Engineering and transformation of ultra-compact CRISPR / Cas12j-8 and its applications

Abstract

The present invention belongs to the field of molecular biology technology, and specifically relates to an engineered ultra-compact CRISPR / Cas12j‑8 and its application. The technical problem to be solved by the present invention is that the editing efficiency of the CRISPR / Cas12j‑8 system is low. The technical solution of the present invention is to engineer an ultra-compact CRISPR / Cas12j‑8, whose nucleotide sequence is shown in SEQ ID No.3 or SEQ ID No.5. The present invention has engineered crRNA and Cas12j‑8 proteins, and developed an en4Cas2j‑8 / crRNA‑Rz genome editing system, which exhibits strong editing activity in soybean, rice and human HEK293T cells, and can edit target sites that were previously ineditable.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology technology, and specifically relates to an engineered ultra-compact CRISPR / Cas12j-8 and applications thereof. Background Art

[0002] The advent of CRISPR-Cas systems, especially CRISPR-Cas9 and Cas12a, has dramatically changed the field of genome editing, enabling precise genetic modification in a wide range of organisms, including human and plant genomes. In plants, virus-induced genome editing (VIGE) faces similar limitations due to the large size of nucleases. To address these issues, a large amount of research has been devoted to the development of smaller and more compact Cas proteins. Recent advances in this field have led researchers to discover several promising candidates, including compact Cas9 variants, Cas12f (400-700 amino acids), TnpB (about 400 amino acids), and IscB (about 400 amino acids), whose smaller molecular weight is expected to simplify the construction of viral vectors, thereby expanding the potential of virus-mediated genome editing applications. However, despite these advances, the gene editing efficiency of these smaller nucleases in plant systems remains suboptimal.

[0003] The CRISPR / Cas12j (Casφ) system, recently discovered in bacteriophages, represents an important advance in genome editing tools. The system has attracted widespread attention due to its compact size (700-800 amino acids) and preference for 5′-TBN-3′ protospacer adjacent motifs (PAMs). Despite its small size, the Cas12j system has shown promising genome editing capabilities in mammalian cells. There are currently 10 homologous proteins reported in the CRISPR / Cas12j system, CRISPR / Cas12j-1 to CRISPR / Cas12j-9, and the latest reported CRISPR / Cas12j-SF05. The editing efficiency of wild-type Cas12j-2 in Arabidopsis is as low as 0.3%, while the editing efficiency of engineered Cas12j-2 variants can be increased to 6%. In monocots such as rice (Oryza sativa), the editing efficiency with inheritance is even lower, reaching only 1.2% in stably transformed lines. By combining engineered Cas12j-2 variants with optimized crRNA, genome editing efficiencies in rice ranged from 2% to 78%. In wheat (Triticum aestivum) and rye (Secale cereale), genome editing efficiencies were increased by optimizing crRNA and introducing a nuclear localization signal (NLS), resulting in improved gene knockout and base editing. In addition, the recently reported Cas12j-SF05 has shown potential in plant genome editing, with editing efficiencies of up to 20% in rice. Despite these advances, the overall efficiency of the Cas12j system remains lower than that of more established tools such as SpCas9. In addition to Cas12j-2 and Cas12j-SF05, the CRISPR / Cas12j-8 system has recently demonstrated efficacy in human cells, characterized by low off-target activity and high specificity for single nucleotide mismatches in the PAM-adjacent region. However, its potential in plant genome editing remains underexplored, with validation limited to rice protoplasts, where its editing efficiency was low (<2.4%). In addition, Cas12j lacks an HNH domain, which significantly limits its base editing ability. Therefore, its potential for base editing in plants has not been fully explored. Summary of the invention

[0004] The technical problem to be solved by the present invention is that the editing efficiency of the CRISPR / Cas12j-8 system is low.

[0005] The technical solution of the present invention is to engineer an ultra-compact CRISPR / Cas12j-8, whose amino acid sequence is as shown in SEQ ID No.4 or SEQ ID No.6.

[0006] Furthermore, the nucleotide sequence of the gene encoding the engineered ultra-compact CRISPR / Cas12j-8 is shown in SEQ ID No.3 or SEQ ID No.5.

[0007] The present invention also provides a crRNA suitable for engineering transformation of an ultra-compact CRISPR / Cas12j-8, the nucleotide sequence of which is shown in SEQ ID No. 2, bp 1 to 36.

[0008] The present invention also provides an ultra-compact CRISPR / Cas12j-8 editing system, comprising a Cas12j-8 expression unit and a crRNA expression unit; the structure of the Cas12j-8 expression unit is promoter-Cas12j-8-terminator; the structure of the crRNA expression unit is promoter-crRNA-terminator.

[0009] Furthermore, in the Cas12j-8 expression unit, the 5' or / and 3' end of Cas12j-8 is also fused with NLS.

[0010] Furthermore, in the crRNA expression unit, the 5' or / and 3' end of the crRNA is also fused with HDV ribozyme.

