shown in SEQ ID NO. 24.

[0034] Further, (1) the nucleotide sequence expression construct is connected with (a) and (b) by a 32aa Linker, and one or more UGI and BpNLS are connected at the C-terminus.

[0035] Further, (2) the expression construct further comprises a promoter for driving the guide RNA, (2) the expression construct is OsU3 promoter-N 20 -gRNA scaffold-SUP4 terminator.

[0036] The N 20 Refers to any 20 bases, which are the target points of the editor.

[0037] Further, (2) the nucleotide sequence of the guide RNA expression construct is shown in SEQ ID NO. 25.

[0038] Further, (2) the target nucleotide region targeted by the guide RNA contains a PAM sequence at the 3' end of the target sequence; the PAM sequence is NNN, and the N refers to any one of the four bases ATGC. Therefore, the double-base editing system of the present application has no PAM restriction.

[0039] Further, (2) the target nucleotide region targeted by the guide RNA contains a PAM sequence at the 3' end of the target sequence, so that the target sequence contains as many A and C as possible; the PAM sequence is NNN, and the NNN includes NAN, NGN, NCN or NTN.

[0040] Further, (1) the expression construct and (2) the expression construct are constructed on different vectors and independently driven by their own promoters.

[0041] Specifically, since the technical development process is carried out in rice protoplasts, for convenience, the mode of separating (1) and (2) is adopted, and the two parts are constructed on different vectors and driven by their own promoters.

[0042] Further, (1) the expression construct and (2) the expression construct are independently constructed on the pJIT163-Ubip-Cas9 vector with the nucleotide sequence shown in SEQ ID NO. 29 or the pJIT163-Ubip-SpRY vector with the nucleotide sequence shown in SEQ ID NO. 26.

[0043] Further, (1) the expression construct or (2) the expression construct is independently connected between the Hind III enzyme cutting site and the EcoR I enzyme cutting site of the pJIT163-ubip-Cas9 vector, or the Hind III enzyme cutting site and the EcoR I enzyme cutting site of the pJIT163-Ubip-SpRY vector.

[0044] A second object of the present application is to provide an application of the aforementioned base editing system in rice gene editing, which is to introduce the base editing system into a rice genome to be edited for double-base editing of nucleotides at multiple sites in a target nucleotide region of the rice genome to be edited.

[0045] Further, the editing type is A>G and / or C>T editing.

[0046] Further, the double-base editing system is named STEME8-SpRY variant.

[0047] In some embodiments, the editing window of A of the double-base editing protein of the present application is located at positions 2-12 of the target sequence, that is, the double-base editing protein of the present application can substitute one or more A in positions 2-10 from the 5' end of the target sequence with G.

[0048] In some embodiments, the editing window of C of the double-base editing protein of the present application is located at positions 1-16 of the target sequence, that is, the double-base editing protein of the present application can substitute one or more C in positions 1-16 from the 5' end of the target sequence with T.

[0049] In some embodiments, when the double-base editing protein of the present application performs A&C simultaneous editing, the A editing window is located at positions 2-12 and the C editing window is located at positions 1-16, that is, the double-base editing capability of the double-base editing protein of the present application can substitute one or more A in positions 2-12 from the 5' end of the target sequence with G and substitute one or more C in positions 1-16 from the 5' end of the target sequence with T.

[0050] In some embodiments, the double-base editing protein of the present application can produce A>G&C>T editing in target sequences with PAM sites NAN, NGN, NCN and NTN.

[0051] In some embodiments, the average double base substitution efficiency of the preferred double base editing protein based on the SpRY variant in the present application is significantly higher than that of the double base editing protein based on SpRY. Specifically, the average double base substitution efficiency of the preferred STEME8-SpRY-KK double base editing protein is 2.2 times that of the SpRY double base editing protein. Specifically, at the NAN PAM, the average editing efficiency of STEME8-SpRY-KK is 1.2 times that of STEME8-SpRY; at the NGN PAM, the average editing efficiency of STEME8-SpRY-KK is 1.9 times that of STEME8-SpRY; at the NCN PAM, the average editing efficiency of STEME8-SpRY-KK is 4.8 times that of STEME8-SpRY; at the NTN PAM, the average editing efficiency of STEME8-SpRY-KK is 2.7 times that of STEME8-SpRY.

[0052] STEME8-SpRY-KK can edit adenine in the range of A2-A12 and cytosine in the range of C1-C17, and can produce multiple base substitutions in the target sequence.

[0053] The organism that can be genetically modified by the base editing system of the present application includes any organism suitable for base editing, preferably eukaryotes. Examples of organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; poultry such as chickens, ducks, geese; plants, including monocotyledons and dicotyledons, for example, the plant is a crop plant, including but not limited to wheat, rice, corn, soybean, sunflower, sorghum, oilseed rape, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava and potato. Preferably, the organism is a plant. More preferably, the organism is rice.

[0054] In the method of the present application, the base editing composition can be introduced into the plant by various methods well known to those skilled in the art. Methods that can be used to introduce the base editing system of the present application into plants include, but are not limited to, gene gun method, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube channel method and ovary injection method. Preferably, the base editing composition is introduced into the plant by transient transformation.

[0055] Compared with the prior art, the technical scheme of the present application has the following beneficial effects:

[0056] After the double base editing system of the present application is introduced into the genome to be edited, the base editing protein forms a complex with the guide RNA, which specifically targets the target sequence under the mediation of the guide RNA, and realizes A>G, C>T, A>G&C>T editing within the window of the target sequence.

[0057] The application introduces point mutations in SpRY, develops new variants more efficient than SpRY, and improves the editing efficiency of non-canonical PAM in plants; the application combines high-efficiency deaminases and high-efficiency SpRY variants to effectively expand the range of double-base editing in plants. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 Schematic diagram of SpRY variant point mutation.

[0059] Figure 2 Efficiency of SpRY variant in generating indels, wherein:

[0060] A is the average editing efficiency of SpRY variants in Example 3;

[0061] B is the average editing efficiency of SpRY variants at NAN PAM in Example 3;

[0062] C is the average editing efficiency of SpRY variants at NGN PAM in Example 3;

[0063] D is the average editing efficiency of SpRY variants at NCN PAM in Example 3;

[0064] E is the average editing efficiency of SpRY variants at NTN PAM in Example 3.

[0065] Figure 3 Editing efficiency of different SpRY variant-based double-base editors, wherein:

[0066] A is the average editing efficiency of different double-base editors in Example 5;

[0067] B is the editing efficiency of different double-base editors at NAN PAM in Example 5;

[0068] C is the editing efficiency of different double-base editors at NGN PAM in Example 5;

[0069] D is the editing efficiency of different double-base editors at NCN PAM in Example 5;

[0070] E is the editing efficiency of different double-base editors at NTN PAM in Example 5.

