Optimized omega RNA and gene editing application thereof

Abstract

The invention belongs to the technical field of gene editing, and particularly relates to optimized omega RNA and gene editing application thereof. According to the optimized omega RNA, base mutation is carried out on the basis of wild type omega RNA with a gene sequence shown as SEQ ID NO.1, and by taking the 5'end of the wild type omega RNA, the 36th paired base UA is replaced with GC, the 37th paired base UA is replaced with AU, the 38th paired base UG is replaced with AU, the 50th paired base UG is replaced with CG, and the 51st paired base UA is replaced with GC. Compared with wild type omega RNA, the omega RNA has the advantages that the maximum enhancement multiple of the insertion and deletion activity is 16.7 times, 15 pairs of paired bases from the 40th site to the 54th site of the omega RNA are further deleted, and the editing efficiency is further improved.

Description

Technical Field

[0001] The present invention belongs to the field of gene editing technology, and specifically relates to an optimized ωRNA and its gene editing application. Background Art

[0002] The advent of programmable genome editing technologies, particularly the CRISPR-Cas system, has revolutionized modern biotechnology and medicine. CRISPR effectors, such as Cas9 and Cas12 nucleases, enable precise DNA manipulation across species, making them 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 significant challenges for efficient delivery, particularly for in vivo gene therapy via adeno-associated viruses (AAVs).

[0003] Recently, researchers have discovered and characterized compact CRISPR nucleases from prokaryotes and their ancestral proteins, including the miniature Cas12 effectors (Cas12f, Cas12j, and Cas12n, ranging in length from 400 to 800 amino acids), as well as their progenitor proteins TnpB and IscB (~400 amino acids). 19-30 Furthermore, Fanzor (Fz), a eukaryotic ω RNA-guided endonuclease, is widely found in fungi, algae, protozoa, metazoans, acellular organisms, and some large double-stranded DNA viruses, representing a unique class of RNA-programmed genome editing enzymes that have significant evolutionary divergence from prokaryotic systems. Notably, through phylogenetic and structural studies, the newly discovered prokaryotic mandatory mobile element guide activity (OMEGA) protein TnpB is considered to be the evolutionary precursor of eukaryotic Fz proteins and prokaryotic CRISPR-Cas12 nucleases. Fanzor proteins are mainly divided into two major classes: Fz1 and Fz2. The Fz1 protein ranges from 600 to 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 virally delivered therapeutic genome editing. However, the native Fz2 system has extremely low activity in mammalian cells (<1% editing efficiency), likely due to suboptimal ωRNA scaffolding and protein-DNA / RNA interactions. These limitations highlight the importance of systematic engineering optimization of ωRNA scaffolds to realize practical genome editing tools. Summary of the Invention

[0004] Based on the above technical problems, the present invention provides an optimized ωRNA and applications thereof.

[0005] The present invention specifically adopts the following technical solutions: An optimized ωRNA is prepared by performing base mutations on the basis of a wild-type ωRNA having a gene sequence as shown in SEQ ID NO.1. Starting from the 5′ end of the wild-type ωRNA, the 36th pairing base UA is replaced by GC (UA36GC), the 37th pairing base UA is replaced by AU (UA37AU), the 38th pairing base UG is replaced by AU (UG38AU), the 50th pairing base UG is replaced by CG (UG50CG), and the 51st pairing base UA is replaced by GC (UA51GC).

[0006] The present invention is optimized based on the wild-type ωRNA. Among them, the ωRNA-V3 variant containing five substitutions (UG50CG, UA51GC, UA36GC, UA37AU and UG38AU) has a maximum enhancement of insertion and deletion activity of 16.7 times compared with the wild-type ωRNA, significantly improving the efficiency of gene editing.

[0007] Furthermore, the optimized ωRNA has 15 pairs of paired bases deleted. Starting from the 5′ end of the wild-type ωRNA, the 15 pairs of paired bases from position 40 to position 54 are deleted.

[0008] Based on the same inventive concept, the present invention also provides a gene editing system, in which the optimized ωRNA guides MmeFz2 to recognize target sites.