[0011] Specifically, the structure of the crRNA expression unit is promoter-crRNA-target sequence-HDV-PolyT.

[0012] Preferably, the nucleotide sequence of the crRNA expression unit is shown in SEQ ID No.2.

[0013] Among them, in the crRNA expression unit, the 5' and / or 3' ends of the crRNA are also fused with a transfer RNA precursor (pre-tRNA).

[0014] Specifically, the structure of the crRNA expression unit is promoter-tRNA-crRNA-target sequence-tRNA-terminator.

[0015] In particular, the promoter is a constitutive promoter, an inducible promoter or a tissue-specific promoter.

[0016] Wherein, the constitutive promoter is ZmUbi1, CaMV35S, CMV, EF-1aα, AtU6, OsU3 or HsU6.

[0017] Specifically, the terminator is pinII, AtHSP, NOS, T35S or bGH Ploy (A) signal.

[0018] The present invention also provides a vector, a cell or a host containing the editing system.

[0019] Furthermore, the vector is a plant or animal expression vector.

[0020] Beneficial effects of the present invention: The present invention has engineered crRNA and Cas12j-8 proteins and developed the en4Cas2j-8 / crRNA-Rz genome editing system, which exhibits strong editing activity in soybean, rice and human cells (taking HEK293T as an example), and is able to edit target sites that were previously uneditable. Its editing activity is comparable to that of SpCas9. In addition, a cytosine base editor based on en4Cas2j-8 / crRNA-Rz was introduced, which exhibited efficient base editing capabilities (C to T) in plants, with a maximum efficiency of 91.90%, an average increase of 5.36 to 6.85 times, compared to unengineered systems, and no insertions or deletions (indels) were observed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows the evaluation of gene editing by Cas12j-8 nuclease in soybean hairy roots. a. Schematic diagram of the vector expressing Ruby, Cas12j-8 and crRNA. b. Gene editing efficiency of 10 endogenous sites mediated by Cas12j-8 in soybean hairy roots. Each point represents a biological replicate of an independent experiment (n>3). Data are expressed as mean ± standard deviation (SD). c. Location of deletion sites of representative targets. PAM and spacer regions are represented in red and blue, respectively. Dotted lines indicate deletions. d. Deletion size distribution of representative targets.

[0022] Figure 2 shows that the engineering design of crRNA enhances gene editing activity. a. Single crRNA backbone structure of Cas12j-8. The PAM sequence is marked with a box, the protospacer is marked in red, the crRNA is marked in gray, and the stem-loop structure of the crRNA is marked in blue. b. Effect of the length of the protospacer on the gene editing efficiency of the GmPDS2-T1 site. c. Editing efficiency of different crRNA constructs targeting the same GmPDS2-T1 site. The engineered crRNA region is shaded in blue, the stem-loop structure of the wild-type crRNA is bolded, and the mutant bases are marked in red and represented in lowercase letters. Each point represents a biological replicate of an independent experiment (n>3). The data were analyzed using one-way analysis of variance (ANOVA), assuming that the residuals followed a Gaussian distribution and that the standard deviations of the groups were equal. ns indicates P>0.05; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001.

[0023] Figure 3 shows that the engineering design of Cas12j-8 nuclease enhances gene editing activity. a. Identification of potential interaction sites with the crRNA backbone in Cas12j-8. The protein structures of Cas12j-2 (green) and Cas12j-8 (red) are shown. The crRNA backbone is represented in gray, and the blue-green highlights indicate the specific interaction sites of Cas12j-2 with the crRNA backbone and the potential interaction sites of Cas12j-8. b. The domain organization of Cas12j-8 and the location of its predicted interaction sites with the crRNA backbone, PI: PAM interaction domain; RecI: recognition domain I; RecII: recognition domain II; OBD: oligonucleotide binding domain; RuvC: RuvC endonuclease domain; ZR: zinc ribbon domain. c. Structural alignment of Cas12j-8 with Cas12j-2 and Cas12j-SF05. The protein structures of Cas12j-2 (green), Cas12j-8 (red), and Cas12j-SF05 (light cyan) are shown. The mutation sites of nCas12j-2 and vCas12j-2 and their corresponding positions in Cas12j-8 are highlighted in blue. The sites that interact with PAM in Cas12j-SF05 and their corresponding positions in Cas12j-8 are indicated in green. The amino acid fragment missing in en5Cas12j-2 is marked in yellow. d, Alignment of the four Cas12j-8 variants with Cas12j-2 and Cas12j-8. The mutation sites are marked with triangles. e, Editing efficiency of Cas12j-8 variants. Each point represents a biological replicate of an independent experiment (n>3). The data were analyzed using one-way analysis of variance (ANOVA), assuming that the residuals followed a Gaussian distribution and that the standard deviations of the groups were equal. ns indicates P>0.05; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001.