[0071] Figure 4 Editing product proportion of different variants at NNN PAM, wherein:

[0072] A is the editing product of different double-base editors at NAN PAM in Example 5;

[0073] B is the editing product of different double-base editors at NGN PAM in Example 5;

[0074] C is the editing product of different double-base editors at NCN PAM in Example 5;

[0075] D is the editing product of different double-base editors at NTN PAM in Example 5;

[0076] E is the editing product of different double-base editors at NNN PAM in Example 5.

[0077] Figure 5 The editing window of different double-base editors, wherein:

[0078] A is the cytosine editing range of different double-base editors in Example 5;

[0079] B is the adenine editing range of different double-base editors in Example 5. DETAILED DESCRIPTION

[0080] The embodiments of the present application will be described below in conjunction with the accompanying drawings. It should be noted that the following embodiments are only used to illustrate the exemplary implementation of the present application, and do not limit the present application in any way. Those skilled in the art can make certain equivalent changes and obvious improvements to the present application.

[0081] In the absence of other specific descriptions, the operations in the following detailed description are all carried out by using the conventional operations in the art. Those skilled in the art can easily obtain the teachings about such conventional operations from the prior art. The medicinal material raw materials, reagents, materials, etc. used in the following embodiments, if not specifically stated, are all commercially available products.

[0082] In order to improve the editing efficiency of SpRY in plants and broaden its editing range in plants, SpRY was modified to construct seven variants of SpRY-AK, SpRY-K1, SpRY-K2, SpRY-K3, SpRY-KK, SpRY-V and SpRY-KV. Figure 1

[0083] ​(1) Vector construction to obtain vector skeleton. The pJIT163-Ubip-SpRY vector shown in nucleotide SEQ ID NO. 26 was double digested with Hind III and Mlu I to obtain a 6142 bp pJIT163 skeleton (37°C, 2.5 h). Using pJIT163-Ubip-SpRY as a template, Hind III-RY-F and R221K-R, R221K-F and Mlu I-RY-R were used for amplification to construct SpRY-K2; using pJIT163-Ubip-SpRY as a template, Hind III-RY-F and R394K-R, N394K-F and Mlu I-RY-R were used for amplification to construct SpRY-K3; using pJIT163-Ubip-SpRY-K2 as a template, Hind III-RY-F and R394K-R, N394K-F and Mlu I-RY-R were used for amplification to construct SpRY-KK.

[0084] (2) Vector construction to obtain vector skeleton. The pJIT163-Ubip-SpRY vector was double digested with Hind III, EcoR I and Mlu I to obtain a pJIT163 skeleton (37°C, 2.5 h). Using pJIT163-Ubip-SpRY as a template, Hind III-RY-F and R61A-R, R61A-F and G1104K-R, G1104K-F and EcoR I-R were used for amplification to construct SpRY-AK; using pJIT163-Ubip-SpRY as a template, Mlu I-RY-F and G1104K-R, G1104K-F and EcoR I-R were used for amplification to construct SpRY-K1; using pJIT163-Ubip-SpRY as a template, Mlu I-RY-F and L1245V-R, L1245V-F and EcoR I-R were used for amplification to construct SpRY-V; using pJIT163-Ubip-SpRY-K3 as a template, Mlu I-RY-F and L1245V-R, L1245V-F and EcoR I-R were used for amplification to construct SpRY-KV.

[0085] The primer sequences are as follows:

[0086] Hind III-RY-F: gtttggtgttacttctgcaaagcttATGGATTACAAGGACCACGACG (SEQ ID NO. 30);

[0087] Mlu I-RY-F: ACCACCATGCCCATGACGCGTACCTGAACGCGGTGG (SEQ ID NO. 31);

[0088] MluI-RY-R: CCACCGCGTTCAGGTACGCGTCATGGGCATGGTGGT (SEQ ID NO. 32);

[0089] EcoRl-R: ctggtgatttcagcgtaccgaattcTCACTTCTTCTTCTTCGCCTG (SEQ ID NO. 33);

[0090] R61A-F: GACGGCTGAGGCTACGCGGCTCAA (SEQ ID NO. 34);

[0091] R61A-R: TTGAGCCGCGTAGCCTCAGCCGTC (SEQ ID NO. 35);

[0092] R221K-F: GAAGTCTCGGAAGCTCGAGAACCTGATC (SEQ ID NO. 36);

[0093] R221K-R: TCTCGAGCTTCCGAGACTTCGAGAGCCTAG (SEQ ID NO. 37);

[0094] N394K-F: GGTGAAGCTCAAGCGCGAGGACCTCCTGA (SEQ ID NO. 38);

[0095] N394K-R: CCTCGCGCTTGAGCTTCACCAGCAGCTC (SEQ ID NO. 39);

[0096] G1104K-F: GGTCCAGACTGGCAAGTTCTCTAA (SEQ ID NO. 40);

[0097] G1104K-R: TTAGAGAACTTGCCAGTCTGGACC (SEQ ID NO. 41);

[0098] L1245V-F: CTACGAGAAGGTTAAGGGCAGCCCCGAG (SEQ ID NO. 42);

[0099] L1245V-R: TGCCCTTAACCTTCTCGTAGTGGGAGGCC (SEQ ID NO. 43).

[0100] In the above primers, the F primer is used to amplify the 5' end of the fragment with homologous arms, and the R primer is used to amplify the 3' end of the fragment with homologous arms. The pJIT163-Ubip-SpRY is a carrier already available in the laboratory, and the nucleotide sequence of pJIT163-Ubip-SpRY is shown in SEQ ID NO. 26, and the nucleotide sequence of SpRY is shown in SEQ ID NO. 27.

[0101] The PCR amplification system of the above fragment is as follows:

[0102] Template carrier: 1 µL (30-50 ng), Primer-F: 1.5 µL (10 µM), Primer-R: 1.5 µL (10 µM), 2 × PCR Buffer for KOD FX: 25 µL, 2 mM dNTPs: 10 µL, KOD FX (TOYOBO KFX-101): 1 µL, ddH2O: 10 uL.

[0103] The PCR program is as follows:

[0104] Stage 1 (×1): 95℃, 3 min (pre-denaturation); Stage 2 (×35): 95℃, 15 s (denaturation); Tm−5℃, 15 s (annealing); 72℃, 1 kb / min (extension); Stage 3 (×1): 72℃, 5 min (complete extension); 4℃, ∞.

[0105] (3) The fragments obtained by amplification for constructing SpRY-AK, SpRY-K1, SpRY-K2, SpRY-K3, SpRY-KK, SpRY-V and SpRY-KV were respectively connected with the pJIT163 skeleton obtained by enzyme digestion for homologous recombination, the connection product was subjected to heat shock transformation of E. coli, and was coated on a solid LB plate and cultured at 37℃ for 12-16 h.

[0106] The above homologous recombination system is as follows:

[0107] Skeleton carrier fragment: length (bp) × 0.02 / recovered product concentration (ng / µL).

