Reverse transcriptase mrt3 variants and their use in prime editing

Abstract

The application provides a reverse transcriptase MRT3 variant and application thereof in prime editing. Specifically, a mutant of reverse transcriptase MRT3 and a prime editor containing the mutant are provided. The prime editor can realize accurate replacement and insertion of bases. The reverse transcriptase MRT3 variant of the application can be used in prime editing of genomes of animals and plants, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of gene editing, and more specifically, to a reverse transcriptase MRT3 variant and its application in guided editing. Background Technology

[0002] In guided editing and other reverse transcription-dependent systems, Moloney murine leukemia virus reverse transcriptase (M-MLV RT) is currently the most extensively studied and widely used reverse transcriptase. However, M-MLV RT is relatively large (692 aa), which is very unfavorable for the delivery of guided editing tools and easily leads to the appearance of by-editing products, resulting in low purity of the edited products; while the reported smaller reverse transcriptases have the drawback of very low efficiency. Therefore, screening for small, active candidate enzymes with further engineering potential from RTs from different sources has important application value.

[0003] Therefore, there is an urgent need in the field for a reverse transcriptase that is smaller in size while maintaining or improving upon the original editing activity. Summary of the Invention

[0004] The purpose of this invention is to provide a reverse transcriptase MRT3 variant and its application in guided editing.

[0005] In a first aspect, the present invention provides a reverse transcriptase having, relative to the amino acid sequence shown in SEQ ID NO:1, a mutation selected from the group consisting of: F457K, T361R, Q59R, L58K, Q358R, Q55R, V105K, N365R, L359K, L310K, L235K, Q231R, S155R, N132R, Q129R, I119K, L117K, I107K, E106Q, L103K, A66K, V61K, V60K, N234R, N63R, N111R, N153K, N51R, or combinations thereof.

[0006] In another preferred embodiment, the amino acid sequence of the reverse transcriptase has a mutation selected from the group consisting of N234R, N63R, N111R, N153K, N51R, or a combination thereof, relative to the amino acid sequence shown in SEQ ID NO:1.

[0007] In another preferred embodiment, the amino acid sequence of the reverse transcriptase has a mutation selected from the group consisting of, relative to the amino acid sequence shown in SEQ ID NO:1: (a1) N234R and N63R; (a2) N111R, N234R and N63R; (a3) N111R and N234R; or (a4) N63R and N111R.

[0008] In another preferred embodiment, the amino acid sequence of the reverse transcriptase has a mutation selected from the group consisting of the following, relative to the amino acid sequence shown in SEQ ID NO:1: F457K, T361R, Q59R, L58K, Q358R, Q55R, V105K, N365R, L359K, L310K, L235K, Q231R, S155R, N132R, Q129R, I119K, L117K, I107K, E106Q, L103K, A66K, V61K, V60K, N234R, N63R, or combinations thereof.

[0009] In another preferred embodiment, the amino acid sequence of the reverse transcriptase has a mutation selected from the group consisting of, relative to the amino acid sequence shown in SEQ ID NO:1: (b1) F457K, N234R and N63R; (b2) T361R, N234R and N63R; (b3) Q59R, N234R and N63R; (b4) L58K, N234R and N63R; (b5) Q358R, N234R and N63R; (b6) Q55R, N234R and N63R; (b7) V105K, N234R and N63R; (b8) L103K, N234R and N63R; (b9) N365R, N234R and N63R; (b10) E106Q, N234R and N63R; (b11) V61K, N234R and N63R; (b12) I107K, N234R and N63R; (b13) Q129R, N234R and N63R; (b14) A66K, N234R and N63R; (b15) L359K, N234R and N63R; (b16) S155R, N234R and N63R; (b17) I119K, N234R and N63R; (b18) V60K, N234R and N63R; (b19) L117K, N234R and N63R; (b20) L310K, N234R and N63R; (b21) L235K, N234R and N63R; (b22) Q231R, N234R and N63R; or (b23) N132R, N234R and N63R.

[0010] In another preferred embodiment, the amino acid sequence of the reverse transcriptase has a mutation selected from the group consisting of, relative to the amino acid sequence shown in SEQ ID NO:1: F457K, N234R, and N63R; T361R, N234R and N63R; Q59R, N234R, and N63R; L58K, N234R, and N63R; Q358R, N234R and N63R; Q55R, N234R, and N63R; V105K, N234R, and N63R; N234R and N63R; N63R; or N234R.

[0011] In another preferred embodiment, the editing efficiency of the reverse transcriptase is ≥1%, such as ≥2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, more preferably ≥15%, 20%, and even more preferably ≥24%.

[0012] In another preferred embodiment, the editing efficiency of the reverse transcriptase is significantly higher than that of the wild-type reverse transcriptase.

[0013] In another preferred embodiment, "significantly higher than wild-type reverse transcriptase" means that, compared with the editing efficiency M0 of wild-type MRT3, the editing efficiency M2 of the reverse transcriptase satisfies M2 / M0 ≥ 100%, 105%, 110%, 115%, 120%, preferably ≥ 125%, 130%, and more preferably ≥ 200%, 300%, 400%, 500%.

[0014] In another preferred embodiment, the editing efficiency of the reverse transcriptase is significantly higher than that of known engineered reverse transcriptases.

[0015] In another preferred embodiment, "significantly higher than known engineered reverse transcriptases" means that, compared with the editing efficiency V0 of MarathonRT-5m, the editing efficiency V1 of the reverse transcriptase satisfies V1 / V0 ≥ 100%, 105%, 110%, 115%, 120%, preferably ≥ 125%, 130%, and more preferably ≥ 200%.

[0016] In another preferred embodiment, the Marathon-RT 5M has a nucleotide sequence as shown in SEQ ID NO:3.

[0017] In another preferred embodiment, the purity of the reverse transcriptase mutant product is ≥50%, such as ≥60%, ≥65%, 70%, 75%, 80%, 85%, more preferably ≥90%, 95%, and even more preferably ≥96%, 97%, 98%, 99%, or 100%.

[0018] In a second aspect, the present invention provides a fusion protein comprising the reverse transcriptase and Cas protein described in the first aspect of the present invention.

[0019] In another preferred embodiment, the fusion protein has the structure shown in formula (I): Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I), In the formula, "-" represents a peptide bond or a linking peptide. Z1 is the nuclear localization signal sequence; Z2 is a Cas protein; Z3 is a connection sequence; Z4 is the viral nucleocapsid protein sequence; Z5 is the nuclear positioning signal sequence; Z6 is the reverse transcriptase; Z7 is the nuclear positioning signal sequence.

[0020] In another preferred embodiment, the fusion protein has the structure shown in formula (II): Z1-Z2-Z3-Z4-Z5 (I), In the formula, "-" represents a peptide bond or a linking peptide. Z1 is the nuclear localization signal sequence; Z2 is a Cas protein; Z3 is the nuclear localization signal sequence; Z4 is the reverse transcriptase; Z5 is the nuclear positioning signal sequence.

