An optimized omega rna and its gene editing applications

Abstract

The application belongs to the technical field of gene editing, and particularly relates to an optimized omega RNA and gene editing application thereof. The optimized omega RNA is based on a wild-type omega RNA shown in a gene sequence such as SEQ ID NO. 1, and the 36th pairing base UA is replaced by GC, the 37th pairing base UA is replaced by AU, the 38th pairing base UG is replaced by AU, the 50th pairing base UG is replaced by CG, and the 51st pairing base UA is replaced by GC at the 5' end of the wild-type omega RNA. Compared with the wild-type omega RNA, the maximum enhancement multiple of the insertion and deletion activity of the omega RNA is 16.7 times, and the editing efficiency is further improved by deleting 15 pairs of pairing bases from the 40th to the 54th of the omega RNA.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, specifically relating to an optimized ωRNA and its gene editing applications. Background Technology

[0002] The advent of programmable genome editing technologies, especially the CRISPR-Cas system, has revolutionized modern biotechnology and medicine. CRISPR effectors, such as Cas9 and Cas12 nucleases, enable precise DNA manipulation across species, becoming powerful tools for biological research, gene therapy, and agricultural breeding. However, widely used Cas nucleases (such as Cas9 and Cas12a) typically exceed 1,000 amino acids, posing a significant challenge to efficient delivery, particularly in in vivo gene therapy via adeno-associated virus (AAV).

[0003] Recently, researchers discovered and characterized compact CRISPR nucleases and their ancestral proteins from prokaryotes, including miniature Cas12 effectors (Cas12f, Cas12j, and Cas12n, ranging in length from 400 to 800 amino acids), and their ancestral proteins TnpB and IscB (~400 amino acids)19-30. Furthermore, Fanzor (Fz), a eukaryotic ωRNA-guided endonuclease widely found in fungi, algae, protozoa, metazoa, cell-free organisms, and certain large double-stranded DNA viruses, represents a unique class of RNA-programmed genome editing enzymes with significant evolutionary differences compared to the prokaryotic system. Notably, through phylogenetic and structural studies, the newly discovered prokaryotic forced movement element guided activity (OMEGA) protein TnpB is considered an evolutionary precursor to both eukaryotic Fz proteins and prokaryotic CRISPR-Cas12 nucleases. Fanzor proteins are mainly divided into two classes: Fz1 and Fz2. The Fz1 protein is between 600 and 900 amino acids in length, while the Fz2 protein is more compact (~480 amino acids) and structurally more similar to TnpB. The compact structure of Fz2 makes it an ideal candidate for viral delivery of therapeutic genome editing. However, the natural Fz2 system exhibits extremely low activity in mammalian cells (<1% editing efficiency), likely due to suboptimal ωRNA scaffolds and protein-DNA / RNA interactions. These limitations highlight the importance of systems engineering optimization of ωRNA scaffolds to realize practical genome editing tools. Summary of the Invention

[0004] To address the aforementioned technical issues, this invention provides an optimized ωRNA and its applications.

[0005] The present invention specifically adopts the following technical solution:

[0006] An optimized ωRNA is based on the wild-type ωRNA with the gene sequence shown in SEQ ID NO.1, by making base mutations. Taking the 5′ end of the wild-type ωRNA as the reference, the 36th pairing base UA is replaced with GC (UA36GC), the 37th pairing base UA is replaced with AU (UA37AU), the 38th pairing base UG is replaced with AU (UG38AU), the 50th pairing base UG is replaced with CG (UG50CG), and the 51st pairing base UA is replaced with GC (UA51GC).

[0007] This invention optimizes wild-type ωRNA. The ωRNA-V3 variant containing five substitutions (UG50CG, UA51GC, UA36GC, UA37AU, and UG38AU) exhibits a maximum fold increase in insertion / deletion activity of 16.7-fold compared to wild-type ωRNA, significantly improving gene editing efficiency.

[0008] Furthermore, the optimized ωRNA also has the deletion of 15 pairs of paired bases, with the 5′ end of the wild-type ωRNA representing the deletion of 15 pairs of paired bases from position 40 to position 54.

[0009] Based on the same inventive concept, the present invention also provides a gene editing system, wherein the gene editing system is guided by the optimized ωRNA to recognize target sites by MmeFz2.

[0010] Based on the same inventive concept, the present invention also provides a polynucleotide encoding the gene editing system.

[0011] Based on the same inventive concept, the present invention also provides a recombinant vector containing the polynucleotide.