[0108] Insert fragment: length (bp) × 0.04 / recovered product concentration (ng / µL).

[0109] 2 × MultiF Seamless Assembly Mix (ABclonal RK21020): 5 µL.

[0110] ddH2O: to 10 µL.

[0111] PCR program settings

[0112] 2-3 fragment assembly: 50°C, 30 min; 4°C, ∞.

[0113] 4-6 fragment assembly: 50°C, 60 min; 4°C, ∞.

[0114] E. coli heat shock transformation procedure: 10 μL of ligation product was added to 50 μL of DH5α E. coli competent cells, 30 min on ice, 42°C heat shock for 45 s, 2 min on ice, 700 μL of antibiotic-free LB was added, 37°C, 220 rpm shaker incubation for 30 min, 6000 rpm centrifugation for 1 min to collect the bacteria, and then evenly coated on ampicillin-resistant LB plates, and incubated in a 37°C constant temperature incubator for 12-16 h.

[0115] Colony PCR identification: a pair of primers was used for PCR on smaller single colonies, and 12 single colonies were picked from each vector for detection, and the reaction system was as follows:

[0116] 2x Rapid Taq Master Mix (Vazyme P222-01): 5 μL, Primer-F: 0.5 μL, Primer-R: 0.5 μL, ddH2O: 4 μL.

[0117] PCR program settings:

[0118] Stage 1 (x1): 94°C, 30 s (pre-denaturation);

[0119] Stage 2 (x30): 98°C, 10 s (denaturation); Tm-5°C, 30 s (annealing); 72°C, 1 kb / min (extension);

[0120] Stage 3 (x1): 72°C, 2 min (complete extension); 4°C, ∞.

[0121] The primer sequences are:

[0122] F1: gcatatgcagcagctatatgtgga (SEQ ID NO. 44).

[0123] R1: CGTAGGGTACTTCTCGTGGTAGG (SEQ ID NO. 45).

[0124] F2: ATCCGAAGAAGTACGGCGGGTTC (SEQ ID NO. 46).

[0125] R2: ccttatcgggaaactactcacacat (SEQ ID NO. 47).

[0126] F1 and R1 were combined for colony PCR of SpRY-AK, SpRY-K2, SpRY-K3 and SpRY-KK; F2 and R2 were combined for colony PCR of SpRY-AK, SpRY-K1 and SpRY-V. 2 correct monoclonal colonies were picked for each vector for sequencing by Sangon Biotech (Shanghai) Co., Ltd. Finally, 7 SpRY nuclease editing vectors with different point mutations were obtained.

[0127] SpRY nuclease editing vectors obtained by ligating SpRY variants such as SpRY-AK, SpRY-K1, SpRY-K2, SpRY-K3, SpRY-KK, SpRY-V, SpRY-KV, etc. shown in SEQ ID NO. 1~7 into the Hind III and EcoR I double enzyme digestion sites of pJIT163-Ubip-SpRY vector.

[0128] For the SpRY nuclease editing vectors constructed in Example 1, suitable endogenous target sites of rice were selected. The target sequence selected was first determined as a 20 bp-NNN PAM in the endogenous gene of rice, and the sequence was ensured to have as many A and C as possible.

[0129] A total of 16 endogenous target sites of rice were screened in this experiment for testing the efficiency of different SpRY variants, including OsMPK13 -NAA, OsGS1- NAT, OsPDS- NAC and O sCOI2 -NAG; OsCDC48 -NGA, OsCDC48 -NGT, OsCDC48- NGC and OsCPK5 -NGG; OsPDS -NCA, OsCDC48 -NCT, OsSPL7 -NCC and OsPDS -NCG; OsWaxy -NTA, OsWaxy -NTT, OsCPK28 -NTC and OsSPL7 -NTG.

[0130] In the following target sequences, the PAM sequence is bolded and underlined.

[0131] An expression construct containing a nucleotide sequence encoding a guide RNA (nucleotide sequence shown as SEQ ID NO. 25) was ligated between the Hind III and EcoR I restriction sites of the pJIT163-ubip-Cas9 vector (nucleotide sequence shown as SEQ ID NO. 29) to form the pOsU3-sgRNA vector. At the same time, a sgRNA primer pair (SgF / SgR) corresponding to the target sequence was prepared, i.e., a primer required for constructing a guide RNA, for ligation between the two Bsa I restriction sites of the pOsU3-sgRNA vector. The upper case portion of the following sgRNA primer pair corresponds to 20 bp of the target sequence, and the lower case portion is used to generate a sticky end and ligate the vector backbone.

[0132] Target information for testing editing efficiency:

[0133] (1) OsMPK13 -NAA

[0134] Target sequence: CGGACATGGAGTTCTTTACG GAA (SEQ ID NO 122)

[0135] sgF: ggcgCGGACATGGAGTTCTTTACG (SEQ ID NO. 48)

[0136] sgR: aaacCGTAAAGAACTCCATGTCCG (SEQ ID NO. 49)

[0137] (2) OsGS1 -NAT

[0138] Target sequence: CACACCAACTACAGGTGAGG GAT (SEQ ID NO. 50)

[0139] sgF: ggcgCACACCAACTACAGGTGAGG (SEQ ID NO. 51)

[0140] sgR: aaacCCTCACCTGTAGTTGGTGTG (SEQ ID NO. 52)

[0141] (3) OsPDS -NAC

[0142] Target sequence: ATCCTGATGGAACAGTGAAA CAC (SEQ ID NO. 53)

[0143] sgF: ggcgAGGAATGGGTTGGACGGAGT (SEQ ID NO. 54)

[0144] sgR: aaacACTCCGTCCAACCCATTCCT (SEQ ID NO. 55)

[0145] (4) OsCOI2 - NAG

[0146] Target sequence: TGAGCGCGTCGACCCTGCAC CAG (SEQ ID NO. 56)

[0147] sgF: ggcgTGAGCGCGTCGACCCTGCAC (SEQ ID NO. 57)

[0148] sgR: aaacGTGCAGGGTCGACGCGCTCA (SEQ ID NO. 58)

[0149] (5) OsCDC48 -NGA

[0150] Target sequence: GGCTCACTTCGAGGAGTCGA TGA (SEQ ID NO. 59)

[0151] sgF: ggcgGGCTCACTTCGAGGAGTCGA (SEQ ID NO. 60)

[0152] sgR: aaacTCGACTCCTCGAAGTGAGCC (SEQ ID NO. 61)

[0153] (6) OsCDC48 -NGT

[0154] Target sequence: TCACTTCGAGGAGTCGATGA AGT (SEQ ID NO. 62)

[0155] sgF: ggcgTCACTTCGAGGAGTCGATGA (SEQ ID NO. 63)

[0156] sgR: aaacTCATCGACTCCTCGAAGTGA (SEQ ID NO. 64)

[0157] (7) OsCDC48- NGC

[0158] Target sequence: TTCGAGGAGTCGATGAAGTA CGC (SEQ ID NO. 65)