[0009] Based on the same inventive concept, the present invention also provides a polynucleotide encoding the gene editing system.

[0010] Based on the same inventive concept, the present invention also provides a recombinant vector comprising the polynucleotide.

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

[0012] 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.

[0013] Based on the same inventive concept, the present invention also provides the use of the mutant gene editing system, the polynucleotide, the recombinant vector or the cell in gene editing.

[0014] Based on the same inventive concept, the present invention also provides the use of the mutant gene editing system, the polynucleotide, the recombinant vector or the cell in preparing gene-edited preparations.

[0015] Compared with the prior art, the present invention has the following beneficial effects: By integrating structural insights predicted by AlphaFold3 and rational ωRNA engineering, the present invention achieves synergistic modification, resulting in a nearly 20-fold increase in insertion / deletion efficiency at multiple genomic sites while reducing the ωRNA scaffold length by 30%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Engineering MmeFz2 ωRNA to improve genome editing efficiency. a: Predicted secondary structure of the MmeFz2 ωRNA binding to the B2M target double-stranded DNA. The S1, S2, and PK regions are highlighted for optimization. b: Experimental workflow for testing the genome editing activity of MmeFz2 at the endogenous B2M locus by optimizing the ωRNA scaffold in HEK293T cells. c: Effects of AU or GU base substitutions in the S1, S2, and PK regions on editing efficiency. The optimal ωRNA (GU33GC) is marked with a red triangle. d: Effects of uracil substitutions within the uracil-rich region (U49–U52) of S2 on editing efficiency. e: Combined effects of the top four modifications at positions U49–U52 in S2 on genome editing efficiency. The top two combinations—UA49CG+UG50CG and UG50CG+UA51GC—showed >4.5-fold enhancement at positions U49–U52 of S2 and were selected for further optimization, indicated by red triangles. f: Effect of replacing uracil within the uracil-rich region (U36–U40) of S2 on editing efficiency. Highly efficient ωRNA variants (UA36GC, UA37CG, UA37AU, UG38GC, UG38AU, UA39CG, UA39AU, and UA40CG) were selected for further optimization. g: Combined effect 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 greater than 9.5-fold enhancement at positions U36 to U40 in the S2 region. These combinations were selected for further optimization and are highlighted with red triangles. h: 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 the editing efficiency of the ωRNA variant to that of the WT ωRNA.

[0017] Figure 2Validation results for optimizing the ωRNA scaffold to reduce size and enhance activity. a: Schematic diagram of 2- and 3-base pair truncations in the S2 region. b: Gene editing efficiency of the MmeFz2-ωRNA system using 2- and 3-base pair truncations at the B2M locus in HEK293T cells. The dashed line represents the indel efficiency of ωRNA-V3. c: Editing efficiency of ωRNA variants with 1 to 19 base pair truncations in the S2 region at the B2M locus in HEK293T cells. The dashed line represents the indel efficiency of ωRNA-V3. Three highly efficient truncation variants (Del-14bp, Del-15bp, and Del-16bp) were selected for further validation and are marked with red triangles. d: Schematic diagram of 14- to 16-base pair truncations in the S2 region. e: Comparison of the average gene editing efficiency of the three highly efficient truncation variants at eight endogenous loci in HEK293T cells. A 15-base pair truncated variant (designated en-ωRNA) was selected for further study due to its highest editing activity and is marked with a red triangle. Each data point represents the average gene editing efficiency per target site. f: Schematic diagram of the predicted ternary complex consisting 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 ratio of the editing efficiency of the ωRNA variant to that of ωRNA-V3.

[0018] Figure 3 The structure of the MmeFz2-ω RNA-dsDNA ternary complex predicted by AlphaFold3. A: AlphaFold3-predicted structure of the MmeFz2 ribonucleoprotein (RNP) bound to the double-stranded DNA at the B2M target site. B: Overall structure of the MmeFz2 nuclease. C: Schematic diagram of the domain organization of the MmeFz2 nuclease.