[0012] Based on the same inventive concept, the present invention also provides cells comprising the recombinant vector.

[0013] Based on the same inventive concept, the present invention also provides a gene editing composition comprising the mutated gene editing system, the polynucleotide, the recombinant vector, or the cell.

[0014] Based on the same inventive concept, the present invention also provides the application of the mutated gene editing system, the polynucleotide, the recombinant vector, or the cell in gene editing.

[0015] Based on the same inventive concept, the present invention also provides the use of the mutated gene editing system, the polynucleotide, the recombinant vector, or the cell in the preparation of gene editing formulations.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention achieves synergistic modification by integrating structural insights predicted by AlphaFold3 with rational ωRNA engineering, resulting in a nearly 20-fold increase in insertion and deletion efficiency at multiple genomic sites, while reducing the length of the ωRNA scaffold by 30%. Attached Figure Description

[0018] Figure 1 To engineer MmeFz2 ωRNA to improve genome editing efficiency. a: The predicted secondary structure of MmeFz2 ωRNA binding to B2M target double-stranded DNA; the S1, S2, and PK regions are highlighted for optimization. b: The experimental procedure for detecting the genome editing activity of MmeFz2 at endogenous B2M sites by optimizing the ωRNA scaffold in HEK293T cells. c: The effect of replacing AU or GU bases in the S1, S2, and PK regions on editing efficiency; the optimal ωRNA (GU33GC) is marked with a red triangle. d: The effect of replacing uracil in the uracil-rich region (U49–U52) within S2 on editing efficiency. e: The combined effect of the first four modifications at the U49–U52 position in S2 on genome editing efficiency. The first two combinations—UA49CG+UG50CG and UG50CG+UA51GC—enhanced by more than 4.5-fold at the U49–U52 position in S2 and were selected for further optimization, indicated by a red triangle. f: The impact of replacing uracil in the uracil-rich region (U36–U40) within S2 on editing efficiency; highly efficient ωRNA variants (UA36GC, UA37CG, UA37AU, UG38GC, UG38AU, UA39CG, UA39AU, and UA40CG) were selected for further optimization. g: The combined impact of the first eight modifications at positions U36–U40 in S2 on genome editing efficiency. The top seven combinations—UG38GC+UA39AU, UA36GC+UA37AU+UG38GC, UA36GC+UA37AU+UG38AU, UA37AU+UA39CG+UA40CG, UA36GC+UA37AU+UG38GC+UA39AU, UA36GC+UA37AU+UA39AU+UA40CG, and UA37AU+UG38GC+UA39CG+UA40CG—showed an enhancement of over 9.5-fold at positions U36 to U40 in the S2 region. These combinations were selected for further optimization and are marked with red triangles. h: The effect of combining the ten modifications described in MS1 ​​and MS2 on gene editing efficiency. Data are mean ± standard error (n = 3). fold change represents the ratio of editing efficiency of the ωRNA variant to that of WT-ωRNA.

[0019] Figure 2Validation results for optimizing the ωRNA scaffold to reduce volume and enhance activity. a: Schematic diagram of 2- and 3-base-pair truncation in the S2 region. b: Gene editing efficiency of the MmeFz2-ωRNA system at the B2M site using 2- or 3-base-pair truncated ωRNA variants in HEK293T cells. Dashed lines indicate the insertion / deletion efficiency of ωRNA-V3. c: Editing efficiency of the S2 region at the B2M site using 1 to 19-base-pair truncated ωRNA variants in HEK293T cells. Dashed lines indicate the insertion / deletion efficiency of ωRNA-V3. Three highly efficient truncated variants (Del-14bp, Del-15bp, and Del-16bp) were selected for further validation and marked with red triangles. d: Schematic diagram of 14 to 16-base-pair truncation in the S2 region. e: Comparison of average gene editing efficiency of the three highly efficient truncated variants at eight endogenous sites in HEK293T cells. Fifteen-base-pair truncated variants (named en-ωRNA) were selected for further study due to their highest editing activity and are marked with red triangles. Each data point represents the average gene editing efficiency at each target site. f: Schematic diagram of the predicted ternary complex composed of MmeFz2 protein, en-ωRNA, and B2M target double-stranded DNA generated using AlphaFold3. Data are mean ± standard error (n = 3). Fold change represents the editing efficiency ratio of the ωRNA variant to ωRNA-V3.