[0159] sgF: ggcgTTTGAGGAGTCGATGAAGTA (SEQ ID NO. 66)

[0160] sgR: aaacTACTTCATCGACTCCTCGAA (SEQ ID NO. 67)

[0161] (8) OsCPK5- NGG

[0162] Target sequence: AAGTACTTCGCCAGCTTCCG CGG (SEQ ID NO. 68)

[0163] sgF: ggcgAAGTACTTCGCCAGCTTCCG (SEQ ID NO. 69)

[0164] sgR: aaacCGGAAGCTGGCGAAGTACTT (SEQ ID NO. 70)

[0165] (9) OsPDS -NCA

[0166] Target sequence: CTTAAATGGTGAGATCATAT GCA (SEQ ID NO. 71)

[0167] sgF: ggcgCTTAAATGGTGAGATCATAT (SEQ ID NO. 72)

[0168] sgR: aaacATATGATCTCACCATTTAAG (SEQ ID NO. 73)

[0169] (10) OsCDC48 -NCT

[0170] Target sequence: ACACCATCTGCATTGTTCTT GCT (SEQ ID NO. 74)

[0171] sgF: ggcgACACCATCTGCATTGTTCTT (SEQ ID NO. 75)

[0172] sgR: aaacAAGAACAATGCAGATGGTGT (SEQ ID NO. 76)

[0173] (11) OsSPL7- NCC

[0174] Target sequence: ACAGAAGAGAGAGAGCACCA GCC(SEQ ID NO. 77)

[0175] sgF: ggcgACAGAAGAGAGAGAGCACCA (SEQ ID NO. 78)

[0176] sgR: aaacTGGTGCTCTCTCTCTTCTGT (SEQ ID NO. 79)

[0177] (12) OsPDS - NCG

[0178] Target sequence: AACAAGCCAGGAGAATTCAG CCG (SEQ ID NO. 80)

[0179] sgF: ggcgAACAAGCCAGGAGAATTCAG (SEQ ID NO. 81)

[0180] sgR: aaacCTGAATTCTCCTGGCTTGTT (SEQ ID NO. 82)

[0181] (13) OsWaxy - NTA

[0182] Target sequence: CTACCTCAGCCACAACGCTG GTA (SEQ ID NO. 83)

[0183] sgF: ggcgCTACCTCAGCCACAACGCTG (SEQ ID NO. 84)

[0184] sgR: aaacCAGCGTTGTGGCTGAGGTAG (SEQ ID NO. 85)

[0185] (14) OsWaxy - NTT

[0186] Target sequence: CGAACACAACATCCTCACCT GTT (SEQ ID NO. 86)

[0187] sgF: ggcgCGAACACAACATCCTCACCT (SEQ ID NO. 87)

[0188] sgR: aaacAGGTGAGGATGTTGTGTTCG (SEQ ID NO. 88)

[0189] (15) OsCPK28 - NTC

[0190] Target sequence: GCGAGTACGCGTGCAAGTCC ATC (SEQ ID NO. 89)

[0191] sgF: ggcgGCGAGTACGCGTGCAAGTCC (SEQ ID NO. 90)

[0192] sgR: aaacGGACTTGCACGCGTACTCGC (SEQ ID NO. 91)

[0193] (16) OsSPL7 -NTG

[0194] Target sequence: ACGAGAATCAGTCGGGGCAC GTG (SEQ ID NO. 92)

[0195] sgF: ggcgACGAGAATCAGTCGGGGCAC (SEQ ID NO. 93)

[0196] sgR: aaacGTGCCCCGACTGATTCTCGT (SEQ ID NO. 94)

[0197] Each pair of SgF / R above was annealed separately, with the following program: 98°C, 5 min; 95°C, 1 min; 90°C, 1 min; 80°C, 1 min; 70°C, 1 min; 60°C, 1 min; 50°C, 1 min; 40°C, 1 min; 30°C, 1 min; 20°C, 1 min; 10°C, 1 min; 4°C hold, and then OsMPK13 -NAA, OsGS1- NAT, OsPDS- NAC, O sCOI2 -NAG; OsCDC48 -NGA, OsCDC48 -NGT, OsCDC48- NGC, OsCPK5 -NGG; OsPDS -NCA, OsCDC48 -NCT, OsSPL7 -NCC, OsPDS -NCG; OsWaxy -NTA, OsWaxy -NTT, OsCPK28 -NTC and OsSPL7 -NTG were ligated to the pOsU3-sgRNA backbone after Bsal enzyme inactivation (37°C, 2.5 h; 80°C, 20 min).

[0198] The annealing product was connected with the pOsU3-sgRNA enzyme digestion backbone connection system:

[0199] Target annealing product: 5 μL, enzyme digestion vector: 10-20 ng, T4 DNA Ligase (NEB M0202L): 0.5 μL, 10×T4 DNA Ligase Buffer: 1 μL, ddH2O: to 10 μL.

[0200] The reaction system was 16°C for 30 min.

[0201] The ligation product was transformed into E. coli, identified by colony PCR and sequencing, and the pOsU3-sgRNA vector with Figure 2 -NAA, Figure 2 NAT, Figure 2 NAC, O Figure 2 -NAG; Figure 2 -NGA, Figure 2 -NGT, OsGS1 NGC, OsGSK4 -NGG; OsCOI2 -NCA, OsPDS -NCT, OsMPK10 -NCC, OsCOI2 -NCG; OsCPK1 -NTA, OsCDC48 -NTT, OsCDC48 -NTC and OsCDC48 -NTG target sequence.

[0202] The seven vectors constructed in Example 1 were subjected to efficiency verification with the target points in Example 2 in rice protoplasts, and the highest efficiency variant was screened out.

[0203] Extraction of protoplasts:

[0204] Rice seedlings cultured for 15-20 days were selected, and the stem and leaf sheath parts were cut into 0.5 mm strips and transferred to a 0.6 M Mannitol solution, and placed in the dark for 10 min; the Mannitol solution was filtered out with a Miracloth filter cloth, and the strips were transferred to a 150 mL triangular flask containing enzyme solution, and placed in the dark for 5 h with 10 rpm shaking;

[0205] After enzyme digestion, an equal volume of W5 solution was added, and the protoplasts were released by gently shaking horizontally for 10-20 s; the released protoplasts were filtered into a 50 mL round-bottom centrifuge tube with a Miracloth filter cloth; to obtain more protoplasts, 100 mL of W5 solution was added along the wall, and the protoplasts were released again and filtered.

[0206] 250 g horizontal centrifugation for 3 min (3 for lift, 3 for drop), carefully aspirate the supernatant with 5 mL pipette;

[0207] Add 15 mL W5 to resuspend, gently suspend all protoplast cells, repeat centrifugation, and discard the supernatant;

[0208] Add an appropriate amount of MMG solution to gently resuspend, so that the protoplast concentration is 2 × 10 6 / mL;

[0209] The 7 SpRY variants constructed in Example 1 were respectively added to the bottom of a 2 ml EP tube at a ratio of 10 μg + 10 μg to the corresponding pOsU3-sgRNA vector constructed in Example 2.