[0024] Figure 4 shows that the combination of en4Cas12j-8 and crRNA-Rz produces efficient gene editing activity. a. Editing efficiency of the combination of en4Cas12j-8 and crRNA-Rz. Data were analyzed by two-way analysis of variance (ANOVA) multiple comparison test, ns indicates P>0.05; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001. Each point represents a biological replicate of an independent experiment (n>3). Data are expressed as mean ± standard deviation (SD). b. Gene editing efficiency of en4Cas12j-8 / crRNA-Rz at 8 endogenous sites in soybean hairy roots, which were not edited by Cas12j-8 / crRNA. Each point represents a biological replicate of an independent experiment (n>3). Data are expressed as mean ± standard deviation (SD). c, Schematic diagram of the common target sites of Cas12j-8 and SpCas9. The PAM and target sites of Cas12j-8 and SpCas9 are indicated in red and blue, respectively, and the PAM sequences are marked in bold. d, Comparison of mutation rates of 7 endogenous sites in soybean hairy roots between the engineered Cas12j-8 system and SpCas9. Data were analyzed using an unpaired t-test with two-tailed P values, ns indicates P>0.05; * indicates P<0.05; ** indicates P<0.01. Each point represents a biological replicate of an independent experiment (n>3).

[0025] Figure 5 shows that the Cas12j-8 base editor achieves efficient base editing activity. a, Schematic diagram of four versions of base editors. b, d Editing window and efficiency of Cas12j-8 / crRNA / CBE at GmPDS1-T1 and GmPDS2-T1 sites. c, Editing efficiency of four versions of base editors at C8 position of GmPDS1-T1 and GmPDS2-T1 sites. Data were analyzed by two-way analysis of variance (ANOVA) multiple comparison test, ns indicates P>0.05; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001. Each point represents a biological replicate of an independent experiment (n>3). Data are expressed as mean ± standard deviation (SD). d and e, Base editing spectra of two endogenous sites in the GmCCD4 gene. PAM and spacer regions are indicated in red and blue, respectively, and the mutant bases are indicated in lowercase letters. f and g, Editing window and efficiency of en4Cas12j-8 / crRNA-Rz / CBE at two endogenous sites in the GmCCD4 gene. C-to-T base editing at both sites occurred between positions 6 and 11. Each point represents a biological replicate of an independent experiment (n>3). Data are presented as mean ± standard deviation (SD). Source data are provided in the source data file.

[0026] Figure 6 shows gene editing in stable transgenic rice mediated by the engineered Cas12j-8 system. a. Schematic diagram of Cas12j-8 / crRNA and en4Cas12j-8 / crRNA-Rz vectors. b. Gene editing efficiency of Cas12j-8 / crRNA and en4Cas12j-8 / crRNA-Rz systems in rice OsNRAMP5 gene. Bi: biallelic mutation; Ho: homozygous; He: heterozygous. c. Sequencing results of different Osnramp5 mutants, with PAM and spacer regions represented by red and blue, respectively, and dotted lines representing deletions. d. Sequencing peaks of OsNRAMP5 gene in edited plants.

[0027] Figure 7 shows gene editing in human HEK293T cells mediated by en4Cas12j-8 / crRNA-Rz. a. Schematic diagram of gene editing by Cas12j-8 / crRNA and en4Cas12j-8 / crRNA-Rz constructs in human HEK293T cells. b. Types of PCSK9 gene mutations mediated by en4Cas12j-8 / crRNA-Rz in HEK293T cells. PAM and spacer regions are indicated in red and blue, respectively, dotted lines indicate deletions, and insertions are indicated in green. c and d. Gene editing efficiencies of Cas12j-8 / crRNA and en4Cas12j-8 / crRNA-Rz at PCSK9-site1, AAVS1-site1, and AAVS1-site2. Data were analyzed using a two-tailed unpaired t-test. Each point represents a biological replicate of an independent experiment (n>3). Data are presented as mean ± standard deviation (SD). ns indicates P>0.05; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001. DETAILED DESCRIPTION

[0028] In the following examples, data were analyzed using GraphPad Prism 8.0 software, and graphs were further processed using Adobe Photoshop and Adobe Illustrator software. PCR product sequencing was provided by Sangon Biotech (Shanghai, China).

[0029] The main equipment used in the following examples: Hi-TOM sequencing platform (Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou).

[0030] Example 1 Characteristic Analysis of Cas12j-8-Mediated Genome Editing in Soybean Hairy Roots

[0031] The Cas12j-8 gene (SEQ ID No. 1) was synthesized by a commercial company (Sangon Biotech) and cloned into the 35S-Ruby vector (He Y, Zhang T, Sun H, Zhan H, Zhao Y. A reporter for noninvasively monitoring gene expression and plant transformation. Horticulture Research, 2020, 7(1): 152) to obtain 35S-Ruby-Cas12j-8. The Ruby gene was used as a marker for selecting transgenic roots. The final vector was confirmed by sequencing ( Figure 1 a). And used crRNA driven by Arabidopsis U6 (AtU6) promoter, aiming to target 10 specific endogenous sites in GmBADH1, GmCCD4a, GmCCD4b, GmPDS1, GmPDS2, GmSweet15a, GmSweet15b and GmFAD2-1A genes (Table 1). The above crRNA expression cassettes were assembled into the restriction endonuclease Pme I linearized 35S-Ruby-Cas12j-8 backbone vector by seamless cloning (TransGen Biotech), and the final vector was confirmed by sequencing. The successfully constructed vector was transformed into Agrobacterium rhizogenes K599 for use.