[0021] In another preferred embodiment, the Cas protein includes: Cas9 or a variant thereof, Cas12 or a variant thereof, Cas13 or a variant thereof, Cas14 or a variant thereof, and TnpB.

[0022] In another preferred embodiment, the Cas protein is Cas9 or a variant thereof.

[0023] In another preferred embodiment, the amino acid sequence of the Cas9 is the sequence shown in SEQ ID NO:4.

[0024] In another preferred embodiment, the Cas9 variants include Cas9-NG and Cas9-SpRY.

[0025] In another preferred embodiment, the Cas9 variant has only mutations selected from the group consisting of (m1) R221K, N394K and H840A relative to the amino acid sequence shown in SEQ ID NO:4; or (m2) H840A.

[0026] In another preferred embodiment, the fusion protein further includes a tag sequence.

[0027] A third aspect of the present invention provides a CRISPR-Cas composition comprising: (1) Guide RNA, or one or more DNA molecules encoding said guide RNA; and (2) The fusion protein described in the second aspect of the present invention, or the nucleic acid molecule encoding the fusion protein described in the second aspect of the present invention.

[0028] In another preferred embodiment, the CRISPR-Cas composition is used for single base substitution, multi-base substitution, and base insertion.

[0029] In a fourth aspect, the present invention provides a polynucleotide encoding the reverse transcriptase described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention.

[0030] In another preferred embodiment, the polynucleotide is a DNA molecule codon-optimized according to the codon preference of the host cell.

[0031] In another preferred embodiment, the host cell includes eukaryotic cells and prokaryotic cells.

[0032] In a fifth aspect, the present invention provides a carrier containing the polynucleotide described in the fourth aspect of the present invention.

[0033] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0034] In another preferred embodiment, the vector is a eukaryotic expression vector.

[0035] A sixth aspect of the present invention provides a delivery system comprising the reverse transcriptase described in the first aspect of the present invention, or the fusion protein described in the second aspect of the present invention, or the CRISPR-Cas composition described in the third aspect of the present invention, or the polynucleotide described in the fourth aspect of the present invention, or the vector described in the fifth aspect of the present invention, and a delivery medium.

[0036] In another preferred embodiment, the delivery medium includes nanoparticles, plasmids, exosomes, microbubbles, gene guns, or electroporation devices.

[0037] In a seventh aspect, the present invention provides a host cell that expresses the fusion protein described in the second aspect of the present invention, or whose genome is integrated with the polynucleotide described in the fourth aspect of the present invention, or contains the vector described in the fifth aspect of the present invention.

[0038] In an eighth aspect, the present invention provides a kit comprising the reverse transcriptase described in the first aspect of the present invention, or the fusion protein described in the second aspect of the present invention, or the CRISPR-Cas composition described in the third aspect of the present invention, or the host cell described in the seventh aspect of the present invention.

[0039] In another preferred embodiment, the kits are combined in the same or different containers.

[0040] In another preferred embodiment, the kit includes one or more buffer solutions.

[0041] In another preferred embodiment, the kit also includes a label or instructions.

[0042] In another preferred embodiment, the kit is used for one or more of the following: gene or genome editing, plant breeding, targeting a target gene, cutting a target gene or a non-target gene, single base substitution, multi-base substitution, and base insertion.

[0043] In a ninth aspect, the present invention provides a method for sequence optimization of reverse transcriptase MRT3, the method comprising: superimposing an RNA / DNA template-primer complex with a predicted structure of MRT3 to obtain a superimposed model; selecting residues in the superimposed model that are ≤10 Å away from the RNA / DNA double strand and have a certain surface accessibility as sequence optimization sites; wherein, MRT3 has an amino acid sequence as shown in SEQ ID NO:1.

[0044] In another preferred embodiment, the RNA / DNA template-primer complex is an RNA / DNA template-primer complex of type IIC reverse transcriptase of Bacillus stearothermophilus (GsI-IIC RT-RNA / DNA template-primer complex).

[0045] In another preferred embodiment, residues in the superposition model that are ≤5 Å away from the RNA / DNA double strand and have a certain surface accessibility are selected as sequence optimization sites.

[0046] In another preferred embodiment, the sequence optimization site is located around: (i) N234R, N63R, N111R or N153K; (ii) the C-terminal functionally relevant segment in MRT3.

[0047] In a tenth aspect of the present invention, the use of the reverse transcriptase described in the first aspect of the present invention, or the fusion protein described in the second aspect of the present invention, or the CRISPR-Cas composition described in the third aspect of the present invention, or the polynucleotide described in the fourth aspect of the present invention, or the vector described in the fifth aspect of the present invention, or the delivery system described in the sixth aspect of the present invention, or the host cell described in the seventh aspect of the present invention, for the preparation of a drug or formulation for guided editing is provided.

[0048] In an eleventh aspect of the present invention, a method for editing a target nucleic acid is provided, the method comprising contacting the target nucleic acid with the CRISPR-Cas composition described in a third aspect of the present invention.

[0049] A twelfth aspect of the present invention provides a pharmaceutical composition comprising the CRISPR-Cas composition of the third aspect of the present invention, or the delivery system of the sixth aspect of the present invention, or the host cell of the seventh aspect of the present invention; and a pharmaceutically acceptable excipient.

[0050] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0051] Figure 1 The predicted three-dimensional structure and spatial distribution of the mutated residues of the MarathonRT 5 mutant are shown. (a) Predicted three-dimensional structure of the MarathonRT 5 mutant; (b) Spatial distribution of the mutated residues of the MarathonRT 5 mutant.

[0052] Figure 2 The diagrams show candidate reverse transcriptase guided editing vectors constructed based on the ePPEmax backbone. (a) Schematic diagram of a guided editing vector containing a single eepegRNA based on the ePPEmax backbone; (b) Schematic diagram of a guided editing vector containing two eepegRNAs based on the ePPEmax backbone.

[0053] Figure 3The comparison of the target editing efficiency of different candidate reverse transcriptases at three endogenous target sites in rice protoplasts is shown. (a) In OsSLR1 (a) Editing diagram and target editing efficiency; (b) In OsGl2 (c) Editing diagram and target editing efficiency; OsEhd1 The above shows the editing diagram and the target editing efficiency.

[0054] Figure 4 A schematic diagram of candidate reverse transcriptase-guided editing vectors constructed based on the PE4max backbone is shown. R221K, N394K, and H840A are Cas9 mutants. Among them, R221K and N394K can increase the nuclease activity of Cas9, while H840A is a nickase variant of Cas9 that only cleaves the non-target strand.

[0055] Figure 5 The comparison of the targeted editing efficiency of different candidate reverse transcriptases at three endogenous target sites in HEK293T cells is shown. (a) In FANCF (a) Editing diagram and target editing efficiency; (b) In PRNP (c) Editing diagram and target editing efficiency on HEK3.

[0056] Figure 6 A phylogenetic tree of candidate reverse transcriptases constructed using the neighbor-joining method based on amino acid sequences is shown.

[0057] Figure 7 The study compares the target editing efficiency of the MRT3 single-point mutant and the five-site mutant at three endogenous target sites in HEK293T cells. (a) In FANCF (a) Target editing efficiency on HEK3; (b) Editing diagram and target editing efficiency on HEK3; (c) On RNF2 The target editing efficiency.