[0020] Figure 3 The structure of the MmeFz2-ωRNA-dsDNA ternary complex predicted by AlphaFold3 is shown below. A: The structure of the complex formed by the binding of the MmeFz2 ribonucleoprotein (RNP) to the B2M target site double-stranded DNA, as predicted by AlphaFold3. B: The overall structure of the MmeFz2 nuclease. C: A schematic diagram of the domain composition of the MmeFz2 nuclease.

[0021] Figure 4 To validate the results of improving gene editing efficiency by sequentially replacing five consecutive uridines (U36–U40) within the S2 sequence, uridines at each site in the five-uridine region (U36–U40) of the S2 sequence were replaced with non-uridine nucleotides to assess their impact on gene editing efficiency. Further analysis was performed on eight optimal replacement combinations (UA36GC, UA37CG, UA37AU, UG38GC, UG38AU, UA39CG, UA39AU, and UA40CG) to explore their synergistic effects on editing efficiency within this region. Data are presented as mean ± standard error (n = 3).

[0022] Figure 5To validate the results of reducing the molecular size and improving the gene editing efficiency of MmeFz2 ωRNA in mammalian cells by truncating it, we compared the gene editing efficiency of three highly efficient truncated versions (Del-14bp, Del-15bp, and Del-16bp) against eight sites (KRAS-guide1, CXCR4-guide1, CXCR4-guide2, DYRK1A-guide5, B2M-guide4, VEGFA-guide1, EMX1-guide6, and DYRK1A-guide1) in HEK293T cells. Data are presented as mean ± standard error (n = 3). Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0024] The methods and materials involved in the following embodiments of the present invention:

[0025] 1. Structural prediction using AlphaFold3

[0026] The amino acid sequence of the wild-type MmeFz2 protein (SEQ ID NO.2), its corresponding / engineered full-length ωRNA (containing a 20-nucleotide B2M guide sequence; the gene sequence of the wild-type ωRNA is shown in SEQ ID NO.1), and a 40 bp endogenous B2M target DNA sequence were submitted to the AlphaFold3 online server (https: / / golgi.sandbox.google.com / ) to predict the structure of the ternary complex. The resulting structure was then refined using COOT. Molecular visualization images were generated using CueMol software (http: / / www.cuemol.org).

[0027] SEQ ID NO. 1: UUCGGGUUCGAUUCUAUCCCCAGGGCUCGAAUGCAUUUUUGUCACAGAUUUUGCCAAUGCAAGAUCUGGGGGCAAGAAUGUCUCCGGGUGAAAAGAGUCAG.

[0028] SEQ ID NO.2: MKRKREQMTLWKAAFVNGQETFKSWIDKARMLELNCDVSSASSTHYSDLNLKTKCAKTDDKFMCNYSVCIRPTSKQKRTLNQMLKVSNYAYNWCNYLVKEKDFKPKQFDLQRIVAKTNSTDVPAEYRLPGDDWFFDNKMSSIKLTACKNFCTMYKSTQTNQKKTKVDLRNKDIVQLREGSFEVQSKYVRLLTEKDIPGERIRQSRIALMPDSFSKSKKDWKERFLRLSKNVSKIPPLSHDMKVCKRPNGKFILQISCDPICTRQIQVQTSDSICSIDPGGRTFATCYDPSNIKTFQIGPEADKKEIIHEFHNKIDYVHRLLSHAQEKKQTQAVQDRIGQLKKLHLKLKTYVDDVHLKLCSYLVKNYKLVVLGKISVSSIVRKDRPNHLAKSANRDLLCWQHYRFRQRLLHRVRGTDCEVIIQDERYTSKTCGNCGEKNNKLGGKETFTCESCNYKTHRDVNGARNILCKYLGLFPFAA。