[0210] Because the present application takes wild-type SpRY as a control, 10 μg of GFP vector is also transformed as a fluorescence observation group, and a control group (CK) without any vector is added.

[0211] The pJIT163-GFP vector is the GFP sequence connected between the Hind III and EcoR I enzyme cutting sites of the pJIT163-ubip-Cas9 vector (the GFP nucleotide sequence is shown in SEQ ID NO. 28).

[0212] After centrifugation of each group of samples, 200 μL of protoplasts were added, gently mixed, and then 220 μL of PEG solution was added. After gentle mixing, the sample was placed at room temperature in the dark for induction of transformation; 20 min later, 880 μL of W5 solution was added, gently mixed, and centrifuged at 250 g for 3 min, and the supernatant was aspirated.

[0213] Add 1 mL of WI solution to resuspend all cells, place the centrifuge tube flat and incubate at 30°C in the dark overnight, observe the GFP control group fluorescence, and evaluate the transformation efficiency; after 48-60 h of culture, the protoplast genomic DNA was extracted.

[0214] The rice protoplast DNA was extracted using a kit, and the target sequence was amplified by designing 2 rounds of corresponding target primers. The length of the first round of PCR product was 500-700 bp, and the length of the second round of PCR product with different Barcodes was 150-200 bp. Barcodes were used to distinguish different SpRY. The PCR stock solution after amplification was mixed and recovered in the library, and was sent to Jinweizhi for second-generation sequencing.

[0215] Through cluster splitting, the average editing efficiency of the 7 SpRY variants was obtained by analyzing and arranging the data. OsPDS-

[0216] ​The average editing efficiency of SpRY variants at six endogenous target sites tested for editing efficiency was as follows, from highest to lowest: SpRY-KK (4.50%) > SpRY-K1 (2.88%) > SpRY-K2 (2.74%) > SpRY-AK (2.45%) > SpRY-KV (2.28%) > SpRY-K3 (2.13%) > SpRY-V (1.99%) > SpRY (1.31%). This indicates that introducing different point mutations into SpRY improves its average editing efficiency in plants. Compared to SpRY, SpRY-KK showed a significantly higher efficiency, being 3.4 times that of SpRY; ​​followed by SpRY-K1, with a 2.2-fold increase; and then SpRY-K2, with a 2.1-fold increase. OsPDS A).

[0217] At NAN PAM ( OsPDS- B), average editing efficiency SpRY-KK (7.39%) > SpRY-K1 (3.88%) > SpRY-KV (3.28%) > SpRY-AK (3.19%) > SpRY-K2 (2.77%) > SpRY (2.23%) = SpRY-K3 (2.23%) > SpRY-V (1.92%); at NGN PAM ( OsSPL7 C), average editing efficiency: SpRY-KK (6.46%) > SpRY-K2 (4.18%) > SpRY-K1 (3.94%) > SpRY-KV (3.58%) > SpRY-AK (3.14%) > SpRY-V (2.48%) > SpRY-K3 (2.04%) > SpRY (1.63%); at NCN PAM ( LOC_OS08g37790- D), average editing efficiency SpRY-KK (3.44%) > SpRY-K3 (2.99%) > SpRY-K2 (2.98%) > SpRY-V (2.49%) > SpRY-AK (2.47%) > SpRY-KV (1.43%) > SpRY (1.22%); at NTN PAM ( OsSPL7 E), the average editing efficiency is SpRY-K1 (1.56%)>SpRY-K3 (1.24%)>SpRY-V (1.09%)>SpRY-K2 (1.03%)>SpRY-AK (1.00%)>SpRY-KV (0.82%)>SpRY-KK (0.71%)>SpRY (0.25%).

[0218] The average editing efficiency of SpRY-K3 at NYN PAM (NCN PAM and NTN PAM) is 1.2 times and 1.9 times of SpRY-AK and SpRY-KV, respectively. Considering comprehensively, SpRY-K1 (SEQ ID NO. 2), SpRY-K2 (SEQ ID NO. 3), SpRY-K3 (SEQ ID NO. 4) and SpRY-KK (SEQ ID NO. 5) are selected for further exploration of high-efficiency PAM-less double-base editing system.

[0219] 1. Constructing double-base editing system based on SpRY, SpRY-K1, SpRY-K2, SpRY-K3 and SpRY-KK

[0220] (1) The pJIT163-Ubip-Cas9 represented by the nucleotide sequence of SEQ ID NO. 29 was subjected to enzyme digestion using restriction endonuclease Hind III and EcoR I, and a 4943 bp vector skeleton was obtained by recovery. The synthesized APOBEC3A-TadA8e (nucleotide sequence of SEQ ID NO. 162) and 32aa linker-Cas9-NLS-UGI-bpNLS (nucleotide sequence of SEQ ID NO. 163) were used as templates for fragment amplification, and fragments with homologous arms were obtained by recovery. The fragments were constructed into the pJIT163-Ubip-Cas9 skeleton by homologous recombination, and the E. coli DH5α competent cells were heat-shocked and transformed. After picking single colony bacteria P, it was sent for measurement, and pJIT163-Ubip-STEME8 was obtained. The specific method is shown in Example 1, and the structure of pJIT163-Ubip-STEME8 is Ubi promoter, APOBEC3A-TadA8e, 32aa Linker, Cas9, nucleoplasmin NLS, UGI, BpNLS, CaMV terminator arranged in turn.

[0221] The primer sequence for amplifying A3A-TadA8e is:

[0222] A3A-F: gtttggtgttacttctgcaaagcttATGATGGAGGCCAGCCCG (SEQ ID NO. 95);

[0223] TadA8e-R: CGAGTACTTCTTGTCcttaagGCTCCCTCCAGAGCT (SEQ ID NO. 96);

[0224] The primer sequence for amplifying Cas9-NLS-UGI-bpNLS is:

[0225] Cas9-F: AGCTCTGGAGGGAGCcttaagGACAAGAAGTACTCG (SEQ ID NO. 97);

[0226] bpNLS-R: ctggtgatttcagcgtaccgaattcTCACACCTTCCTTTTCTTCTTCGG (SEQ ID NO. 98).

[0227] Among the above primers, A3A-F and TadA8e-R are used to amplify the A3A-TadA8e fragment with homologous arms; Cas9-F and bpNLS-R are used to amplify the Cas9-NLS-UGI-bpNLS fragment with homologous arms.