[0058] Figure 8 The purity of target products at multiple endogenous target sites in HEK293T cells was compared between the MRT3 single-point mutant and the five-point mutant.

[0059] Figure 9 The study compared the target editing efficiency of MRT3 dual-site and three-site combination mutants on endogenous targets in HEK293T cells.

[0060] Figure 10 This study provides supplemental validation of MRT3 (N63R-N234R) at the HEK3 (+1 CTT insertion) target site in HEK293T cells.

[0061] Figure 11The purity of the target product of the MRT3 candidate mutant at the HEK293T cell site is compared between (a) FANCF and (b) HEK3.

[0062] Figure 12 The structure-guided screening of the second round of candidate optimized sites for MRT3 is shown.

[0063] Figure 13 The editing efficiency and relative efficiency improvement of the second round of candidate mutants are shown. (a) Target editing efficiency of the second round of candidate optimized sites for MRT3; (b) fold change in the efficiency of guided editing by introducing other mutations based on N63R-N234R.

[0064] Figure 14 The purity analysis of the target product of representative candidate variants is shown. Detailed Implementation

[0065] Through extensive and in-depth research, the inventors used MarathonRT as a starting point for bioinformatics mining, identifying various candidate reverse transcriptases, and ultimately selecting MRT3 reverse transcriptase, which exhibits editing activity in both plant and animal cells. The amino acid sequence length of MRT3 is significantly shorter than that of the commonly used M-MLV reverse transcriptase. Experimental results demonstrate that various mutants formed by single-point and multi-site mutations on this MRT3 reverse transcriptase exhibit excellent editing activity, producing highly pure edited products capable of precise editing. Based on these findings, this invention was completed.

[0066] It should be understood that the specific methods and experimental conditions of the invention described below in varying degrees of detail are intended to provide a substantive understanding of the invention. Definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0067] the term As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made of” or “made of”.

[0068] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0069] As used in this article, the term "significant" means that, in a hypothesis test, the observed effect (such as the difference between the experimental and control groups) is unlikely to be caused solely by random error. A hypothesis test includes: the null hypothesis (H0), which assumes that the observed effect does not exist (such as no difference between the experimental and control groups); the p-value, which is the probability of observing the current or more extreme effect when H0 is true; and the significance threshold (α). The significance threshold is typically used to determine whether a hypothesis test is significant. Generally, the significance threshold is 0.05. If the p-value ≤ α, then H0 is rejected, meaning the observed effect exists, and the result is called "significant."

[0070] As used herein, the term "MarathonRT" refers to the marathon reverse transcriptase encoded by an intron in Group II of *Eubacterium rectale*. Previous studies have shown that engineered five-mutant versions of MarathonRT exhibit higher editing efficiency in the PE system than the wild type, thus this family member has the potential for further engineered optimization. This study used the MarathonRT five-mutant (D14R-N26R-D74R-N116K-N197R) as a search template. The amino acid sequence of this mutant was used for sequence homology retrieval, and its AlphaFold predicted structure was used for structural similarity retrieval.

[0071] As used in this article, the term "GsI-IIC RT-RNA / DNA template-primer complex" is a universal complex that reverse transcriptase uses as a template to reverse transcribe RNA into single-stranded DNA during its operation. The reverse transcription process is usually initiated by a short primer, which is then converted into a PBS (primer binding site) sequence in the guide editing tool.

[0072] As used herein, a protein “fragment,” “variant,” or “homologous” may optionally be characterized as having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with a reference protein (such as a reference isoform). In some preferred embodiments, fragments, variants, isoforms, and homologs of a reference protein may be characterized by their ability to perform the functions performed by the reference protein.

[0073] Typically, protein derivatization does not adversely affect the protein's desired activity (e.g., activity binding to guide RNA, endonuclease activity, activity of binding to and cleaving a target sequence at a specific site guided by guide RNA); that is, the protein derivative has the same activity as the original protein. Modified forms of "derivatives" include the deletion, insertion, modification, and / or substitution of one or more amino acids in the protein. The term "artificial evolutionary modification" indicates artificial involvement.

[0074] Those skilled in the art will recognize that the structure of a protein can be altered without adversely affecting its activity and functionality, for example, by introducing one or more conserved amino acid substitutions into the protein's amino acid sequence without adversely affecting the protein molecule's activity and / or three-dimensional structure.

[0075] Those skilled in the art will recognize examples and implementations of conserved amino acid substitutions. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted; that is, a nonpolar amino acid residue can replace another nonpolar amino acid residue, a polar, uncharged amino acid residue can replace another polar, uncharged amino acid residue, a basic amino acid residue can replace another basic amino acid residue, and an acidic amino acid residue can replace another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions, where an amino acid is replaced by another amino acid belonging to the same group, fall within the scope of this invention, provided that the substitution does not lead to the inactivation of the protein's biological activity. Therefore, the proteins of this invention can contain one or more conserved substitutions in their amino acid sequence, preferably generated by substitutions according to Table A. Furthermore, this invention also covers proteins that also contain one or more other nonconservative substitutions, provided that such nonconservative substitutions do not significantly affect the desired function and biological activity of the proteins of this invention.

[0076] Conserved amino acid substitutions can occur at one or more predicted non-essential amino acid residues. “Non-essential” amino acid residues are those that can be altered (deleted, substituted, or replaced) without changing biological activity, while “essential” amino acid residues are required for biological activity. A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Amino acid substitutions can occur in non-conserved regions of Cas enzymes. Generally, such substitutions are not performed on conserved amino acid residues, or on amino acid residues located within conserved motifs, where such residues are required for protein activity. However, those skilled in the art will understand that functional variants may have fewer conserved or non-conserved alterations in conserved regions.

[0077] In some implementations, the selected group of amino acids considered to be mutually conserved substitutions includes: Table A Those skilled in the art will recognize that one or more amino acid residues can be altered (replaced, deleted, truncated, or inserted) from the N and / or C-terminus of a protein while retaining its functional activity. Therefore, proteins that have one or more amino acid residues altered from the N and / or C-terminus of the fusion protein of this invention, or a fusion protein including a Cas protein, while retaining their desired functional activity, are also within the scope of this invention. These alterations may include those introduced by modern molecular methods such as PCR, which includes PCR amplification that alters or lengthens the protein-coding sequence by means of oligonucleotides containing amino acid-coding sequences used in PCR amplification.

[0078] It should be recognized that proteins can be altered in various ways, including amino acid substitution, deletion, truncation, and insertion, and methods for such operations are generally known to those skilled in the art. For example, amino acid sequence variants of proteins can be prepared by mutating DNA. This can also be accomplished through other forms of mutagenesis and / or directed evolution, for example, by using known mutagenesis, recombination, and / or shuffling methods, combined with relevant screening methods, to perform one or more amino acid substitutions; or the deletion and / or insertion of one or more amino acids.