[0029] 2. Plasmid vector construction

[0030] Plasmid cloning employed standard molecular cloning techniques. Wild-type MmeFz2 with optimized human codons and the ωRNA scaffold were synthesized by Huajin Biotechnology Co., Ltd. PCR amplification was performed using Phanta Max Super-Fidelity DNA polymerase (Vazyme) during the construction of the MmeFz2-ωRNA plasmid, followed by fragment assembly using the Basic Seamless cloning and Assembly Kit (TransGen). Each plasmid contains a CBh promoter, a 3×FLAG tag, an SV40 nuclear localization signal, MmeFz2 protein, ribosomal NLS, bGH poly(A) signal, a U6 promoter, and ωRNA. Target oligonucleotides for the ωRNA were ordered from Qingke Biotechnology Co., Ltd., and ligated to a BsaI-digested backbone vector using T4 DNA ligase (Thermo) after annealing. The backbone vector was synthesized using the Cas12i editing system plasmid (from the literature "An engineeredxCas12i with high activity, high specificity, and broad PAM range"; this plasmid contains the CBh promoter to initiate Cas12i protein expression, the U6 promoter to initiate sgRNA expression, and the CMV promoter to initiate red fluorescent protein expression, thus enabling fluorescence flow cytometry sorting). The nucleotide sequences encoding the wild-type MmeFz2 protein and the wild-type ωRNA scaffold were obtained by seamless cloning. The nucleotide sequences encoding the Cas12i protein (only the Cas12i protein in the expression cassette was replaced, retaining the NLS sequences directly connected upstream and downstream: the 5' nuclear localization signal sequence SV40NLS and the 3' nuclear localization signal sequence nucleoplasmin NLS) and the nucleotide sequence encoding the sgRNA backbone were replaced within the Cas12i editing system plasmid to obtain the wild-type MmeFz2-ωRNA editing system backbone plasmid. The ωRNA spacer sequences (i.e., guide sequences) used in this invention are listed in Table 1.

[0031] Table 1: Target sites and related information

[0032]

[0033] like Figure 1 As shown in figure a, the wild-type MmeFz2 ωRNA scaffold has a secondary structure consisting of two different stem-loop elements (S1 and S2) and a pseudoknot (PK) motif. Base substitution and deletion modifications were performed on a regionally segmented basis starting from the 5′ end of the wild-type MmeFz2 ωRNA scaffold to obtain engineered mutants of MmeFz2 ωRNA with highly efficient genome editing activity.

[0034] For example, MmeFz2 ωRNA AU11CG (or abbreviated as AU11CG, and all other mutants are abbreviated in this way) replaces the 11th pair of paired bases AU at the 5′ end of the wild-type MmeFz2 ωRNA scaffold sequence with CG; MmeFz2 ωRNAUG50CG+UA51GC+UA36GC+UA37AU+UG38AU (ωRNA-V3) replaces the 36th pair of paired bases UA at the 5′ end of the wild-type MmeFz2 ωRNA scaffold sequence with GC, the 37th pair of paired bases UA with AU, the 38th pair of paired bases UG with AU, the 50th pair of paired bases UG with CG, and the 51st pair of paired bases UA with GC.

[0035] MmeFz2 ωRNA Del2.1 is based on ωRNA-V3 with the deletion of two pairs of paired bases. The deleted base pairs are the first and second paired bases from the left (towards the 5′ end) of the “Loop” (CAAU) at the top of the S2 stem-loop element, corresponding to the two pairs of paired bases at positions 54 and 53 of SEQ ID NO.1. MmeFz2 ωRNA Del2.2 deletes the second and third paired bases from the left (towards the 5′ end) of the “Loop” (CAAU) at the top of the S2 stem-loop element, and so on.

[0036] MmeFz2 ωRNA Del3.1 is based on ωRNA-V3 with the deletion of 3 pairs of paired bases. The deleted base pairs are the 1st, 2nd, and 3rd paired bases from the left (towards the 5′ end) of the "Loop" (CAAU) at the top of the S2 stem-loop element, corresponding to the 3 pairs of paired bases at positions 54, 53, and 52 of SEQ ID NO.1. MmeFz2 ωRNA Del3.2 deletes the 2nd, 3rd, and 4th paired bases from the left (towards the 5′ end) of the "Loop" (CAAU) at the top of the S2 stem-loop element, and so on.

[0037] MmeFz2 ωRNA Del-1bp is based on ωRNA-V3 with the deletion of one pair of paired bases. The deleted base pair is the first paired base from the left (towards the 5′ end) of the "Loop" (CAAU) at the top of the S2 stem-loop element. MmeFz2 ωRNA Del-2bp deletes the first and second paired bases from the left (towards the 5′ end) of the "Loop" (CAAU) at the top of the S2 stem-loop element, and so on.