[0228] (2) The pJIT163-Ubip-STEME8 is double enzyme cut by using restriction endonuclease Afl II and EcoR I, and a 6191 bp vector skeleton is obtained by recovery; the SpRY, SpRY-K1, SpRY-K2, SpRY-K3 and SpRY-KK corresponding (D10A) variant are used as templates for fragment amplification, and a fragment with homologous arms is obtained by recovery; the fragment is constructed into the pJIT163 skeleton by seamless cloning, and the double base editing vectors pJIT163-STEME8-SpRY, pJIT163-STEME8-SpRY-K1 (hereinafter referred to as STEME8-SpRY-K1), pJIT163-STEME8-SpRY-K2 (hereinafter referred to as STEME8-SpRY-K2), pJIT163-STEME8-SpRY-K3 (hereinafter referred to as STEME8-SpRY-K3) and pJIT163-STEME8-SpRY-KK (hereinafter referred to as STEME8-SpRY-KK) with point mutations are constructed.

[0229] The structure of pJIT163-Ubip-STEME8-SpRY is: Ubi promoter, APOBEC3A-TadA8e, 32aa Linker, SpRY(D10A), nucleoplasmin NLS, UGI, BpNLS, CaMV terminator (the nucleotide sequence except the promoter and terminator is shown as SEQ ID NO. 20, and the promoter and terminator are on the pJIT163 vector skeleton); the structure of pJIT163-Ubip-STEME8-SpRY-K1 is: Ubi promoter, APOBEC3A-TadA8e, 32aa Linker, SpRY-K1(D10A), nucleoplasmin NLS, UGI, BpNLS, CaMV terminator (the nucleotide sequence except the promoter and terminator is shown as SEQ ID NO. 21); the structure of pJIT163-Ubip-STEME8-SpRY-K2 is: Ubi promoter, APOBEC3A-TadA8e, 32aa Linker, SpRY-K2(D10A), nucleoplasmin NLS, UGI, BpNLS, CaMV terminator (the nucleotide sequence except the promoter and terminator is shown as SEQ ID NO. 22); the structure of pJIT163-Ubip-STEME8-SpRY-K3 is: Ubi promoter, APOBEC3A-TadA8e, 32aa Linker, SpRY-K3(D10A), nucleoplasmin NLS, UGI, BpNLS, CaMV terminator (the nucleotide sequence except the promoter and terminator is shown as SEQ ID NO. 23); the structure of pJIT163-Ubip-STEME8-SpRY-KK is: Ubi promoter, APOBEC3A-TadA8e, 32aa Linker, SpRY-KK(D10A), nucleoplasmin NLS, UGI, BpNLS, CaMV terminator (the nucleotide sequence except the promoter and terminator is shown as SEQ ID NO. 24).

[0230] The primer sequence used for amplification is:

[0231] Cas9-F: AGCTCTGGAGGGAGCcttaagGACAAGAAGTACTCG (SEQ ID NO. 99);

[0232] bpNLS-R: ctggtgatttcagcgtaccgaattcTCACACCTTCCTTTTCTTCTTCGG (SEQ ID NO. 100).

[0233] Among the above primers, Cas9-F and bpNLS-R are used to amplify different SpRY variant fragments with homologous arms.

[0234] The specific vector construction method is shown in Example 1.

[0235] For the double-base editing vector constructed in the above Example 3, a suitable endogenous target site of rice is selected. The selected target sequence is first determined as a 20 bp-NNN PAM in the endogenous gene of rice as a target sequence, and it is ensured that there are as many A and C as possible in the sequence.

[0236] In this experiment, 16 endogenous target sites of rice were screened for testing the efficiency of different SpRY variants, including OsSPL14 -NAT, OsWaxy -NAC, OsWaxy -NAG, OsCOI2 -NAC and OsGS1 -NAT; GAT -NGC, OsGSK4 -NGA, GAC -NGT, OsCOI2 -NGC and CAG -NGC; OsPDS NCA, CAC -NCG, OsMPK10 NCG, AAT -NCC and OsCOI2 NCA; GGC -NTG, OsCPK1 -NTC, GGC -NTT, OsCDC48 -NTG and AGT -NTG.

[0237] In the following target sequences, the PAM sequence is bolded and underlined.

[0238] At the same time, the sgRNA primer pair (SgF / SgR) corresponding to the target sequence is prepared, that is, the primer required for constructing the guide RNA is used for connecting to the pOsU3-sgRNA vector. In the following sgRNA primer pair, the capital part is 20 bp corresponding to the target sequence, and the lowercase part is used to generate a sticky end and connect the vector skeleton.

[0239] Target information for testing editing efficiency:

[0240] (1) OsCDC48- -NAT

[0241] Target sequence: CACACCAACTACAGGTGAGG CGC (SEQ ID NO. 101);

[0242] sgF: ggcgCACACCAACTACAGGTGAGG (SEQ ID NO. 102);

[0243] sgR: aaacCCTCACCTGTAGTTGGTGTG (SEQ ID NO. 103).

[0244] (2) OsCDC48 -NAC

[0245] Target sequence: ATATAGCTAATAGTCTAGAG GGC (SEQ ID NO. 104);

[0246] sgF: ggcgATATAGCTAATAGTCTAGAG (SEQ ID NO. 105);

[0247] sgR: aaacCTCTAGACTATTAGCTATAT (SEQ ID NO. 106).

[0248] (3) OsPDS -NAG

[0249] Target sequence: TGAGCGCGTCGACCCTGCAC GCA (SEQ ID NO. 107);

[0250] sgF: ggcgTGAGCGCGTCGACCCTGCAC (SEQ ID NO. 108);

[0251] sgR: aaacGTGCAGGGTCGACGCGCTCA (SEQ ID NO. 109).

[0252] (4) OsPDS -NAC

[0253] Target sequence: ATCCTGATGGAACAGTGAAA GCG (SEQ ID NO. 110);

[0254] sgF: ggcgATCCTGATGGAACAGTGAAA (SEQ ID NO. 111);

[0255] sgR: aaacTTTCACTGTTCCATCAGGAT (SEQ ID NO. 112).

[0256] (5) OsPDS- -NAT

[0257] Target sequence: CACACTGGAGAGAAAGTGGC CCG (SEQ ID NO. 113);

[0258] sgF: ggcgCACACTGGAGAGAAAGTGGC (SEQ ID NO. 114);

[0259] sgR: aaacGCCACTTTCTCTCCAGTGTG (SEQ ID NO. 115).

[0260] (6) OsSPL7 -NGC

[0261] Target sequence: GTCGACCCTGCACCAGTGGC GCC (SEQ ID NO. 116);

[0262] sgF: ggcgGTCGACCCTGCACCAGTGGC (SEQ ID NO. 117);

[0263] sgR: aaacGCCACTGGTGCAGGGTCGAC (SEQ ID NO. 118).

[0264] (7) LOC_OS08g37790 -NGA

[0265] Target sequence: GTCGACCCTGCACCAGTGGC GCA (SEQ ID NO. 119);

[0266] sgF: ggcgAAGACCGAAGTCAGTGGCCT (SEQ ID NO. 120);

[0267] sgR: aaacAGGCCACTGACTTCGGTCTT (SEQ ID NO. 121).