[0032] SEQ ID No.1 Cas12j-8

[0033]

[0034] Table 1 Target names and sequences in each example

[0035] .

[0036] Healthy soybean seedlings of seven days old with fully expanded true leaves were selected for the experiment. The main root was removed using sterile scissors, leaving the top part of the hypocotyl of 0.7-1 cm. In the inoculation medium, a sterile scalpel was used to make an oblique cut of about 0.5 cm in length on this part. The oblique cut surface of the remaining hypocotyl was scraped on a plate containing the K599 strain. Then, the seedlings were immediately transplanted into pots containing sterile vermiculite, which had been pre-moistened with water. Alternatively, each seedling was inoculated with about 5 mL of K599 inoculum. After inoculation, the seedlings were placed in a high humidity environment. The development of hairy roots was observed after two weeks. The hairy roots with red color were selected to extract DNA, and PCR amplification primers were designed according to the target sequence (Table 2). PCR amplification was performed using the above DNA as templates, and the PCR products were sent to the Hi-TOM sequencing platform (Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou) for high-throughput sequencing. The results showed that among these target sites, the average editing efficiencies of vectors targeting the same sequence of GmPDS1-T1 and GmPDS2-T1 were 15.39% and 30.82%, respectively, indicating that Cas12j-8 has genome editing activity in plants ( Figure 1 b). Further analysis of editing types showed that deletions were the major editing form, ranging from 4 bp to 12 bp ( Figure 1 c), these deletions are mainly concentrated around the 10th nucleotide position ( Figure 1 d).

[0037] Table 2 Primer sequences

[0038]

[0039] .

[0040] Example 2 Engineering of crRNA improves genome editing activity

[0041] Previous studies have determined that the crRNA sequence plays a key role in determining the editing efficiency of the Cas12a system. This effect may be attributed to the differences in expression and stability between eukaryotic and prokaryotic environments. In order to explore whether the editing efficiency of Cas12j-8 can be improved by optimizing the crRNA framework, a series of experiments were conducted. First, the effect of the length of the spacer sequence on the editing efficiency in the plant system was evaluated. After the constructed vectors with different spacer sequence lengths were transferred into Agrobacterium rhizogenes, positive transgenic hairy roots were screened, and then the Hi-TOM sequencing platform was used to complete high-throughput sequencing of the PCR products at the target site. The results showed that the 18 nt and 16 nt spacer sequences showed higher editing activity ( Figure 2 a and Figure 2 b), and finally 18 nt was selected for subsequent optimization. Analysis of the crRNA structure of the nine Cas12j variants showed significant conservation ( Figure 2 a). Given the high conservation of crRNA structure among these nine Cas12j variants, 18 crRNA variants were designed by modifying the stem-loop region of the 25-nt mature crRNA and introducing a highly stable hairpin structure to enhance its stability ( Figure 2 c). In addition, studies have shown that adding the "U4AU6" motif, inserting a 3'-terminal self-cleaving ribozyme (such as HDV ribozyme) downstream of crRNA, or introducing a transfer RNA precursor (pre-tRNA) sequence upstream and downstream of crRNA can enhance the activity of the CRISPR-Cas12a system. Therefore, the present invention adopts a similar strategy ( Figure 2 c). To screen crRNA frameworks with higher editing efficiency, each crRNA framework was cloned into an all-in-one expression system (the vector construction was carried out according to the method in Example 1, except that the structure or sequence of crRNA was replaced). The results showed that the insertion of the self-cleaving HDV ribozyme at the 3ʹ end significantly enhanced the genome editing activity by an average of 2.0 times. This optimized crRNA framework was named crRNA-Rz ( Figure 2 c).

[0042] SEQ ID No.2 crRNA-Rz sequence: crRNA scaffold-HDV ribozyme-Poly T, the 1st to 36th bp are the crRNA backbone sequence, the 37th bp n represents the target sequence of the gene to be edited, and to edit different genes, only n needs to be replaced, the 38th to 107th bp are the HDV ribozyme sequence, and the 108th to 114th bp are the PolyT sequence;

[0043] CTTTCAAGACTAATAGATTGCTCCTTACGAGGAGACnggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgcttcggcatggcgaatgggacTTTTTTTT.