[0038] MmeFz2 ωRNA engineered mutants with highly efficient genome editing activity include:

[0039] MmeFz2 ωRNA AU11CG;MmeFz2 ωRNA UG12CG;MmeFz2 ωRNA UA13GC;MmeFz2 ωRNA UA15GC;MmeFz2 ωRNA UG27CG;MmeFz2 ωRNA GU33GC (ωRNA-V1.1);MmeFz2 ωRNAAU35GC;MmeFz2 ωRNA UG42CG;MmeFz2 ωRNA AU46GC;MmeFz2 ωRNA AU48GC;MmeFz2 ωRNA UA49CG;MmeFz2 ωRNA UA49GC;MmeFz2 ωRNA UG50CG;MmeFz2 ωRNA UG50GC;MmeFz2ωRNA UA51CG;MmeFz2 ωRNA UA51GC;MmeFz2 ωRNA UA52CG;MmeFz2 ωRNA UA52GC;MmeFz2 ωRNA UA36AU;MmeFz2 ωRNA UA36CG;MmeFz2 ωRNA UA36GC;MmeFz2 ωRNAUA37AU;MmeFz2 ωRNA UA37CG;MmeFz2 ωRNA UA37GC;MmeFz2 ωRNA UG38AU;MmeFz2 ωRNA UG38CG;MmeFz2 ωRNA UG38GC;MmeFz2 ωRNA UA39AT; MmeFz2 ωRNA UA39CG; MmeFz2 ωRNA UA39GC; MmeFz2 ωRNA UA40AU; MmeFz2 ωRNA UA40CG; MmeFz2 ωRNA UA40GC; MmeFz2 ωRNA UA49CG+UG50CG (ωRNA-V2.1); MmeFz2 ωRNA UA49CG+UA51GC; MmeFz2 ωRNA UA49CG+UA52GC; MmeFz2 ωRNA UG50CG+UA51GC (ωRNA-V2.2); MmeFz2 ωRNA UG50CG+UA52GC; MmeFz2 ωRNA UA51GC+UA52GC;MmeFz2 ωRNA UG50CG+UA51GC+UA52GC;MmeFz2ωRNA UA49CG+UA51GC+UA52GC;MmeFz2 ωRNA UA49CG+UG50CG+UA52GC;MmeFz2 ωRNAUA49CG+UG50CG+UA51GC;MmeFz2 ωRNA UA49CG+UG50CG+UA51GC+UA52GC;MmeFz2 ωRNAUG38GC+UA39AU (ωRNA-V2.3); MmeFz2 ωRNA UA36GC+UA37AU+UG38GC (ωRNA-V2.4); MmeFz2 ωRNA UA36GC+UA37AU+UG38AU (ωRNA-V2.5); MmeFz2 ωRNA UA37AU+UA39CG+UA40CG (ωRNA-V2.6); MmeFz2 ωRNA UA36GC+UA37AU+UG38GC+UA39AU (ωRNA-V2.7); MmeFz2 ωRNA UA36GC+UA37AU+UA39AU+UA40CG (ωRNA-V2.8); MmeFz2 ωRNA UA37AU+UG38GC+UA39CG+UA40CG (ωRNA-V2.9); MmeFz2 ωRNA GU33GC+UA49CG+UG50CG; MmeFz2 ωRNA GU33GC+UG50CG+UA51GC; MmeFz2 ωRNA GU33GC+UG38GC+UA39AU; MmeFz2 ωRNA GU33GC+UA36GC+UA37AU+UG38GC; MmeFz2 ωRNA GU33GC+UA36GC+UA37AU+UG38AU; MmeFz2 ωRNA GU33GC+UA37AU+UA39CG+UA40CG; MmeFz2 ωRNA GU33GC+UA36GC+UA37AU+UG38GC+UA39AU;MmeFz2 ωRNA GU33GC+UA36GC+UA37AU+UA39AU+UA40CG;MmeFz2 ωRNA GU33GC+UA37AU+UG38GC+UA39CG+UA40CG;MmeFz2 ωRNA UA49CG+UG50CG+UG38GC+UA39AU;MmeFz2ωRNA UA49CG+UG50CG+UA36GC+UA37AU+UG38GC;MmeFz2 ωRNA UA49CG+UG50CG+UA36GC+UA37AU+UG38AU;MmeFz2 ωRNA UA49CG+UG50CG+UA37AU+UA39CG+UA40CG;MmeFz2 ωRNAUA49CG+UG50CG+UA36GC+UA37AU+UG38GC+UA39AU;MmeFz2 