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CN-121991922-A - SpuFz variant derived from eukaryote and application of SpuFz variant in gene editing

CN121991922ACN 121991922 ACN121991922 ACN 121991922ACN-121991922-A

Abstract

The invention provides SpuFz variants derived from eukaryotes and application thereof in gene editing, in particular SpuFz variants and modified omega RNA, and the invention discovers for the first time that the combination of the modified omega RNA and SpuFz mutant can remarkably improve editing efficiency and greatly reduce off-target rate.

Inventors

  • ZHAO GUOLI
  • HUANG JINHAI
  • ZHOU XINGTAO
  • LIU YULING

Assignees

  • 复旦大学附属眼耳鼻喉科医院

Dates

Publication Date
20260508
Application Date
20241108

Claims (13)

  1. 1. A SpuFz mutant, characterized in that the mutant is a non-native protein and the variant is mutated at a core amino acid position of wild-type SpuFz1 corresponding to the amino acid sequence shown in SEQ ID No.3, which is associated with gene editing activity as follows: C310, D487, T513, W219, Q259, E425 and N385.
  2. 2. An isolated polynucleotide, characterized in that, the polynucleotide encodes the SpuFz mutant of claim 1.
  3. 3. A modified omega RNA backbone characterized in that the modified omega RNA backbone is replaced with base C with respect to base U at position 26 of SEQ ID No.5 of the wild type omega RNA backbone and further truncates No. 35%, preferably No. 50%, preferably No. 70%, more preferably No. 80%, more preferably No. 85%, more preferably No. 90%, more preferably No. 95%, such as 100% of the nucleotides at positions 41-49 and 62-69.
  4. 4. An omega RNA, wherein the omega RNA comprises: i) The omega RNA scaffold of claim 3; ii) a targeting segment that specifically binds to the target sequence.
  5. 5. A polynucleotide encoding the omega RNA backbone of claim 3 or the omega RNA of claim 4.
  6. 6. A vector comprising the polynucleotide of claim 2, and/or claim 5.
  7. 7. A composition or complex for gene editing, the composition or complex comprising: a) Omega RNA according to claim 4, polynucleotide according to claim 5 or vector comprising polynucleotide according to claim 5, and B) The SpuFz1 mutant, or expression vector thereof, of claim 1.
  8. 8. A kit comprising one or more components selected from the group consisting of the SpuFz mutant of claim 1, the polynucleotide of claim 2, the engineered omega RNA backbone of claim 3, the omega RNA of claim 4, the polynucleotide of claim 5, the vector of claim 6, and the composition or complex of claim 7.
  9. 9. An enzyme preparation comprising the SpuFz mutant of claim 1, the composition or complex of claim 7.
  10. 10. A medicine box, which comprises a medicine box body, characterized by comprising the following steps: A first container, and a drug comprising the SpuFz mutant of claim 1, the polynucleotide of claim 2, the engineered omega RNA backbone of claim 3, the omega RNA of claim 4, the polynucleotide of claim 5, the vector of claim 6, the composition or complex of claim 7, the enzyme preparation of claim 9, or an enzyme preparation comprising the SpuFz1 mutant of claim 1, the polynucleotide of claim 2, the engineered omega RNA backbone of claim 3, the omega RNA of claim 4, the polynucleotide of claim 5, the vector of claim 6, the composition or complex of claim 7, the enzyme preparation of claim 9, in the first container.
  11. 11. Use of the SpuFz mutant of claim 1, the polynucleotide of claim 2, the engineered ωrna scaffold of claim 3, the ωrna of claim 4, the polynucleotide of claim 5, the vector of claim 6, the composition or complex of claim 7, the enzyme preparation of claim 9 in the preparation of i) a medicament for the treatment of a disease or ii) a kit for gene editing.
  12. 12. A method of gene editing using the SpuFz mutant of claim 1, the polynucleotide of claim 2, the engineered ωrna scaffold of claim 3, the ωrna of claim 4, the polynucleotide of claim 5, the vector of claim 6, the composition or complex of claim 7, the kit of claim 8, the enzyme preparation of claim 9, and the kit of claim 10.
  13. 13. A method for preparing the engineered omega RNA scaffold of claim 3, comprising the steps of: 1) Providing a wild-type omega RNA backbone as shown in SEQ ID NO. 5; 2) The modified omega RNA backbone is obtained by replacing base U at position 26 of the wild type omega RNA backbone with base C and further truncating at least 35%, preferably at least 50%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, such as 100% of the nucleotides at positions 41-49 and 62-69.