[0044] Example 3 Engineering of Cas12j-8 nuclease improves genome editing activity

[0045] The protein structures of Cas12j-8 and Cas12j-SF05 were predicted by Alphafold v2.2.0. The cryo-electron microscopy (cryo-EM) map and model coordinates of Cas12j-2 were downloaded from the Protein Data Bank (PDB) using accession numbers 7LYS and 7M5O, respectively. Multiple sequence alignment and phylogenetic tree construction were performed using TBtools v2.012 (Chen, C., Wu, Y., Li, J., Wang, X., Zeng, Z., Xu, J., Liu, Y., Feng, J., Chen, H. and He, Y. (2023) TBtools-II: A “one for all, all for one” bioinformatics platformfor biological big-data mining. Molecular plant, 16, 1733-1742.). Visualization and diagrams of protein structures were generated using PyMOL (DeLano, WL (2002) Pymol: An open-source molecular graphics tool. CCP4 Newsl. Protein Crystallogr, 40, 82-92.).

[0046] In addition to crRNA engineering, engineering of nuclease proteins is also a key method to improve editing efficiency. An effective strategy to improve the nuclease activity of Cas proteins is to strengthen their interaction with crRNA. Phylogenetic tree analysis and structural analysis performed by AlphaFold2 found that although the amino acid sequence homology between Cas12j-8 and Cas12j-2 (31.5%) and Cas12j-SF05 (33.5%) was low, Cas12j-2 and Cas12j-SF05 still showed shorter branch lengths, indicating that they are closely related. In contrast, the branches of Cas12j-8 are farther, indicating that it has greater evolutionary differences. However, Cas12j-8 has a high structural similarity with these proteins, with RMSD values ​​of 5.76 and 4.8 ( Figure 3 Based on the reported structure of Cas12j-2 protein, the corresponding residues that may mediate its interaction with crRNA were identified in Cas12j-8 ( Figure 3 a). To enhance the electrostatic interaction between these residues and the negatively charged crRNA, they were replaced with positively charged arginine (R). Therefore, a library of 22 Cas12j-8 variants (Table 3) was constructed, each containing a single R substitution, for subsequent screening ( Figure 3 b). Based on the structural analysis, three mutants were designed, namely en1Cas12j-8(Δ149–170 (GSSG)), en2Cas12j-8 (N156R / E157R / I158R / A159R / K161R / N162R) and en3Cas12j-8(E157A) (Figure 3c and 3d).

[0047] Table 3 Mutation sites

[0048] .

[0049] Protein structure analysis of Cas12j-SF05 showed that the amino acids at positions 127 and 202 are involved in PAM recognition, and mutations in these residues may seriously affect genome editing activity. By comparing the amino acid sequences of 10 reported Cas12j homologous proteins, it was found that this region is significantly conserved ( Figure 3 d). However, Cas12j-8 differs from other homologous proteins at positions 127 and 202. Therefore, it was hypothesized that replacing these two amino acids might enhance genome editing activity, and a new variant was generated, named en4Cas12j-8 (A121Q / S196Q) ( Figure 3 d). Although Cas12j-8 is only 700 amino acids in size, its expression is still beyond the capacity of some plant viruses. To facilitate the application of Cas12j-8 in virus-induced genome editing (VIGE), we used AlphaFold2 to predict the structure of Cas12j-8. The results showed that there was a sequence at the C-terminus that might not be involved in the function of the nuclease. Therefore, a nuclease variant en5Cas12j-8 (Δ672–717) lacking this amino acid segment was constructed ( Figure 3 c).

[0050] In order to screen Cas12j-8 variants with higher editing efficiency, each variant type was cloned into the XhoI site of the 35S-Ruby vector by homologous recombination method according to Example 1 (He Y, Zhang T, Sun H, Zhan H, Zhao Y. A reporter for noninvasively monitoring gene expression and plant transformation. Horticulture Research, 2020, 7(1): 152), and then transferred into Agrobacterium rhizogenes and infected soybean to obtain transgenic hairy roots. The editing efficiency was determined by next-generation sequencing of the target site. The results showed that the activity of Cas12j-8 (W299R) and en4Cas12j-8 (A121Q / S196Q) was enhanced, with an average increase of 1.38 times and 2.1 times ( Figure 3 e).

[0051] SEQ ID No.3 en4Cas12j-8 (A121Q / S196Q) encoding gene, underlined amino acid codons A121Q and S196Q that are different from the wild type:

[0052]

[0053] SEQ ID No.4 en4Cas12j-8(A121Q / S196Q) protein:

[0054] *。

[0055] SEQ ID No.5 Cas12j-8(W299R):

[0056]

[0057] SEQ ID No.6 en4Cas12j-8 (W299R) protein:

[0058] MIKPTVSQFLTPGFKLIRNHSRTAGLKLKNEGEEACKKFVRENEIPKDECPNFQGGPAIANIIAKSREFTEWEIYQSSLAIQEVIFTLPKDKLPEPILKEEWRAQWLSEHGLDTVPYKEAAGLNLIIKNAVNTYKGVQVKVDNKNKNNLAKINRKNEIAKLNGEQEISFEEIKAFDDKGYLLQKPSPNKSIYCYQSVSPKPFITSKYHNVNLPEEYIGYYRKSNEPIVSPYQFDRLRIPIGEPGYVPKWQYTFLSKKENKRRKLSKRIKNVSPILGIICIKKDWCVFDMRGLLRTNHRKKYHKPTDSINDLFDYFTGDPVIDTKANVVRFRYKMENGIVNYKPVREKKGKELLENICDQNGSCKLATVDVGQNNPVAIGLFELKKVNGELTKTLISRHPTPIDFCNKITAYRERYDKLESSIKLDAIKQLTSEQKIEVDNYNNNFTPQNTKQIVCSKLNINPNDLPWDKMISGTHFISEKAQVSNKSEIYFTSTDKGKTKDVMKSDYKWFQDYKPKLSKEVRDALSDIEWRLRRESLEFNKLSKSREQDARQLANWISSMCDVIGIENLVKKNNFFGGSGKREPGWDNFYKPKKENRWWINAIHKALTELSQNKGKRVILLPAMRTSITCPKCKYCDSKNRNGEKFNCLKCGIELNADIDVATENLATVAITAQSMPKPTCERSGDAKKPVRARKAKAPEFHDKLAPSYTVVLREAV*。

[0059] Example 4 The combination of crRNA-Rz and en4Cas12j-8 further improves genome editing activity

[0060] Although the editing efficiency of the Cas12j-8 (W299R) variant was increased by 1.38 times, in order to determine whether the combination of optimized crRNA and Cas12j-8 can further improve the gene editing efficiency, the en4Cas12j-8 variant was selected to be combined with crRNA-Rz for further analysis. The editing efficiency of the GmPDS1-T1 and GmPDS2-T1 target sites was first examined. Referring to Example 1, the method for detecting the genome editing efficiency in soybean is the same, constructing the vector-transferring into Agrobacterium rhizogenes-infecting soybeans to obtain transgenic hairy roots-high-throughput sequencing to determine the genome editing efficiency.

[0061] The results showed that the en4Cas12j-8 / crRNA-Rz combination maintained a high editing efficiency, increasing the efficiency by 2.19 to 3.86 times on average ( Figure 4 a). Subsequently, the editing efficiency of the engineering system at other target sites was evaluated. The results showed that the engineering system was able to successfully edit 6 of the 8 previously uneditable target sites with high efficiency ( Figure 4 b).

[0062] Example 5 Comparison of editing efficiency between engineered Cas12j-8 system and SpCas9

[0063] SpCas9 is a widely used Cas nuclease with high editing efficiency in a variety of plant species. The vectors were constructed with reference to Example 1, and CRISPR / Cas9 genome editing vectors were constructed respectively. Soybean transgenic hairy roots were obtained by Agrobacterium rhizogenes. The genome editing efficiency was obtained by high-throughput sequencing. The genome editing efficiency was compared with the editing efficiency of the engineered Cas12j-8 system at seven independent sites in the soybean genome, GmCCD4a-T1, GmCCD4b-T1, GmCCD4a-T3, FmSweet15a-T2, GmFAD2-1A-T2, GmPDS1-T1, and GmPDS2-T1 (Figure 4c). The results showed that the editing efficiency of en4Cas12j-8 / crRNA-Rz was similar to that of SpCas9 (Figure 4d), and there was no significant difference in the editing efficiency of the four sites. Notably, en4Cas12j-8 / crRNA-Rz outperformed SpCas9 at one site but showed no detectable editing activity at another site (Fig. 4d). Overall, these results suggest that the engineered Cas12j-8 system can achieve editing efficiencies comparable to SpCas9 when targeting the same sequence and meeting compatible PAM requirements.

[0064] Example 6 Cas12j-8 base editor achieves efficient base editing activity without causing insertion or deletion mutations

[0065] Next, the potential of Cas12j-8 for base editing in plants was explored. To this end, E567 was first mutated to alanine to generate catalytically inactive Cas12j-8 (dCas12j-8). dCas12j-8 was synthesized by commissioning Sangon Biotech (Shanghai) Co., Ltd. with the newly discovered cytosine deaminase mini-Sdd7 (Huang et al. https: / / www.cell.com / cell / fulltext / S0092-8674(23)00593-7) and uracil glycosylase inhibitor (UGI) sequences. The vector was constructed with reference to Example 1 and named dCas12j-8 / crRNA / CBE ( Figure 5 a). The fusion base editing protein consists of only 1016 amino acids. Referring to Example 1, the base editing activity of dCas12j-8 / crRNA / CBE was evaluated at the GmPDS1-T1 and GmPDS2-T1 sites. The results showed that the cytosine base editing (C to T) efficiencies at these two sites were 9.15% and 8.58%, respectively ( Figure 5 b). Notably, no insertion or deletion mutations (indels) were observed in these mutations ( Figure 5 b). These findings highlight the important potential of Cas12j-8 in advancing plant genome and base editing applications.