[0268] (8) OsSPL7 -NGT

[0269] Target sequence: TCACTTCGAGGAGTCGATGA GTG (SEQ ID NO. 123);

[0270] sgF: ggcgTCACTTCGAGGAGTCGATGA (SEQ ID NO. 124);

[0271] sgR: aaacTCATCGACTCCTCGAAGTGA (SEQ ID NO. 125).

[0272] (9) OsSPL14 NGC

[0273] Target sequence: TTCGAGGAGTCGATGAAGTA TTC (SEQ ID NO. 126);

[0274] sgF: ggcgTTCGAGGAGTCGATGAAGTA (SEQ ID NO. 127);

[0275] sgR: aaacTACTTCATCGACTCCTCGAA (SEQ ID NO. 128).

[0276] (10) OsWaxy -NGC

[0277] Target sequence: GCCTGGTACTTGCGGATGTC GTT (SEQ ID NO. 129);

[0278] sgF: ggcgGCCTGGTACTTGCGGATGTC (SEQ ID NO. 130);

[0279] sgR: aaacGACATCCGCAAGTACCAGGC (SEQ ID NO. 131).

[0280] (11) OsWa -NCA

[0281] Target sequence: CTTAAATGGTGAGATCATAT ATG (SEQ ID NO. 132);

[0282] sgF: ggcgCTTAAATGGTGAGATCATAT (SEQ ID NO. 133);

[0283] sgR: aaacATATGATCTCACCATTTAAG (SEQ ID NO. 134).

[0284] (12) OsCOI2 -NCG

[0285] Target sequence: CGAACCTCACCACCCAAAGA CTG (SEQ ID NO. 135);

[0286] sgF: ggcgAACAAGCCAGGAGAATTCAG (SEQ ID NO. 139);

[0287] sgR: aaacCTGAATTCTCCTGGCTTGTT (SEQ ID NO. 140).

[0288] (13) OsGS1 NCG

[0289] Target sequence: AACAAGCCAGGAGAATTCAG OsGSK4 (SEQ ID NO. 138);

[0290] sgF: ggcgAACAAGCCAGGAGAATTCAG (SEQ ID NO. 139);

[0291] sgR: aaacCTGAATTCTCCTGGCTTGTT (SEQ ID NO. 140).

[0292] (14) OsCOI2 -NCC

[0293] Target sequence: ACAGAAGAGAGAGAGCACCA OsPDS (SEQ ID NO. 141);

[0294] sgF: ggcgACAGAAGAGAGAGAGCACCA (SEQ ID NO. 142);

[0295] sgR: aaacTGGTGCTCTCTCTCTTCTGT (SEQ ID NO. 143).

[0296] (15) OsMPK10 -NCA

[0297] Target sequence: CACCACGGACATCTGGAGGA OsCOI2 (SEQ ID NO. 144);

[0298] sgF: ggcgCACCACGGACATCTGGAGGA (SEQ ID NO. 145);

[0299] sgR: aaacTCCTCCAGATGTCCGTGGTG (SEQ ID NO. 146).

[0300] (16) OsCPK1 -NTG

[0301] Target sequence: ACGAGAATCAGTCGGGGCAC OsCDC48 (SEQ ID NO. 147);

[0302] sgF: ggcgACGAGAATCAGTCGGGGCAC (SEQ ID NO. 148);

[0303] sgR: aaacGTGCCCCGACTGATTCTCGT (SEQ ID NO. 149).

[0304] (17) OsCDC48 -NTC

[0305] Target sequence: TTCATACCAGTTCCCCCCTT OsCDC48 (SEQ ID NO. 150);

[0306] sgF: ggcgTTCATACCAGTTCCCCCCTT (SEQ ID NO. 151);

[0307] sgR: aaacAAGGGGGGAACTGGTATGAA (SEQ ID NO. 152).

[0308] (18) OsPDS- -NTT

[0309] Target sequence: CGAACACAACATCCTCACCT OsPDS (SEQ ID NO. 153);

[0310] sgF: ggcgCGAACACAACATCCTCACCT (SEQ ID NO. 154);

[0311] sgR: aaacAGGTGAGGATGTTGTGTTCG (SEQ ID NO. 155).

[0312] (19) OsPDS- xy-NTG

[0313] Target sequence: GTACACTACTACACATTTAC OsSPL7 (SEQ ID NO. 156);

[0314] sgF: ggcgGTACACTACTACACATTTAC (SEQ ID NO. 157);

[0315] sgR: aaacGTAAATGTGTAGTAGTGTAC (SEQ ID NO. 158).

[0316] (20) LOC_OS08g37790- -NTG

[0317] Target sequence: ACTCCACCACGCCCGACCGG OsSPL7 (SEQ ID NO. 159);

[0318] sgF: ggcgACTCCACCACGCCCGACCGG (SEQ ID NO. 160);

[0319] sgR: aaacCCGGTCGGGCGTGGTGGAGT (SEQ ID NO. 161).

[0320] Each pair of SgF / R above was annealed respectively, OsSPL14 -NAT, OsWaxy -NAC, OsWaxy -NAG, OsCOI2 -NAC, Figure 3 -NAT; Figure 3 -NGC, Figure 3 -NGA, Figure 3 -NGT, Figure 3 -NGC, Figure 4 -NGC, Figure 5 NCA, ​ -NCG, ​ NCG, ​ -NCC, ​ NCA, ​ -NTG, ​ -NTC, ​ -NTT, ​ -NTG and ​ -NTG to the pOsU3-sgRNA backbone (see Example 2 for construction method).

[0321] The five vectors constructed in Example 4 were subjected to efficiency verification with the target points in Example 5 in rice protoplasts, and the best double-base editor with the best comprehensive performance was screened.

[0322] The extraction and transformation of protoplasts are shown in Example 3.

[0323] Through the analysis of the second generation sequencing data, the five SpRY and SpRY variant based double base editors constructed in Example 3 can produce single base A>G, single base C>T and double base A>G&C>T substitutions at the non-canonical PAM of NAN, NGN, NCN and NTN. The average efficiency STEME8-SpRY-KK (6.77%)>STEME8-SpRY-K1 (4.77%)>STEME8-SpRY-K2 (3.58%)>STEME8-SpRY-K3 (3.39%)>STEME8-SpRY (3.08%), the average editing efficiency of STEME8-SpRY-KK is the highest, which is 2.2 times of SpRY ​ A).

[0324] At NAN PAM (A), the average editing efficiency STEME8-SpRY-KK (5.93%)>STEME8-SpRY (5.02%)>STEME8-SpRY-K3 (3.80%)>STEME8-SpRY-K1 (3.34%)>STEME8-SpRY-K2 (2.59%), the average editing efficiency of STEME8-SpRY-KK is the highest, which is 1.2 times of STEME8-SpRY ​ B).