[0079] Those skilled in the art will understand that minor amino acid changes (e.g., naturally occurring mutations) or (e.g., using r-DNA technology) may occur in the fusion proteins of this application or fusion proteins including Cas proteins without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domains of the protein, the properties of the polypeptide may be altered, but the polypeptide may retain its activity. If the mutations are not located near the catalytic domain, active site, or other functional domains, a smaller impact can be expected.

[0080] Those skilled in the art can identify the essential amino acids of fusion proteins using methods known in the art, such as localized mutagenesis, protein evolution, or bioinformatics analysis. The catalytic domains, active sites, or other functional domains of a protein can also be determined through physical structural analysis, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations in presumed key site amino acids.

[0081] reverse transcriptase This invention identifies a reverse transcriptase, MRT3, based on the amino acid sequence of the MarathonRT 5 mutant through sequence homology and structural similarity. The size of MRT3 is significantly smaller than that of the existing M-MLV reverse transcriptase. Based on MRT3, single-point and multi-site mutations were further performed to construct mutants. Experimental results show that some mutants exhibit good base editing activity and can yield edited products with high purity.

[0082] The amino acid sequence of the reverse transcriptase mutant, relative to the amino acid sequence shown in SEQ ID NO:1, has mutations selected from the group consisting of: F457K, T361R, Q59R, L58K, Q358R, Q55R, V105K, N365R, L359K, L310K, L235K, Q231R, S155R, N132R, Q129R, I119K, L117K, I107K, E106Q, L103K, A66K, V61K, V60K, N234R, N63R, N111R, N153K, N51R, or combinations thereof.

[0083] Preferably, the amino acid sequence of the reverse transcriptase mutant has mutations selected from the group consisting of the following groups relative to the amino acid sequence shown in SEQ ID NO:1: (a1) N234R and N63R; (a2) N111R, N234R and N63R; (a3) ​​N111R and N234R; or (a4) N63R and N111R.

[0084] Preferably, the amino acid sequence of the reverse transcriptase mutant has mutations selected from the group consisting of, relative to the amino acid sequence shown in SEQ ID NO:1: (b1) F457K, N234R, and N63R; (b2) T361R, N234R, and N63R; (b3) Q59R, N234R, and N63R; (b4) L58K, N234R, and N63R; (b5) Q358R, N234R, and N63R; (b6) Q55R, N234R, and N63R; or (b7) V105K, N234R, and N63R.

[0085] Preferably, the editing efficiency of the reverse transcriptase mutant is ≥1%, such as ≥2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, more preferably ≥15%, 20%, and even more preferably ≥24%. In the reverse transcriptase containing a single mutation of the present invention, the MRT3 mutant containing N234R can achieve an editing efficiency of ≥4% at a specific editing target site. In the reverse transcriptase containing a double mutation of the present invention, the MRT3 mutant containing N63R-N234R can achieve an editing efficiency of ≥9% at a specific editing target site. In the reverse transcriptase containing a triple mutation of the present invention, the MRT3 mutant containing N63R-N234R-F457K can achieve an editing efficiency of ≥24% at a specific editing target site.

[0086] Preferably, the editing efficiency of the reverse transcriptase mutant of the present invention is significantly higher than that of the wild-type reverse transcriptase. "Significantly higher than the wild-type reverse transcriptase" means that, compared with the editing efficiency M0 of wild-type MRT3 or MarathonRT, the editing efficiency M2 of the reverse transcriptase mutant of the present invention satisfies the following conditions: M2 / M0 ≥ 100%, 105%, 110%, 115%, 120%; more preferably, ≥ 125%, 130%; and even more preferably, ≥ 200%, 300%, 400%, 500%.

[0087] Preferably, the editing efficiency of the reverse transcriptase mutant of the present invention is significantly higher than that of known engineered reverse transcriptases. "Significantly higher than known engineered reverse transcriptases" means that, compared to the editing efficiency V0 of MarathonRT-5m, the editing efficiency V1 of the reverse transcriptase mutant of the present invention satisfies the following conditions: V1 / V0 ≥ 100%, 105%, 110%, 115%, 120%; more preferably, ≥ 125%, 130%; and even more preferably, ≥ 200%.

[0088] In CRISPR editing, metrics for product purity typically include indel (insertion / deletion) content / frequency, homology-directed repair (HDR) efficiency, and base editing purity. Those skilled in the art are familiar with methods for detecting and evaluating product purity, such as gel electrophoresis-based methods and sequencing-based methods.

[0089] Preferably, the product purity of the reverse transcriptase mutant is ≥50%, such as ≥60%, ≥65%, 70%, 75%, 80%, 85%, more preferably ≥90%, 95%, and even more preferably ≥96%, 97%, 98%, 99%, or 100%. In the reverse transcriptase containing a single mutation of the present invention, the product purity of the MRT3 mutant containing N63R or N234R can reach ≥75%. In the reverse transcriptase containing a double mutation of the present invention, the product purity of the MRT3 mutant containing N63R-N234R can reach 100%. In the reverse transcriptase containing a triple mutation of the present invention, the product purity of the MRT3 mutant containing N63R-N234R-F457K can reach 100%.

[0090] Fusion protein This invention provides a fusion protein comprising the reverse transcriptase and Cas protein of this invention. As used herein, the term "Cas protein" is used in the broadest sense, including wild-type Cas protein, its derivatives or variants, analogs, and functional fragments such as oligonucleotide-binding fragments.

[0091] The term “wild type” has the meaning commonly understood by those skilled in the art as referring to the typical form of an organism, strain, gene, or protein, or the characteristic that distinguishes it from mutant or variant forms when it exists in nature, which can be isolated from its natural source and has not been intentionally modified by humans.

[0092] Preferably, the Cas protein may include: Cas9 or a variant thereof, Cas12 or a variant thereof, Cas13 or a variant thereof, Cas14 or a variant thereof, TnpB, etc. Preferably, the Cas protein is Cas9 or a variant thereof. A "variant" or "homology" of a Cas protein refers to a polypeptide that substantially retains the function or activity of the Cas protein. These Cas protein variants may contain mutations or modifications that enhance the function or activity of the Cas protein.

[0093] Preferably, the Cas protein is a Cas protein nickase variant.

[0094] CRISPR system As used herein, the terms “CRISPR-Cas system” or “CRISPR system” are used interchangeably and have the meaning commonly understood by those skilled in the art, which typically includes transcripts or other elements relating to the expression of a CRISPR-related (“Cas”) gene, or transcripts or other elements capable of directing the activity of said Cas gene.

[0095] The present invention also provides a CRISPR-Cas composition, the composition comprising: (1) Protein component: the fusion protein of the present invention; or the nucleic acid molecule encoding the fusion protein of the present invention; (2) RNA component: guide RNA, or one or more nucleic acids encoding the guide RNA, or precursor RNA of the guide RNA, or nucleic acid encoding the precursor RNA of the guide RNA; the protein component and the nucleic acid component bind to each other to form a complex. In the guided editing system, the guide RNA is epegRNA.