ωRNA UA49CG+UG50CG+UA36GC+UA37AU+UA39AU+UA40CG;MmeFz2 ωRNA UA49CG+UG50CG+UA37AU+UG38GC+UA39CG+UA40CG;MmeFz2 ωRNA UG50CG+UA51GC+UG38GC+UA39AU;MmeFz2 ωRNA UG50CG+UA51GC+UA36GC+UA37AU+UG38GC;MmeFz2 ωRNA UG50CG+UA51GC+UA36GC+UA37AU+UG38GC;MmeFz2 ωRNA UG50CG+UA51GC+UA37AU+UA39CG+UA40CG;MmeFz2 ωRNA UG50CG+UA51GC+UA36GC+UA37AU+UG38GC+UA39AU;MmeFz2 ωRNA UG50CG+UA51GC+UA36GC+UA37AU+UA39AU+UA40CG;MmeFz2 ωRNA UG50CG+UA51GC+UA36GC+UA37AU+UG39AU;MmeFz2 ωRNA UG50CG+UA51GC+UA36GC+UA37AU+UA39AU+UA40CG;MmeFz2 ωRNA UG50CG+UA51GC+UA37AU+UG38GC+UA39CG+UA40CG;MmeFz2 ωRNAGU33GC+UA49CG+UG50CG+UG38GC+UA39AU;MmeFz2 ωRNA GU33GC+UA49CG+UG50CG+UA36GC+UA37AU+UG38GC;MmeFz2 ωRNA GU33GC+UA49CG+UG50CG+UA36GC+UA37AU+UG38AU;MmeFz2ωRNA;MmeFz2 ωRNA MmeFz2 ωRNA GU33GC+UG50CG+UA51GC+UA36GC+UA37AU+UG38GC;MmeFz2 ωRNA GU33GC+UG50CG+UA51GC+UA36GC+UA37AU+UG38AU;MmeFz2 ωRNA GU33GC+UG50CG+UA51GC+UA37AU+UA39CG+UA40CG;MmeFz2ωRNA GU33GC+UG50CG+UA51GC+UA36GC+UA37AU+UG38GC+UA39AU;MmeFz2 ωRNA GU33GC+UG50CG+UA51GC+UA36GC+UA37AU+UA39AU+UA40CG;MmeFz2 ωRNA GU33GC+UG50CG+UA51GC+UA37AU+UG38GC+UA39CG+UA40CG;MmeFz2 ωRNA Del2.1;MmeFz2 ωRNA Del2.2;MmeFz2 ωRNA Del2.3;MmeFz2 ωRNA Del2.4;MmeFz2 ωRNA Del2.5;MmeFz2 ωRNA Del2.6;MmeFz2ωRNA Del2.7;MmeFz2 ωRNA Del2.8;MmeFz2 ωRNA Del2.9;MmeFz2 ωRNA Del2.10;MmeFz2 ωRNA Del2.11;MmeFz2 ωRNA Del2.12;MmeFz2 ωRNA Del2.13;MmeFz2 ωRNADel2.14;MmeFz2 ωRNA Del2.15;MmeFz2 ωRNA Del2.16;MmeFz2 ωRNA Del2.17;MmeFz2ωRNA Del3.1;MmeFz2 ωRNA Del3.2;MmeFz2 ωRNA Del3.3;MmeFz2 ωRNA Del3.4;MmeFz2 ωRNA Del3.5;MmeFz2 ωRNA Del3.6;MmeFz2 ωRNA Del3.7;MmeFz2 ωRNADel3.8;MmeFz2 ωRNA Del3.9;MmeFz2 ωRNA Del3.10;MmeFz2 ωRNA Del3.11;MmeFz2ωRNA Del3.12;MmeFz2 ωRNA Del3.13;MmeFz2 ωRNA Del3.14;MmeFz2 ωRNA Del3.15;MmeFz2 ωRNA Del3.16;MmeFz2 ωRNA Del-1bp;MmeFz2 ωRNA Del-2bp;MmeFz2 ωRNADel-3bp;MmeFz2 ωRNA Del-4bp;MmeFz2 ωRNA Del-5bp;MmeFz2 ωRNA Del-6bp;MmeFz2 ωRNA Del-7bp; MmeFz2 ωRNA Del-8bp; MmeFz2 ωRNA Del-9bp; MmeFz2 ωRNA Del-10bp; MmeFz2 ωRNA Del-11bp; Del-15bp (en-ωRNA); MmeFz2 ωRNA Del-16bp; MmeFz2ωRNA Del-17bp; MmeFz2 ωRNA Del-18bp; MmeFz2 ωRNA Del-19bp. ;