[0066] SEQ ID No.7 dCas12j-8 / CBE: the 1st to 3rd bases are the start codon, the 4th to 24th bases are the nuclear localization signal NLS, the 25th to 549th bases are the cytosine deaminase mini-Sdd7 sequence, the 550th to 2724th bases are the dCas12j-8 sequence, the 2725th to 2772th bases are the nuclear localization signal sequence, the 2773th to 3054th bases are the uracil glycosylase inhibitor (UGI) sequence, and the 3055th to 3075th bases are the nuclear localization signal sequence;

[0067]

[0068] In order to further improve the base editing efficiency of Cas12j-8, we explored whether the engineered inactivated en4Cas12j-8 and crRNA-Rz could improve the base editing efficiency alone or in synergy. E567 of en4Cas12j-8 was mutated to alanine to inactivate it and named den4Cas12j-8. Three vectors were designed: den4Cas12j-8 / crRNA / CBE, dCas12j-8 / crRNA-Rz / CBE and den4Cas12j-8 / crRNA-Rz / CBE ( Figure 5 a). The results showed that both den4Cas12j-8 and crRNA-Rz significantly improved base editing efficiency, and their combination further enhanced the single-base editing efficiency (den4Cas12j-8 / crRNA-Rz / CBE), with the highest efficiency reaching 91.90%, an average increase of 5.36 to 6.85 times ( Figure 5 c).

[0069] The structure of den4Cas12j-8 / CBE is similar to dCas12j-8 / CBE, except that the dCas12j-8 sequence from base 550 to base 2724 in dCas12j-8 / CBE is replaced by the den4Cas12j-8 sequence.

[0070] Next, we evaluated whether the den4Cas12j-8 / crRNA-Rz / CBE system could edit other targets and assess its editing properties. We selected three cytosine-rich sites: GmCCD4a-T1, GmCCD4a-T2, and GmCCD4b-T1. The results showed that the system was able to achieve cytosine to thymine conversion (C to T) at these sites ( Figure 5 d and Figure 5 e). The editing window is mainly located at the +6 to +10 position of the protospacer region. The highest editing efficiency of the GmCCD4a-T2 site reached 71.76%, and the average efficiency was 36% ( Figure 5 f~g). Consistent with previous results, no insertion or deletion mutations (indels) were detected ( Figure 5 f~g).

[0071] Example 7 CRISPR / Cas12j-8-mediated genome editing in stable transgenic rice plants

[0072] In order to evaluate whether en4Cas12j-8 / crRNA-Rz can also improve the genome editing efficiency in monocotyledonous plants, its performance in transgenic rice was studied. First, Cas12j-8 and en4Cas12j-8 products were obtained by PCR amplification and cloned into the BamHI site of the pCXUN vector by homologous recombination (Sun Y, Zhang X, Wu C, et al. Engineering herbicide-resistant rice plants through CRISPR / Cas9-mediated homologous recombination of acetolactate synthase[J]. Molecularplant, 2016, 9(4): 628-631.), respectively obtaining pCXUN-Cas12j-8 and pCXUN-en4Cas12j-8 ( Figure 6 a). Target sequences targeting OsNRAMP5, OsDEP1, OsSweet14, OsSPL16, LOC_Os03g40840, LOC_Os08g03620 and LOC_Os11g4426 were designed respectively (Table 1). The promoter used was OsU3 promoter, and the crRNA expression cassette structure was OsU3-crRNA-HDV-PolyT. After obtaining the above expression cassettes by overlapping PCR, they were cloned into the PmeI site of the above pCXUN-en4Cas12j-8 vector by homologous recombination to obtain the corresponding targeted editing vector. In addition, the crRNA targeting the OsNRAMP5 gene was cloned into the PmeI restriction site of the pCXUN-Cas12j-8 vector by homologous recombination. The above three plasmids were transferred into EHA105 Agrobacterium by electroporation for standby use. When conducting stable transformation of rice, the hulled rice seeds are first disinfected and then cultured on N6D solid medium. The pre-cultured rice embryonic callus is inoculated with the EHA105 strain of Agrobacterium tumefaciens carrying the recombinant vector. After 3 days of co-culture of rice callus with Agrobacterium, the callus is rinsed with sterile water and transferred to N6-S medium for 2 weeks of screening. The newly grown callus is then transferred to RE-III medium and cultured for 2 weeks. The resistant callus is transferred to fresh RE-III medium every 2 weeks until regenerated plants are successfully obtained.

[0073] The results of targeted editing of the OsNRAMP5 gene showed that Cas12j-8 / crRNA failed to induce detectable genome editing ( Figure 6b~d). In contrast, the en4Cas12j-8 / crRNA-Rz system showed an editing efficiency of 52%, and 3 of the 13 edited plants showed bi-allelic or homozygous transformation ( Figure 6 b~d). In addition, the analysis was extended to six additional loci. Successful genome editing was observed at all loci, with the highest editing efficiency reaching 84%. At the OsSPL16 locus, 18 of the 22 edited plants showed biallelic or homozygous transformation (Table 4).