[0019] Figure 4 This study validates the ability of position-by-position substitutions of five consecutive uridine residues (U36–U40) within the S2 sequence to improve gene editing efficiency. Within the pentacylidine region (U36 to U40) within the S2 sequence, uridine residues at each position were replaced with non-uridine nucleotides to assess their impact on gene editing efficiency. The eight top-performing substitution combinations (UA36GC, UA37CG, UA37AU, UG38GC, UG38AU, UA39CG, UA39AU, and UA40CG) were further analyzed to investigate their synergistic effects on editing efficiency within this region. Data are mean ± standard error (n = 3).

[0020] Figure 5This report demonstrates the effectiveness of truncating the MmeFz2 ωRNA to reduce its size and enhance its gene-editing efficiency in mammalian cells. Three highly effective truncated versions (Del-14bp, Del-15bp, and Del-16bp) were compared in HEK293T cells targeting eight endogenous gene loci (KRAS-guide1, CXCR4-guide1, CXCR4-guide2, DYRK1A-guide5, B2M-guide4, VEGFA-guide1, EMX1-guide6, and DYRK1A-guide1). Data are presented as mean ± standard error (n = 3). DETAILED DESCRIPTION

[0021] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0022] The methods and materials involved in the following examples of the present invention are: 1. Structure prediction through AlphaFold3 The amino acid sequence of the wild-type MmeFz2 protein (shown in SEQ ID NO. 2), its corresponding / engineered full-length ω RNA (containing a 20-nucleotide B2M guide sequence; the wild-type ω RNA gene sequence 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 subsequently refined using COOT. Molecular visualization was performed using CueMol software (http: / / www.cuemol.org).

[0023] SEQ ID NO. 1: UUCGGGUUCGAUUCUAUCCCCAGGGCUCGAAUGCAUUUUUGUCACAGAUUUUGCCAAUGCAAGAUCUGGGGGCAAGAAUGUCUCCGGGUGAAAAGAGUCAG.

[0024] SEQ ID NO.2: MKRKREQMTLWKAAFVNGQETFKSWIDKARMLELNCDVSSASSTHYSDLNLKTKCAKTDDKFMCNYSVCIRPTSKQKRTLNQMLKVSNYAYNWCNYLVKEKDFKPKQFDLQRIVAKTNSTDVPAEYRLPGDDWFFDNKMSSIKLTACKNFCTMYKSTQTNQKKTKVDLRNKDIVQLREGSFEVQSKYVRLLTEKDIPGERIRQSRIALMPDSFSKSKKDWKERFLRLSKNVSKIPPLSHDMKVCKRPNGKFILQISCDPICTRQIQVQTSDSICSIDPGGRTFATCYDPSNIKTFQIGPEADKKEIIHEFHNKIDYVHRLLSHAQEKKQTQAVQDRIGQLKKLHLKLKTYVDDVHLKLCSYLVKNYKLVVLGKISVSSIVRKDRPNHLAKSANRDLLCWQHYRFRQRLLHRVRGTDCEVIIQDERYTSKTCGNCGEKNNKLGGKETFTCESCNYKTHRDVNGARNILCKYLGLFPFAA。