[0325] At NGN PAM (C), the average editing efficiency STEME8-SpRY-KK (12.14%)>STEME8-SpRY-K1 (9.14%)>STEME8-SpRY-K3 (6.46%)>STEME8-SpRY-K2 (5.65%)>STEME8-SpRY (4.90%), the average editing efficiency of STEME8-SpRY-KK is significantly higher than that of STEME8-SpRY, which is 1.9 times of STEME8-SpRY; followed by STEME8-SpRY-K1 is 1.9 times of STEME8-SpRY ​ At NCN PAM (D), the average editing efficiency STEME8-SpRY-KK (5.90%)>STEME8-SpRY-K2 (3.25%)>STEME8-SpRY-K3 (1.80%)>STEME8-SpRY-K1 (1.54%)>STEME8-SpRY (1.24%), the average editing efficiency of STEME8-SpRY-KK is significantly higher than that of STEME8-SpRY, which is 4.8 times of it

[0326] ​ At NTN PAM (E), the average editing efficiency STEME8-SpRY-KK (5.90%)>STEME8-SpRY-K2 (3.25%)>STEME8-SpRY-K3 (1.80%)>STEME8-SpRY-K1 (1.54%)>STEME8-SpRY (1.24%), the average editing efficiency of STEME8-SpRY-KK is significantly higher than that of STEME8-SpRY, which is 4.8 times of it

[0327] At NTN PAM (E), the average editing efficiency STEME8-SpRY-KK (5.90%)>STEME8-SpRY-K2 (3.25%)>STEME8-SpRY-K3 (1.80%)>STEME8-SpRY-K1 (1.54%)>STEME8-SpRY (1.24%), the average editing efficiency of STEME8-SpRY-KK is significantly higher than that of STEME8-SpRY, which is 4.8 times of it ​ ​D), the average editing efficiency of STEME8-SpRY-KK (3.09%) > STEME8-SpRY-K1 (2.31%) > STEME8-SpRY-K2 (1.60%) > STEME8-SpRY-K3 (1.52%) > STEME8-SpRY (1.14%), and the average editing efficiency of STEME8-SpRY-KK was significantly higher than that of STEME8-SpRY, which was 2.7 times that of STEME8-SpRY.

[0328] The editing products were analyzed, and STEME8-SpRY-KK had C>T as the main editing product (63.26%), followed by A>G&C>T (34.23%), and finally A>G (2.51%); specifically, C>T editing was mainly at NAN (79.79%) and NGN PAM (77.37%); A>G&C>T double-base simultaneous editing was mainly at NCN (48.65%) and NTN PAM (79.25%). Among different variants, C>T accounted for 57.53%, followed by A>G&C>T (39.40%), and A>G accounted for a small proportion (4.01%) (Table 2). ​ .

[0329] The editing window of different STEME8-SpRY variants was concentrated between A2~A12 and C1~C16. The main editing window of STEME8-SpRY was concentrated between A3~A8 and C5~C10, and the main editing window of STEME8-SpRY-KK was slightly wider than that of STEME8-SpRY, specifically A2~A8 and C4~C15, and the high-efficiency editing positions were A5 and C11, respectively. Compared with the other four variants, the base conversion efficiency in the editing window of STEME8-SpRY-KK was the highest (Table 3). ​ .

[0330] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A SpRY variant-based base editing system, comprising, The base editing system comprises: (1) an expression construct containing a nucleotide sequence of a base editing protein, the base editing protein being selected from a SpRY variant; the structure of the nucleotide sequence expression construct comprising (a) and (b): (a) N-terminal of cytosine deaminase-C-terminal of adenine deaminase; (b) N-terminal of SpRY variant-C-terminal of SpRY variant; the SpRY variant being selected from SpRY-KK including or not including D10A; (2) an expression construct containing a nucleotide sequence encoding a guide RNA; The nucleotide sequence of SpRY-KK not including D10A is shown in SEQ ID NO. 5; The nucleotide sequence of SpRY-KK (D10A) including D10A is shown in SEQ ID NO.

19.

2. The base editing system of claim 1, wherein (b) further comprising a nuclear localization signal of nucleoplasmin, the (b) being a nuclear localization signal of nucleoplasmin connected in sequence to the SpRY variant; the SpRY variant being SpRY-KK, the nucleotide sequence of (b) being shown in SEQ ID NO.

12.

3. The base editing system of claim 1, wherein (a) the cytosine deaminase is APOBEC3A, and the purine deaminase is TadA8e.

4. The base editing system of any one of claims 1-3, wherein (1) the nucleotide sequence expression construct is connected with (a) and (b) by using a 32aa Linker, and one or more UGI and BpNLS are connected at the C-terminal.

5. The base editing system of claim 4, wherein (1) the nucleotide sequence expression construct is connected in the following manner: APOBEC3A-TadA8e-32aa Linker-SpRY variant-nucleoplasmin NLS-UGI-BpNLS.

6. The base editing system of claim 5, wherein The SpRY variant is SpRY-KK (D10A), and the nucleotide sequence of (1) is shown in SEQ ID NO.

24.

7. The base editing system of claim 1, wherein (1) a promoter is arranged at the N-terminal of the nucleotide sequence expression construct, and a terminator is arranged at the C-terminal.

8. The base editing system of claim 1, wherein (2) the expression construct further comprises a promoter for driving the guide RNA, (2) the expression construct is OsU3 promoter-N 20 -gRNA scaffold-SUP4 terminator.

9. The base editing system of claim 8, wherein (2) the nucleotide sequence of the expression construct is shown in SEQ ID NO.

25.

10. The base editing system of claim 1, wherein (2) the target nucleotide region targeted by the guide RNA comprises a PAM sequence at the 3' end of the target sequence; the PAM sequence is NNN, and the N refers to any one of ATGC.

11. The base editing system of claim 1, wherein (1) the expression construct and (2) the expression construct are respectively constructed on different vectors and independently driven by respective promoters for expression.

12. The base editing system of claim 11, wherein (1) the expression construct and (2) the expression construct are respectively independently constructed on a pJIT163-Ubip-Cas9 vector with a nucleotide sequence shown in SEQ ID NO. 29 or a pJIT163-Ubip-SpRY vector with a nucleotide sequence shown in SEQ ID NO.

26.

13. The base editing system of claim 12, wherein (1) the expression construct or (2) the expression construct is respectively independently connected between the Hind III enzyme cutting site and the EcoR I enzyme cutting site of the pJIT163-ubip-Cas9 vector or the pJIT163-Ubip-SpRY vector.

14. Use of the base editing system of any one of claims 1 to 13 in the genetic editing of rice, characterized in that, The application is to introduce the base editing system into a rice genome to be edited for double-base editing of nucleotides at multiple sites in a target nucleotide region of the rice genome to be edited; and the editing type is A>G and / or C>T editing.

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