[0096] In one embodiment, the CRISPR-Cas composition or system includes one or more vectors, said one or more vectors comprising: (1) A first regulatory element, operatively linked to a nucleotide sequence encoding the fusion protein of the present invention; and (2) A second regulatory element, operatively linked to a nucleotide sequence encoding the guide RNA, the guide RNA comprising: (a) A spacer sequence capable of hybridizing with the target sequence of the target nucleic acid; and (b) A direct repeat (DR) sequence attached to the spacer sequence, which guides the Cas protein in the fusion protein of the present invention to bind to the guide RNA to form a CRISPR-Cas complex targeting the target sequence.

[0097] The first control element and the second control element are located on the same or different carriers of the CRISPR-Cas carrier system.

[0098] Preferably, the first or second regulatory element includes a promoter, which includes one or more of an inductive promoter, a constitutive promoter, or a tissue-specific promoter.

[0099] Preferably, the promoter includes one or more of T7, SP6, T3, CMV, EF1a, SV40, PGK1, humanβ-actin, CAG, U6, H1, T7, T7lac, araBAD, trp, lac, or Ptac.

[0100] Preferably, the first control element and the second control element are located on the same or different carriers.

[0101] Preferably, the vector includes a retroviral vector, a lentiviral vector, adenovirus vector, adeno-associated virus vector, herpes simplex vector, or phage vector.

[0102] Preferably, the vector includes a plasmid vector.

[0103] Preferably, the target nucleic acid includes DNA derived from eukaryotes or DNA derived from prokaryotes.

[0104] Preferably, the eukaryote includes an animal or a plant. Preferably, the plant includes rice.

[0105] Guide RNA As used herein, the terms “guide RNA,” “guide sequence,” and “sgRNA” are used interchangeably and have the meanings commonly understood by those skilled in the art. Generally, a guide RNA may comprise a direct repeat (DR) sequence and a spacer sequence, or consist substantially of or composed of a direct repeat sequence and a spacer sequence.

[0106] In some cases, a guide sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence / gene / nucleotide to hybridize with said target sequence / gene / nucleotide and guide the CRISPR-Cas complex to specifically bind to said target sequence / gene / nucleotide. Generally, when optimally aligned, the complementarity between the guide sequence and its corresponding target sequence / gene / nucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0107] Preferably, more than 80% of the spacer sequence is complementary to the target nucleic acid. More preferably, more than 90%, more preferably more than 95%, even more preferably more than 99%, and still more preferably 100% complementary to the target sequence / target gene / target nucleic acid.

[0108] Preferably, the spacer sequence is about or at least about 16 nucleotides in length, for example, about 16-100, about 16-90, about 17-70, about 17-50, or about 18-41 consecutive nucleotides in length; optionally, the spacer sequence is about 20 consecutive nucleotides in length. Preferably, the spacer sequence is about 20 nt in length.

[0109] In a guided editing system, sgRNA (epegRNA) also contains a primer binding site and a reverse transcription template. The primer binding site is used for 3' complementary pairing of the pre-cut DNA strand, and the reverse transcription template is used to carry the target editing sequence to be introduced.

[0110] carrier As used in this article, a "carrier" is a nucleic acid molecule that can transport another nucleic acid molecule linked to it.

[0111] Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, or without free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and a wide variety of other polynucleotides known in the art. Vectors can be introduced into host cells through transformation, transduction, or transfection, thereby enabling the expression of their carried genetic material elements in the host cells. A vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc., as described herein (e.g., CRISPR transcripts, such as nucleic acid transcripts, proteins, or enzymes). A vector may contain a variety of elements controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Vectors may also contain a replication initiation site.

[0112] Vectors include plasmids and viral vectors. A plasmid is a circular double-stranded DNA loop in which another DNA fragment can be inserted, for example, using standard molecular cloning techniques. A viral vector contains a virus-derived DNA or RNA sequence within a vector used to package the virus; viruses include, for example, retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses. Viral vectors also contain polynucleotides carried by a virus intended for transfection into a host cell. Some vectors (e.g., bacterial vectors with bacterial origins of replication and augmented mammalian vectors) are capable of autonomous replication in the host cells into which they are introduced.

[0113] Other vectors (e.g., non-attachment mammalian vectors) integrate into the host cell's genome after introduction and thereby replicate along with the host genome. Furthermore, some vectors can direct the expression of genes they are operatively linked to. Such vectors are called "expression vectors."

[0114] Those skilled in the art will understand that the design of expression vectors can depend on factors such as the selection of host cells to be transformed and the desired expression level.

[0115] Reagent test kit The present invention provides a kit comprising the reverse transcriptase, fusion protein, CRISPR-Cas composition, host cell, or any two or all of the components thereof.

[0116] Preferably, the kit of the present invention further includes a label or instructions for use of one or more components contained therein, and / or a label or instructions for use in combination with one or more additional components that may be available elsewhere or are required.

[0117] Preferably, the kit further includes one or more buffer solutions that can be used to dissolve any of the one or more components contained therein, and / or to provide suitable reaction conditions for one or more of the components. Such buffer solutions may include one or more of the following: PBS, HEPES, Tris, MOPS, Na₂CO₃, NaHCO₃, NaB, or combinations thereof. In some embodiments, reaction conditions include an appropriate pH, such as an alkaline pH. In some embodiments, the pH is between 7 and 10.

[0118] Preferably, any one or more of the reagent kit components can be stored in a suitable container or at a suitable temperature, such as 4 degrees Celsius.

[0119] Target sequence, target gene or target nucleic acid As used herein, the terms “target sequence,” “target gene,” or “target nucleic acid” are used interchangeably and refer to a specific nucleic acid containing a nucleic acid sequence that is wholly or partially complementary to a spacer sequence in the guide RNA. A “target sequence” is a polynucleotide targeted by a spacer sequence in the guide RNA, such as a sequence complementary to that spacer sequence, wherein hybridization between the target sequence and the spacer sequence will promote the formation of a CRISPR-Cas complex (including the Cas protein and the guide RNA). Perfect complementarity between sequences is not required, as long as sufficient complementarity exists to induce hybridization and promote the formation of a CRISPR-Cas complex. Preferably, the target nucleic acid contains a non-coding region (e.g., a promoter or terminator). Preferably, the target nucleic acid is single-stranded or double-stranded.

[0120] Sequence identity As used herein, “sequence identity” refers to the percentage of nucleotide / amino acid residues in the main sequence that are identical to those in the reference sequence after aligning sequences and, where necessary, introducing gaps to achieve the maximum percentage of sequence identity between sequences. To determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences, those skilled in the art are familiar with various methods for performing double and multiple sequence alignments, for example, using publicly available computer software such as ClustalOmega (Söding, J. Bioinformatics (2005) 21, 951-960), T-coffee (Notredame et al. J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)), and MAFFT (Katoh and Standley, Molecular Biology and Evolution (2013) 30(4) 772-780). When using this software, use the default parameters, preferably using features such as open space penalty and extended penalty.