[0025] 2. Plasmid vector construction Plasmid cloning was performed using standard molecular cloning techniques. Wild-type MmeFz2 with human codon optimization and the ω RNA scaffold were synthesized by Huajin Biotechnology Co., Ltd. To construct the MmeFz2-ω RNA plasmid, Phanta Max Super-Fidelity DNA Polymerase (Vazyme) was used for PCR amplification, and fragment assembly was performed using the Basic Seamless Cloning and Assembly Kit (TransGen). Each plasmid contains the CBh promoter, a 3× FLAG tag, an SV40 nuclear localization signal, the MmeFz2 protein, a ribosomal NLS, a bGH poly(A) signal, a U6 promoter, and the ω RNA. Target oligonucleotides for the ω RNA were ordered from Qingke Biotechnology Co., Ltd. and, after annealing, ligated into the BsaI-digested backbone vector using T4 DNA ligase (Thermo). Among them, the backbone vector is a nucleotide sequence encoding the wild-type MmeFz2 protein and the nucleotide sequence encoding the wild-type ω RNA scaffold obtained by synthesis using the Cas12i editing system plasmid (from the literature An engineeredxCas12i with high activity, high specificity, and broad PAM range; this plasmid contains a CBh promoter to drive the expression of Cas12i protein, a U6 promoter to drive the expression of sgRNA, and a CMV promoter to drive the expression of red fluorescent protein to enable fluorescent flow sorting). By using seamless cloning, the nucleotide sequence encoding the Cas12i protein (only the Cas12i protein in the expression cassette is replaced, retaining the NLS sequences directly connected to it upstream and downstream: the nuclear localization signal sequence SV40NLS at the 5' end and the nuclear localization signal sequence nucleoplasmin NLS at the 3' end) and the nucleotide sequence encoding the sgRNA backbone in the Cas12i editing system plasmid were obtained to obtain the wild-type MmeFz2-ω RNA editing system backbone plasmid. The ω RNA spacer sequences (i.e., guide sequences) used in the present invention are listed in Table 1.

[0026] Table 1: Targeting sites and related information like Figure 1 As shown in a, the wild-type MmeFz2 ωRNA scaffold has a secondary structure consisting of two distinct stem-loop elements (S1 and S2) and a pseudoknot (PK) motif. By modifying the 5′-end subregion of the wild-type MmeFz2 ωRNA scaffold with base substitutions and deletions, engineered MmeFz2 ωRNA mutants with efficient genome editing activity were generated.

[0027] Taking MmeFz2 ωRNA AU11CG (or abbreviated as AU11CG, the other mutants are abbreviated in this way) as an example, the 11th pair of paired bases AU at the 5′ end of the wild-type MmeFz2 ωRNA scaffold sequence is replaced by CG; MmeFz2 ωRNA UG50CG+UA51GC+UA36GC+UA37AU+UG38AU (ωRNA-V3) is that the 36th pair of paired bases UA at the 5′ end of the wild-type MmeFz2 ωRNA scaffold sequence is replaced by GC, the 37th pair of paired bases UA is replaced by AU, the 38th pair of paired bases UG is replaced by AU, the 50th pair of paired bases UG is replaced by CG, and the 51st pair of paired bases UA is replaced by GC.

[0028] MmeFz2 ωRNA Del2.1 is based on ωRNA-V3, with two pairs of paired bases deleted. The deleted base pairs are the 1st and 2nd paired bases from the left (towards the 5′ end) of the top "Loop" (CAAU) of the S2 stem-loop element, corresponding to the two pairs of paired bases at positions 54 and 53 of SEQ ID NO.1; the base pairs deleted in MmeFz2 ωRNA Del2.2 are the 2nd and 3rd paired bases from the left (towards the 5′ end) of the top "Loop" (CAAU) of the S2 stem-loop element, and so on.

[0029] MmeFz2 ωRNA Del3.1 is based on ωRNA-V3, with three pairs of paired bases deleted. The deleted base pairs are the 1st, 2nd and 3rd paired bases from the left (towards the 5′ end) of the top "Loop" (CAAU) of the S2 stem-loop element, corresponding to the 54th, 53rd and 52nd pairs of paired bases in SEQ ID NO.1; the base pairs deleted in MmeFz2 ωRNA Del3.2 are the 2nd, 3rd and 4th paired bases from the left (towards the 5′ end) of the top "Loop" (CAAU) of the S2 stem-loop element, and so on.

[0030] MmeFz2 ωRNA Del-1bp deletes one pair of paired bases based on ωRNA-V3. The deleted base pair is the first paired base from the left (towards the 5′ end) of the top "Loop" (CAAU) of the S2 stem-loop element. The deleted base pair in MmeFz2 ωRNA Del-2bp is the first and second paired bases from the left (towards the 5′ end) of the top "Loop" (CAAU) of the S2 stem-loop element, and so on.