[0040] Among them, MmeFz2 ωRNA Del2.2 has the same sequence as MmeFz2 ωRNA Del-2bp, and MmeFz2 ωRNA Del-3bp has the same sequence as MmeFz2 ωRNA Del3.1.

[0041] 3. Cell culture, transfection, and flow cytometry analysis

[0042] Human HEK293T cells were cultured in DMEM (Gibco) medium containing 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin-glutamine. All cell types were cultured at 37°C and 5% CO2 and passaged every 2 days until 80% confluence was reached. To screen for protein and ωRNA variants at endogenous sites, 2 × 10⁶ cells were cultured. 5 HEK293T cells were seeded into 24-well plates. At approximately 80% confluence, 1500 ng of the MmeFz2 expression plasmid was added to each well for transfection with 1500 ng of plasmid and 3 μL of polyethyleneimine (PEI) at a 1:2 ratio of DNA (µg) to PEI (µL). After 60–72 hours, the transfected cells were digested with 0.05% trypsin (Gibco) for fluorescence-activated cell sorting (FACS), and mCherry-positive cells were used for genomic DNA extraction.

[0043] 4. DNA extraction and insertion / deletion efficiency analysis

[0044] Approximately 10,000 flow-cytometry-sorted cells were lysed with 20 μL of lysis buffer (formulation: 10 mM Tris-HCl, pH 8.0; 0.05% SDS; 20 μg / ml proteinase K). The lysis buffer was incubated at 55°C for 30 min, followed by heating at 95°C for 5 min to inactivate the proteinase. Then, 1 μL of the lysis product was used as a template for PCR amplification.

[0045] For targeted amplicon sequencing, nested PCR amplification was performed using Phanta Max high-fidelity DNA polymerase (Vazyme, P505), with amplified fragment lengths of 200–250 bp, using barcode-tagged primers. After mixing, the PCR products were purified using a gel extraction kit (Omega). The amplicon library was constructed using the VAHTS Universal DNA Library Prep Kit (Vazyme), subsequently purified, and then subjected to 150 bp paired-end sequencing on an Illumina NovaSeq 6000 platform.

[0046] Sequencing data were first demultiplexed using Cutadapt (v2.8), and then analyzed using CRISPResso2 software to quantify indel (insertion / deletion) efficiency. Target site sequences and primer information are detailed in Table 1. The optimized ωRNA of this invention and its applications are described in detail below through specific examples.

[0047] Example 1: Structural guidance optimization of ωRNA using AlphaFold3

[0048] Engineered gRNAs and ωRNAs have been shown to significantly improve genome editing efficiency in various CRISPR-Cas and OMEGA systems. However, most compact RNA-guided systems use gRNAs and ωRNAs that are typically over 100 nucleotides (nt) long and form complex tertiary structures when bound to their corresponding RNA-guided nucleases. Current engineering approaches rely primarily on high-resolution structural understanding of natural ternary complexes—complexes consisting of an RNA-guided nuclease, its associated RNA (gRNA or ωRNA), and the target DNA substrate—to guide rational strategy design. The advent of AlphaFold3 fills a key gap in structural biology by accurately predicting not only protein structures but also multi-component biomolecular interactions, including protein-nucleic acid interfaces. Leveraging this breakthrough, we used AlphaFold3 to predict the structure of the ternary complex in the MmeFz2-ωRNA system. Figure 3This provides structural insights into the assembly and interaction dynamics of the ωRNA. The S1 and PK regions likely interact primarily with MmeFz2, while the S2 region forms an elongated stem-loop structure with minimal interaction with MmeFz2 at its distal end and exhibits significant structural irregularities, such as imperfect base pairings and polyuridine chains. Therefore, based on principles from previous gRNA engineering, we implemented two rational modification strategies across the entire ωRNA scaffold: MS1, stabilizing the structure by replacing non-standard GU and AU pairs with standard GC pairs in the stem-loop structure; and MS2, enhancing transcriptional activity by targeting the replacement of uridine residues with non-uridine nucleotides in polyuridine chains (specifically U36–U40 and U49–U52).