[0121] The main advantages of this invention include: (1) This invention obtains a candidate reverse transcriptase MRT3 through sequence homology and structural similarity screening, and constructs five single-site mutants and multi-site mutants based on these five single-site mutants based on MRT3. Compared with wild-type MRT3, N234R, N63R, N111R, N153K and the multi-site mutants all have good editing activity on target nucleic acids.

[0122] (2) Based on the preferred single mutation sites, this invention further constructs two-site mutants and three-site mutants. Compared with wild-type MRT3, all constructed mutants have good editing activity. Among them, MRT3 (N63R-N234R) has the best editing activity.

[0123] (3) This invention focuses on the aforementioned preferred mutation site regions and the C-terminal functionally related segments that experiments suggest may be involved in maintaining PE activity, and screens for candidate mutation sites. Based on the two-site mutants, three-site mutants are further constructed. Compared with wild-type MRT3, all constructed mutants have good editing activity. Among them, MRT3 (N63R-N234R-F457K) can achieve editing activity close to PE4max on the target nucleic acid.

[0124] (4) The guided editing system based on the MRT3 mutant can achieve a high degree of target product retention, so it can be used for precise base editing, and then for gene-based theoretical research, plant breeding, gene therapy and other fields.

[0125] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0126] Example 1: Sequence Homology and Structural Similarity Retrieval and Initial Screening This study used the amino acid sequence of the MarathonRT 5 mutant as the query sequence and performed a BLASTP homology search in the NCBI nr database. It was found that the top-ranked entries were mostly annotated as group II intron reverse transcriptase or maturase-like proteins, and their overall length was close to that of MarathonRT. This indicates that there are relatively abundant MarathonRT-related homologous reverse transcriptase resources in the database (Table 1).

[0127] Table 1. List of candidate reverse transcriptases obtained from sequence homology search Note: Due to the large number of sequences retrieved, only a portion of representative sequences are shown in the table.

[0128] This study did not prioritize entries highly related to the MarathonRT 5 mutant. Instead, it considered factors such as sequence identity, protein length, and annotation completeness, selecting candidate enzymes that retained typical Group II intron RT characteristics while exhibiting certain sequence differences for subsequent validation. Ultimately, the following enzymes were selected: Waltera acetigignens The candidate enzyme from the source was named MRT1; Claveliimonas bilis The candidate enzyme from which the sequence was derived was named MRT2 and used as the test subject obtained through sequence homology retrieval.

[0129] To further supplement candidate reverse transcriptases at the structural level, this study used the predicted structure of the MarathonRT 5 mutant as a template for structural similarity retrieval. AlphaFold prediction results showed that the overall structure of the model was folded intact, with high confidence in the main region and a pTM value of 0.91, indicating that the predicted structure has high reliability at the overall topological level and can be used for subsequent structural similarity analysis. Figure 1 ).

[0130] Based on this, this study used Foldseek to search protein structure databases and predicted structure databases, and manually sorted and screened the results by combining structural similarity, sequence differences, and annotation information. The results yielded a batch of candidate enzymes that were similar in overall folding to the MarathonRT 5 mutant, but with relatively low sequence identity. Ten structurally similar candidate enzymes were initially screened, supplementing the sequence homology search results and further expanding the screening range of candidate reverse transcriptases (Table 2).

[0131] Table 2. List of candidate proteins obtained by structural similarity search Further considering structural alignment quality and sequence integrity, this study selected five representative candidate enzymes from the structural similarity search results for subsequent experimental verification, named MRT3 to MRT7 respectively.

[0132] This study used the MarathonRT 5 mutant as a template to screen candidate reverse transcriptases based on both sequence homology and structural similarity. The results showed that sequence homology analysis yielded a group of closely related candidate reverse transcriptases highly similar to the MarathonRT 5 mutant, while structural similarity analysis further supplemented the pool with candidate enzymes whose sequences were more distant but whose overall folding remained similar. Considering sequence differences, structural similarity, and functional annotation, this study ultimately identified seven representative candidate proteins. MRT1 and MRT2 were derived from sequence homology screening, while MRT3-MRT7 were derived from structural similarity screening. These were collectively named MRT1-MRT7 for subsequent PE functional validation.

[0133] Example 2: Verification of the editing activity of candidate reverse transcriptases in rice protoplasts and mammalian cells To systematically evaluate the compatibility of different candidate reverse transcriptases with the guided editing system and their editing activity in different cellular backgrounds, this example verifies transient expression in rice protoplasts and HEK293T cells.

[0134] To systematically evaluate the editing activity of candidate reverse transcriptases in rice protoplast systems, this study constructed candidate reverse transcriptase editing vectors by replacing the M-MLV-RT portion of the ePPEmax backbone. The original M-MLV reverse transcriptase ePPEmax was retained as a positive control, and MarathonRT and its five-mutant variant (MarathonRT-5m) were used as comparative controls. Figure 2 Subsequently, this study selected three endogenous targets in rice, namely... OsEhd1 (The base at position +10 / +11 is replaced by CG instead of GA) OsSLR1(28 bp fragment replacement) and OsGl2 (Insert C at the +2nd position) Figure 3 ).in, OsSLR1 The target design at the site employs a GRAND editing strategy. Figure 2 (b) This method was used to evaluate the performance of different reverse transcriptases in fragment substitution editing. After the transformation vector was introduced into rice protoplasts via PEG-mediated transformation, genomic DNA was extracted, amplified by PCR, and then quantitatively analyzed for the expected editing results at the target sites using Hi-TOM high-throughput sequencing.

[0135] The results showed that M-MLV achieved high target editing efficiency at all three test sites. Compared with wild-type MarathonRT, MarathonRT-5m showed higher efficiency. OsSLR1 , OsGl2 and OsEhd1 The target editing efficiency at all three sites improved, from 1.30%, 1.38%, and 1.62% to 7.50%, 1.66%, and 2.04%, respectively. Figure 3 The above results indicate that the reported five-mutation optimization can improve the editing activity of MarathonRT in rice protoplasts, but this improvement is target-dependent, and its overall editing efficiency is still lower than that of the M-MLV system.

[0136] Of the candidate reverse transcriptases tested, only MRT1, MRT3, and MRT7 showed the target editing product, while the remaining candidate reverse transcriptases did not show the target editing product at any of the three target sites. Figure 3 ).exist OsSLR1 At the site, the target editing efficiency of MRT3 is approximately 1.44%; OsGl2 site and OsEhd1 At the site, MRT3 did not show editing activity. Overall, MRT1, MRT3, and MRT7 all exhibited detectable guided editing activity in the rice protoplast system, but their editing efficiency and target applicability differed, with MRT1 showing the best overall performance, followed by MRT3. Combined with the aforementioned control results, it is evident that the three candidate reverse transcriptases generally performed weaker than the MarathonRT-5m and M-MLV systems, suggesting that further optimization is needed before they can be used in plant guided editing systems. It should be noted that the candidate reverse transcriptases with detected activity and MarathonRT-5m... OsSLR1 All sites showed relatively higher editing efficiency.