[0031] MmeFz2 ωRNA engineered mutants with efficient genome editing activity include: <h2 style=";text-align:left;direction:ltr">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;MmeFz2ωRNA 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;<h2 style=";text-align:left;direction:ltr">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+UA36GC+UA37AU+UG38AU;MmeFz2ωRNA 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;MmeFz2ωRNA 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. ;

[0032] Among them, the sequence of MmeFz2 ωRNA Del2.2 is identical to that of MmeFz2 ωRNA Del-2bp, and the sequence of MmeFz2 ωRNA Del-3bp is identical to that of MmeFz2 ωRNA Del3.1.

[0033] 3. Cell Culture, Transfection, and Flow Cytometry Analysis Human HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 1% penicillin-streptomycin-glutamine. All cell types were cultured at 37°C in 5% CO2 and passaged every 2 days until they reached 80% confluence. To screen for protein and ωRNA variants at endogenous loci, 2 × 10 5 HEK293T cells were seeded into 24-well plates and, at approximately 80% confluence, transfected with 1500 ng of MmeFz2 expression plasmid at a 1:2 DNA (µg) to PEI (µl) ratio by adding 1500 ng of plasmid and 3 µl of polyethyleneimine (PEI) per well. After 60–72 hours, transfected cells were digested with 0.05% trypsin (Gibco) and used for fluorescence-activated cell sorting (FACS). mCherry-positive cells were used for genomic extraction.

[0034] 4. DNA Extraction and Indel Efficiency Analysis Approximately 10,000 flow-sorted cells were lysed in 20 μL of lysis buffer (10 mM Tris-HCl, pH 8.0; 0.05% SDS; 20 μg / ml proteinase K). The lysate was incubated at 55°C for 30 minutes and then heated at 95°C for 5 minutes to inactivate the proteinase. 1 μL of the lysate was then used as a template for PCR amplification.

[0035] For targeted-amplicon sequencing, nested PCR amplification was performed using Phanta Max High-Fidelity DNA Polymerase (Vazyme, P505) with amplicon lengths of 200–250 bp using barcoded primers. PCR products were pooled and purified using a gel extraction kit (Omega). Amplicon libraries were constructed using the VAHTS Universal DNA Library Prep Kit (Vazyme), purified, and sequenced using 150 bp paired-end sequencing on the Illumina NovaSeq 6000 platform.

[0036] Sequencing data were first processed for linker removal and demultiplexing using Cutadapt (v2.8). CRISPResso2 software was then used to analyze the data and quantify indel (insertion / deletion) efficiency. Target site sequences and primer information are detailed in Table 1. The optimized ωRNA of the present invention and its applications are described in detail below through specific examples.

[0037] Example 1: Structure-guided optimization of ωRNA using AlphaFold3 Engineered gRNAs and ωRNAs have been shown to significantly improve genome editing efficiency in different CRISPR-Cas and OMEGA systems. However, most compact RNA-guided systems use gRNAs and ωRNAs that are typically longer than 100 nucleotides (nt) and form complex tertiary structures when bound to the corresponding RNA-guided nucleases. Current engineering approaches rely heavily on high-resolution structural understanding of natural ternary complexes—comprising RNA-guided nucleases, their associated RNAs (gRNAs or ωRNAs), and target DNA substrates—to guide rational strategy design. The emergence of AlphaFold3 fills a critical gap in structural biology, enabling accurate prediction of not only protein structures but also multicomponent biomolecular interactions, including protein-nucleic acid interfaces. Leveraging this breakthrough, we used AlphaFold3 to predict the ternary complex structure of the MmeFz2-ωRNA system ( Figure 3), providing structural insights into its assembly and interaction dynamics. The S1 and PK moieties likely primarily interact with MmeFz2, while the S2 region forms an extended stem-loop structure, the distal end of which interacts minimally with MmeFz2 and displays significant structural irregularities, such as imperfect base pairing and polyuridine strands. Therefore, building on principles from previous gRNA engineering, we implemented two rational modification strategies across the entire ωRNA scaffold: MS1, which stabilizes the structure by replacing non-canonical GU and AU pairs with canonical GC pairs within the stem-loop structure; and MS2, which enhances transcriptional activity by targeted replacement of uridine residues with non-uridine nucleotides within the polyuridine strand (specifically U36-U40 and U49-U52).