[0049] We first performed MS1 and then used targeted amplicon sequencing in HEK293T cells to evaluate the gene editing efficiency of the endogenous B2M site—previously identified as the most effective target. Figure 1 a, b). Analysis showed that the insertion / deletion efficiency of the GU33GC variant (V1.1) was 3.8 times higher than that of wild-type ωRNA. Figure 1 c). To systematically evaluate the effects of MS2 modification, we introduced nucleotide substitutions on the polyuridine chain. Replacing U49 to U52 nucleotides with GC or CG base pairs improved insertion / deletion efficiency by 2 to 3 times. Figure 1 d). The two combined mutants exhibited a synergistic effect: UA49CG+UG50CG (V2.1) and UG50CG+UA51GC (V2.2) showed an insertion / deletion efficiency more than 4.5 times higher than that of wild-type ωRNA. Figure 1 e). Meanwhile, systematic substitutions in the U36 to U40 regions showed that over half of the single nucleotide variants increased insertion / deletion efficiency by more than 3-fold. Figure 1 f). Of particular note is that all replacements at position UG38 exhibited superior performance, resulting in an insertion loss efficiency improvement of more than 6 times (f). Figure 1 f). Based on these findings, we designed combinatorial mutants by integrating the best-performing replacements. Seven of these mutants (V2.3 to V2.9) showed an insertion / deletion efficiency of more than 9.5 times compared to wild-type ωRNA. Figure 1 g, Figure 4 Subsequently, through systematic integration of all enhancing modifications, a ωRNA-V3 variant containing five substitutions (UG50CG, UA51GC, UA36GC, UA37AU, and UG38AU) was identified, which showed a maximum fold increase in insertion / deletion activity of 16.7-fold compared to wild-type ωRNA. Figure 1 h).

[0050] Structural analysis shows that the interaction between the distal end of the S2 region and MmeFz2 is very limited. Figure 3 A) This suggests that truncating the distal stem loop may enhance cell stability and ωRNA expression while maintaining function. Based on this structural finding, we performed a systematic truncation analysis of the distal stem loop of S2, starting with ωRNA-V3. Initial screening used 33 small fragments of 2-3 bp, and the results showed that most modifications retained editing efficiency comparable to or only slightly reduced compared to ωRNA-V3. Figure 2 a, b). Based on these results, we constructed progressively elongated truncated sections (1-19 bp) by sequentially removing nucleotides from the distal end of S2. Among these, three truncated sections (14-16 bp) not only preserved editing efficiency but also achieved higher editing efficiency at the B2M site than ωRNA-V3. Figure 2 c, d). Subsequent evaluation of eight endogenous sites showed that the 15 bp truncated variant (named en-ωRNA) was superior not only to other truncated variants (14 / 16 bp) but also to ωRNA-V3 ( Figure 2 e, Figure 5 The optimized en-ωRNA structural remodeling revealed a more compact ternary complex compared to MmeFz2 and the target DNA. Figure 2 f). In summary, by integrating structural insights predicted by AlphaFold3 with rational ωRNA engineering, we achieved synergistic modification that resulted in nearly 20-fold increased insertion / deletion efficiency at multiple genomic sites, while reducing ωRNA scaffold length by 30%.

[0051] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

Claims

1. An optimized ωRNA, characterized in that, It involves base mutation based on the wild-type ωRNA gene sequence shown in SEQ ID NO.

1. Taking the 5′ end of the wild-type ωRNA as the reference, the paired bases UA at positions 36 and 78 are replaced with GC, the paired bases UA at positions 37 and 77 are replaced with AU, the paired bases UG at positions 38 and 76 are replaced with AU, the paired bases UG at positions 50 and 63 are replaced with CG, and the paired bases UA at positions 51 and 62 are replaced with GC.

2. The optimized ωRNA according to claim 1, characterized in that, The optimized ωRNA also has 15 pairs of base pairs deleted, with the bases at positions 40 to 54 and 59 to 74 deleted from the 5′ end of the wild-type ωRNA.

3. A gene editing system, characterized in that, The gene editing system comprises the optimized ωRNA and MmeFz2 as described in any one of claims 1 to 2, wherein the amino acid sequence of MmeFz2 is shown in SEQ ID NO.

2.

4. A polynucleotide encoding the gene editing system of claim 3.

5. A recombinant vector comprising the polynucleotide of claim 4.

6. Cells comprising the recombinant vector of claim 5.

7. A gene-editing composition, characterized in that, The gene editing system comprising the mutation of claim 3, the polynucleotide of claim 4, the recombinant vector of claim 5, or the cell of claim 6.

8. The use of the mutated gene editing system of claim 3, the polynucleotide of claim 4, the recombinant vector of claim 5, or the cell of claim 6 in gene editing for purposes other than disease diagnosis or treatment.

9. The use of the mutated gene editing system of claim 3, the polynucleotide of claim 4, the recombinant vector of claim 5, or the cell of claim 6 in the preparation of gene editing formulations.

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