[0137] To further evaluate the editing capabilities of candidate reverse transcriptases in mammalian cells, this study validated their editing activity in the HEK293T cell system. Based on the PE4max backbone, candidate reverse transcriptase editing vectors were constructed by replacing the reverse transcriptase portions within it; PE4max, the original M-MLV reverse transcriptase, was retained as a positive control, and MarathonRT and its five-mutant variant (MarathonRT-5m) were used as comparative controls. Figure 4 Endogenous gene sites were selected as detection targets, and guide editing vectors carrying candidate reverse transcriptases were introduced into HEK293T cells via liposome transfection. Genomic DNA was extracted 48 hours after transfection, and PCR amplification was performed on the target sites. The editing results were then quantitatively analyzed by Hi-TOM sequencing.

[0138] This study selected three endogenous targets in HEK293T cells for testing, namely: FANCF (The +6 base is replaced by C instead of G) PRNP (The +6 base is replaced by T) and HEK3 (CTT is inserted at the +1 position). The results showed that PE4max achieved target editing efficiencies of 23.06%, 15.16%, and 14.40% at the three sites, respectively, while MarathonRT-5m achieved 3.37%, 5.24%, and 4.16%, respectively. FANCF The HEK3 locus is higher than that of wild-type MarathonRT ( Figure 5 ). But PRNP For this locus, the MarathonRT group did not obtain reliable quantitative results due to excessively low transfection efficiency, and therefore was not included in subsequent comparisons. Apart from this case, all other experimental groups obtained sequencing results suitable for analysis.

[0139] Of the candidate reverse transcriptases tested, only MRT1, MRT2, and MRT3 showed detected target editing products, indicating that these three candidate enzymes possess guided editing activity in HEK293T cells, while the remaining candidate reverse transcriptases did not show detected target editing products at most sites. Figure 5 Further comparison revealed that both MRT1 and MRT3 detected the target editing product at all three test sites. MRT3, however, showed... FANCF , PRNPThe editing efficiencies of MRT1 at the HEK3 site were higher than those of MRT1, at 2.53%, 4.03%, and 1.56%, respectively. In contrast, the editing efficiency of MRT2 fluctuated significantly across different target sites, and its overall performance was weaker than that of MRT1 and MRT3. It is worth noting that although both MRT1 and MRT3 exhibited cross-site activity, their editing performances were not the same. The editing efficiency of MRT1 at the three sites was relatively concentrated, indicating that its performance at the current test sites was more balanced; the editing efficiency of MRT3 at all three sites was higher than that of MRT1, especially at… PRNP The sites exhibited higher editing efficiency, suggesting higher overall activity under the current testing conditions, but the differences between different sites were also more pronounced.

[0140] In summary, the validation results in the HEK293T cell system indicate that MRT1, MRT2, and MRT3 all possess certain guided editing activities, with MRT1 and MRT3 being the relatively superior candidate reverse transcriptases in this study. Compared to MRT1, MRT3 showed a higher overall editing level at the currently tested sites and was closer to MarathonRT-5m at some sites; however, compared to PE4max, there is still a significant gap, suggesting that its editing performance has room for further optimization.

[0141] Example 3: MRT3 was identified as the target for subsequent optimization. Based on the validation results from both rice protoplasts and HEK293T cell systems, the candidate reverse transcriptases with guided editing activity are concentrated in a small number of members. Specifically, in the rice protoplast system, MRT1, MRT3, and MRT7 detected the target editing products, with the overall performance roughly in the order of MRT1>MRT3>MRT7; in the HEK293T system, MRT1, MRT2, and MRT3 detected the target editing products, with the overall performance roughly in the order of MRT3>MRT1>MRT2. Therefore, MRT1 and MRT3 are the only candidate reverse transcriptases that showed guided editing activity in both plant and mammalian cells, indicating their good cross-system adaptability and making them priority candidates for subsequent protein optimization.

[0142] To further compare the sequence relationships between candidate reverse transcriptases with editing activity and reference enzymes, and to assist in the selection of subsequent optimization targets, this study constructed a neighbor-joining phylogenetic tree based on amino acid sequences for MRT1-MRT7, MarathonRT, and GsI-IIC RT, which have been used to guide editing studies and served as references in subsequent structural analyses. Figure 6The results showed that MRT1 clustered with MarathonRT in the closest possible branch, while MRT3 was clearly separated from MarathonRT and clustered with GsI-IIC RT in a relatively independent small branch. Further comparison revealed that MRT3 shared only 45.4% amino acid sequence similarity with MarathonRT, and approximately 51% with GsI-IIC RT, indicating a significant sequence differentiation between MRT3 and MarathonRT. In contrast, MRT1 showed higher sequence similarity to MarathonRT, and the two were more closely related.

[0143] Furthermore, although MRT3 and MarathonRT differ significantly at the primary sequence level, structural analysis shows that they retain relatively similar core reverse transcriptase structural features, suggesting that MRT3, while retaining similar core structural features, has sequence composition features different from MarathonRT. Considering the cross-system activity, sequence differences, and structural analysis results, MRT3 possesses both the basic editing activity and cross-system adaptability required for further engineering modification, and exhibits significant sequence differentiation from MarathonRT. Therefore, it is more suitable as a target for subsequent optimization to explore PE optimization pathways for non-M-MLV-derived RTs that are significantly sequence-differentiated from MarathonRT but structurally similar.

[0144] Table 3. Comprehensive comparison of candidate reverse transcriptases with editing activity and screening for subsequent optimization. Example 4: Construction and editing activity analysis of MRT3 single-site and five-site mutants Based on previous experimental results, MRT3 exhibits editing activity and can achieve precise editing in both rice protoplasts and HEK293T cells. However, compared with the optimized MarathonRT-5m, the overall editing efficiency of MRT3 still has room for improvement. Therefore, it is necessary to further optimize MRT3 rationally. To screen candidate optimization sites for MRT3, this study rationally identified five potential active sites: N51, N63, N111, N153, and N234. Five single-point mutants (N51R, N63R, N111R, N153K, and N234R) were designed to test whether these amino acid substitutions could enhance editing activity.

[0145] To systematically evaluate the impact of candidate optimization sites on MRT3-guided editing activity, this study constructed five single-site mutants: MRT3(N51R), MRT3(N63R), MRT3(N111R), MRT3(N153K), and MRT3(N234R), as well as a five-site mutant MRT3-5m containing five candidate sites. The target editing activity was then examined at three endogenous target sites in HEK293T cells: FANCF (G replaced with C at position +6), HEK3 (CTT inserted at position +1), and RNF2 (A replaced with C at position +4). Figure 7 ).

[0146] The results showed that different candidate sites had significantly different effects on MRT3-guided editing activity. Among the five single-point mutants, N234R exhibited the highest target editing efficiency at all three target sites, reaching 4.52% and 4.01% at the FANCF and HEK3 sites, respectively, approximately 3.6 times and 3.0 times that of wild-type MRT3. At the RNF2 site, no target editing was detected in wild-type MRT3, while N234R achieved a target editing efficiency of 2.38%. N63R also showed a relatively stable gain effect, reaching 3.19% and 3.69% at the FANCF and HEK3 sites, respectively, approximately 2.5 times and 2.8 times that of wild-type MRT3, and achieving a target editing efficiency of 1.68% at the RNF2 site. In contrast, N111R and N153K also improved editing activity at the three target sites, but their overall gain magnitude and stability were weaker than those of N234R and N63R; N51R did not show a stable positive optimization effect.