[0074] Table 4 Mutation frequency induced by en4Cas12j-8 / crRNA-Rz in T0 rice plants .

[0075] Example 8 Engineered Cas12j-8 and crRNA enhance genome editing efficiency in human cells

[0076] To evaluate the potential of the en4Cas12j-8 / crRNA-Rz system in improving the efficiency of gene editing in human cells, three target sites were designed based on the sequences of PSCK9 and AAVS1 (Table 1) to evaluate the activity of en4Cas12j-8 / crRNA-Rz in HEK293T cells ( Figure 7a). The specific vector construction method is as follows: Cas12j-8 and en4Cas12j-8 products were obtained by PCR amplification, and cloned into the pCMV vector (purchased from Zhuhai Shutong Medical Technology Co., Ltd.) by homologous recombination to obtain pCMV-Cas12j-8 and pCMV-en4Cas12j-8, respectively. crRNA expression cassettes targeting three sites of PSCK9 and AAVS1 were obtained by in vitro synthesis, wherein each site was designed with a crRNA (crRNA-Rz) expression cassette containing HDV, and the promoters of the above six expression cassettes were all HsU6 promoters. The above six crRNAs were cloned into the pXZ vector (purchased from Zhuhai Shutong Medical Technology Co., Ltd.) by homologous recombination to obtain pXZ-crRNA-PSCK-site1, XZ-crRNA-AAVS1-site1, XZ-crRNA-AAVS1-site2, pXZ-crRNA-PSCK-site1-Rz, XZ-crRNA-AAVS1-site1-Rz and XZ-crRNA-AAVS1-site2-Rz, respectively. The constructed pCMV-Cas12j-8 was combined with pXZ-crRNA-PSCK-site1, XZ-crRNA-AAVS1-site1, and XZ-crRNA-AAVS1-site2, and pCMV-en4Cas12j-8 was combined with pXZ-crRNA-PSCK-site1-Rz, pXZ-crRNA-AAVS1-site1-Rz, and pXZ-crRNA-AAVS1-site2-Rz, respectively, and transferred into human HEK293T cells. The specific method is as follows: human HEK293T cells (ATCC, CRL-3216) were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco) supplemented with 10% (volume / volume) fetal bovine serum (FBS, Gibco) and 1% (volume / volume) penicillin-streptomycin (Gibco). Cells were maintained in a humidified incubator at 37°C and 5% CO2 and seeded in 75 cm² cell culture flasks (NEST) for expansion. To assess the efficiency of cutting and editing in HEK293T cells, cells were plated in 24-well plates (Corning) and grown to approximately 80% confluence before transfection. Plasmids were co-transfected into cells by electrofection. After 72 hours, genomic DNA was extracted using QuickExtract DNA Extraction Solution (Lucigen) according to the manufacturer's instructions.

[0077] High-throughput results of PCR products at different gene target sites showed that en4Cas12j-8 / crRNA-Rz was significantly better than the unengineered system at all three target sites, and the editing efficiency was significantly improved ( Figure 7 b~d). In particular, at sites where Cas12j-8 / crRNA showed limited or no activity, the efficiency of en4Cas12j-8 / crRNA-Rz was increased by up to 18.1 times ( Figure 7 b~d).

Claims

1. An ultra-compact CRISPR / Cas12j-8 editing system, characterized in that: It includes a Cas12j-8 expression unit and a crRNA expression unit; the structure of the Cas12j-8 expression unit is promoter-Cas12j-8-terminator; the structure of the crRNA expression unit is promoter-crRNA-terminator; the amino acid sequence of the Cas12j-8 is as shown in SEQ ID No.4 or SEQID No.6; the nucleotide sequence of the crRNA is as shown in SEQ ID No.2 bp 1 to 36.

2. The editing system according to claim 1, characterized in that: In the Cas12j-8 expression unit, the 5' or / and 3' end of Cas12j-8 is also fused with NLS.

3. The editing system according to claim 1, characterized in that: In the crRNA expression unit, HDV ribozyme is also fused to the 5' or / and 3' end of the crRNA.

4. The editing system according to claim 1, characterized in that: The promoter is a constitutive promoter, an inducible promoter or a tissue-specific promoter.

5. The editing system according to claim 4, characterized in that: The constitutive promoter is ZmUbil, CaMV35S, CMV, EF-1aα, AtU6, OsU3 or HsU6.

6. The editing system according to claim 1, characterized in that: The terminator is pinII, AtHSP, NOS, T35S or bGH Ploy(A) signal.

7. A host comprising the editing system according to any one of claims 1 to 6, characterized in that: The host is a non-plant species.

8. A carrier containing the editing system according to any one of claims 1 to 6, characterized in that: The vector is a plant or animal expression vector.

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