Description

SpuFz variant derived from eukaryote and application of SpuFz variant in gene editing Technical Field The invention relates to the field of gene editing, in particular to SpuFz variant derived from eukaryote and application thereof in gene editing. Background Programmable nucleases represented by the CRISPR-Cas9 and CRISPR-Cass 12 systems are capable of precise gene editing in target DNA. Among the identified Cas proteins, streptococcus pyogenes type II-A Cas9 (SpCas 9) and cryptococcus acidizing type V-A BV3L6 Cas12A (AsCas A) exhibited strong gene editing activity. The maximum packaging capacity of AAV vectors is 4.7 kilobases (kb), which is insufficient to package SpCas9 (1368 amino acids) or AsCas a (1307 amino acids) in a single AAV vector. Researchers have now found a prokaryotic system called obligate mobile element directed activity (OMEGA), which includes microrna-directed endoprotease proteins such as TnpB, iscB and IsrB. ISDra2-TnpB are the first classified and identified TnpB nucleases (408 amino acids). Fanzors is a novel programmable RNA-guided DNA nuclease found in eukaryotes, and also belongs to the OMEGA system. TnpB and Fanzor of OMEGA have been identified as precursors to CRISPR-Cas 12. Importantly, due to the compact size of these systems, they are well suited for single AAV based in vivo delivery. Fanzor nucleases are largely divided into Fanzor and Fanzor. Recently, saito and colleagues reported eukaryotic RNA-directed endonucleases, including two Fz1 orthologs obtained from the soil fungus Spotted spider fungus (S.pubtatus, spuFz 1) and algae Guillardia theta (G.theta, gtFz 1), two Fz2 orthologs obtained from the species lovanisia (N.loviniensis, nlovFz 2) and the multicellular eukaryote marine mollusk MERCENARIA MERCERIA (M.merenaria, mmeFz 2). When plasmids containing Fanzor and its cognate omega RNAs are transfected into human embryonic kidney 293T (HEK 293T) cells, an editing efficiency of more than 10% is achieved that induces insertion and/or deletion of the target site. Jiang et al have studied a variety of eukaryotic genomes and viruses, identifying thousands of RuvC-containing Fanzor nucleases, demonstrating the potential ability of Fanzors to undergo genome editing in human cells. Fanzor nuclease consists of about 600 amino acids, its compact structure and its eukaryotic origin may enhance its editing efficiency in eukaryotic cells, thereby reducing human immunogenicity. However, the currently found Fanzor nucleases have low efficiency in editing human genome, making further application difficult, and additional engineering is required to further increase the activity of these systems in human cells. Therefore, there is an urgent need in the art to develop editing systems that further improve the efficiency of editing in human genomes. Disclosure of Invention The main object of the present invention is to provide an editing system that improves editing efficiency in human genome. Aiming at the defect that Fanzor nuclease has low editing efficiency in human genome, the invention provides a SpuFz variant and application thereof in gene editing. The invention focuses on SpuFz on the first aspect, namely, on the basis of a three-mutation version (C310R, D487K, T K) optimized by the group Zhang Feng on SpuFz protein sequences in 2023 on the protein sequence of SpuFz, lysine (W) at a position 219, glutamine (Q) at a position 259 and glutamic acid (E) at a position 425 are replaced by arginine (R), and asparagine (N) at a position 385 is replaced by glycine (G), so that a novel seven mutant is obtained. On the other hand, the wild type omega RNA of SpuFz1 is optimized and cut, firstly, the 26-bit base U of the omega RNA is replaced by the base C, and 17nt is further cut, so that the omega RNA sequence of the final version is obtained. Compared with SpuFz1 variant (ZF-v) of Zhang Feng, the targeting editing efficiency is improved, the efficiency of programmed Double Strand Breaks (DSBs) is improved by 2-64 times, and the targeting rate is extremely low. To our knowledge, this makes it the most active RNA-guided eukaryotic endoprotease available today, providing a powerful and accurate gene editing capability in mammalian cells. In a first aspect the invention provides a SpuFz-mutant, which is a non-native protein and which is mutated at the core amino acid position of wild-type SpuFz1 corresponding to the sequence shown in SEQ ID NO. 3 as follows in relation to gene editing activity: C310, D487, T513, W219, Q259, E425 and N385. In another preferred embodiment, the SpuFz mutant has increased (e.g., at least 2-64 fold increase) gene editing activity for a target sequence of a target molecule complementary to an omega RNA sequence relative to the gene editing activity of a wild-type SpuFz1 protein. In another preferred embodiment, the SpuFz1 mutant is a polypeptide that has the amino acid sequence set forth in SEQ ID NO. 3: (1) The mutation at C310 is selected from any amino acid, pref