[0038] We first implemented MS1 and used targeted amplicon sequencing in HEK293T cells to assess gene editing efficiency at the endogenous B2M locus, which was previously identified as the most efficient 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 the wild-type ωRNA ( Figure 1 c) To systematically evaluate the effects of MS2 modifications, we introduced nucleotide substitutions along the polyuridine chain. Substituting nucleotides U49 to U52 with GC or CG base pairs increased indel efficiency by 2- to 3-fold ( Figure 1 d). Two combination mutants showed a synergistic effect: UA49CG+UG50CG (V2.1) and UG50CG+UA51GC (V2.2) increased the insertion / deletion efficiency by more than 4.5-fold compared to wild-type ωRNA ( Figure 1 e). Meanwhile, systematic substitutions in the U36 to U40 region showed that more than half of the single nucleotide variants increased the insertion / deletion efficiency by more than 3 times ( Figure 1 f). Of particular note, all substitutions at position UG38 showed excellent performance, resulting in a >6-fold increase in indel efficiency ( Figure 1 f). Based on these findings, we designed combinatorial mutants by integrating the best-performing substitutions. Seven of these mutants (V2.3 to V2.9) showed >9.5-fold improvement in indel efficiency compared to wild-type ωRNA ( Figure 1 g, Figure 4 Subsequently, by systematically combinatorially integrating all enhancing modifications, a ωRNA-V3 variant containing five substitutions (UG50CG, UA51GC, UA36GC, UA37AU, and UG38AU) was identified, which showed a maximum fold enhancement of 16.7-fold indel activity compared to wild-type ωRNA ( Figure 1 h).

[0039] Structural analysis revealed that the distal end of the S2 region interacts very limitedly with MmeFz2 ( Figure 3 A), suggesting that truncation of this distal stem region may enhance cellular stability and ωRNA expression while maintaining function. Based on this structural finding, we systematically analyzed truncation of the distal stem-loop of S2, starting with ωRNA-V3. A preliminary screen using 33 small fragments of 2-3 bp showed that most modifications retained editing efficiencies comparable to or only slightly reduced to those of ωRNA-V3 ( Figure 2 a, b). Based on these results, we constructed progressively longer truncations (1-19 bp) by sequentially removing nucleotides from the distal end of S2. Among them, three truncations (14-16 bp) not only retained the editing efficiency but also achieved higher editing efficiency than ωRNA-V3 at the B2M site ( Figure 2 c, d). Subsequent evaluation of eight endogenous sites showed that the 15 bp truncated variant (named en-ωRNA) outperformed not only the other truncated variants (14 / 16 bp) but also ωRNA-V3 ( Figure 2 e, Figure 5 ). Structural remodeling of the optimized en-ωRNA revealed a more compact ternary complex with MmeFz2 and target DNA ( Figure 2 f). Taken together, by integrating structural insights predicted by AlphaFold3 and rational ωRNA engineering, we achieved synergistic modifications, leading to a nearly 20-fold increase in indel efficiency at multiple genomic loci while reducing ωRNA scaffold length by 30%.

[0040] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. An optimized ωRNA, characterized in that The method is to perform base mutations on the basis of the wild-type ω RNA shown in the gene sequence of SEQ ID NO.

1. Starting from the 5′ end of the wild-type ω RNA, 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.

2. An optimized ωRNA according to claim 1, characterized in that The optimized ωRNA also has 15 pairs of paired bases deleted. Starting from the 5′ end of the wild-type ωRNA, the 15 pairs of paired bases from position 40 to position 54 are deleted.

3. A gene editing system, characterized in that: The gene editing system is guided by the optimized ωRNA according to any one of claims 1 to 2 to enable MmeFz2 to recognize target sites.

4. A polynucleotide encoding the gene editing system according to claim 3.

5. A recombinant vector comprising the polynucleotide according to claim 4. A cell comprising the recombinant vector according to claim 5.

7. A gene editing composition, characterized in that: A 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. 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.

9. 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 a gene-edited preparation.

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