[0147] Compared with the optimized MarathonRT-5m system, the editing activities of some MRT3 single-point mutants are close to those of the optimized MarathonRT system: at the FANCF site, the editing efficiency of N234R is higher than that of MarathonRT-5m (3.37%); at the HEK3 site, the editing efficiencies of N63R, N153K, and N234R are all close to those of MarathonRT-5m (4.32%); at the RNF2 site, except for N51R, all other single-point mutants have obtained detectable target editing activity, with N234R and N63R showing more significant improvement, but still lower than that of MarathonRT-5m (3.11%). These results indicate that N234R and N63R are relatively reliable and advantageous optimization sites in MRT3, and can serve as the main basis for subsequent combination optimization.

[0148] Furthermore, the five-site mutant MRT3-5m did not exhibit a simple additive effect. Its targeted editing efficiencies at the FANCF, HEK3, and RNF2 sites were 2.09%, 2.66%, and 1.33%, respectively. While these were higher than the wild-type MRT3 overall, they were all lower than the better-performing single-site mutants N63R and N234R. These results suggest that different mutation sites may interact with each other, and subsequent optimization should not directly combine all candidate sites, but rather focus on more targeted combination designs around the dominant sites.

[0149] Based on the editing efficiency analysis, the purity of the target product of each mutant was further analyzed to assess whether beneficial single-point mutations can maintain good editing result quality while improving activity. Figure 8 Overall, most dominant single-point mutants maintained high purity of target products at multiple endogenous targets, without showing a stable decreasing trend in purity with increasing target editing efficiency. Specifically, N63R and N234R maintained high purity of target products at different targets, generally approaching that of MarathonRT-5m; N111R and N153K also showed some improvement. In contrast, MRT3-5m did not show a stable advantage over dominant single-point mutants.

[0150] Combination Figure 7 and Figure 8 The results show that N63R and N234R not only significantly improve the target editing efficiency of MRT3, but also do not exhibit a clear trend of decreased target product purity at the current test target sites, making them superior single-point mutants screened in this study. Considering that N111R also showed a certain activity enhancement effect and did not show a significant decrease in target product purity, N63R, N111R, and N234R were identified as priority candidate sites for subsequent combination optimization.

[0151] Example 5: Combination optimization of favorable mutation sites and verification of editing activity Based on the screening results of the aforementioned single-site mutants, in order to further evaluate the impact of the combination of dominant sites on the MRT3-guided editing activity, this embodiment constructed three two-site mutants MRT3 (N63R-N111R), MRT3 (N63R-N234R), and MRT3 (N111R-N234R), as well as one three-site mutant MRT3 (N63R-N111R-N234R), and first tested its editing activity at the FANCF target site in HEK293T cells. Figure 9 ).

[0152] The results showed that the target editing efficiency of each combination mutant was higher than that of wild-type MRT3 (1.64%), reaching 5.32%-9.76%, which is about 3.24-5.95 times that of wild-type MRT3. This indicates that combination optimization based on the advantages of single points can further enhance the guided editing activity of MRT3.

[0153] Among them, MRT3 (N63R-N234R) performed best, with an editing efficiency of 9.76%, which was not only higher than other double and triple mutants, but also exceeded the N63R single-point mutant control (6.41%) in the same batch, suggesting a good combinatorial gain effect between N63R and N234R. In contrast, the editing efficiency of MRT3 (N63R-N111R) was lower than that of the N63R single mutant, and MRT3 (N63R-N111R-N234R) did not exceed that of MRT3 (N63R-N234R), indicating that the introduction of N111R into the current MRT3 backbone did not further enhance the combinatorial activity.

[0154] Among them, MRT3 (N63R-N234R) showed good optimization potential. The editing efficiency of MRT3 (N63R-N234R) was higher than that of MarathonRT (4.57%) and MarathonRT-5m (8.60%), approximately 2.14 times and 1.13 times higher, respectively, but still significantly lower than M-MLV (30.51%). This indicates that combined optimization of favorable sites on MRT3 can significantly improve its guided editing activity, reaching and exceeding the level of MarathonRT-5m at this target site, but still showing a significant gap compared to the M-MLV system.

[0155] To further verify the editing activity of the superior combination MRT3 (N63R-N234R) in this round, supplementary testing was conducted on another endogenous target, HEK3. The results showed that MRT3 (N63R-N234R) maintained a significant activity advantage at this target. Its target editing efficiency was 5.21%, approximately 2.24 times that of wild-type MRT3 (2.33%) and 1.26 times that of MarathonRT-5m (4.14%). This result indicates that the N63R-N234R combination maintains high activity at another editing target and can surpass the optimized MarathonRT-5m, but its overall performance is still lower than the M-MLV system (…). Figure 10 ).

[0156] After confirming the enhanced activity of the combined mutants, the quality of their editing results was further analyzed. Using target product purity as an evaluation index, the editing results of the candidate combined mutants on the two targets, FANCF and HEK3, were compared. Figure 11 ).

[0157] The results showed that at the FANCF site, both double and triple mutants maintained high target product purity overall. The purity level of N63R-N234R was similar to that of MarathonRT-5m and PE4max, with no decrease in purity observed due to increased editing efficiency. Further validation at the HEK3 site showed that the calculated target product purity of N63R-N234R was 100% in all three biological replicates. In the current sequencing results, its target product purity was essentially consistent with MarathonRT-5m and PE4max, and higher than that of wild-type MRT3.

[0158] Combination Figures 9 to 11 The results show that N63R-N234R not only exhibits high target editing efficiency at multiple target sites, but also maintains a level of target product purity comparable to existing optimized systems, indicating that the activity enhancement of this combination does not come at the cost of significantly sacrificing the quality of editing results.

[0159] Example 6: Screening and Design of Second-Round Candidate Optimization Sites for MRT3 In the first round of single-site and combined mutation screening, MRT3 (N63R-N234R) exhibited the best editing activity to date. Based on the above results, this study further conducted a second round of candidate site design for MRT3. The second round of design focused on surface residues adjacent to the predicted nucleic acid binding channel of MRT3 as the main screening scope. Specifically, in this embodiment, the GsI-IIC RT-RNA / DNA template-primer complex was used as a closely related structural reference. The predicted structure of MRT3 was superimposed on this complex, and residues in the superimposed model that were ≤5 Å away from the RNA / DNA double strand and had a certain degree of surface accessibility were preferentially selected. Under this screening principle, candidate sites were mainly concentrated in two types of regions: one is the region around the favorable mutation sites in the first round, such as N63 and N234; the other is the C-terminal functionally related segment that the truncation experiment suggested may be involved in maintaining PE activity ( Figure 12 ).

[0160] After the candidate sites were determined, this study designed mutations based on the physicochemical properties of the original residues and the design concept of local positive charge enhancement, rather than adopting a uniform replacement strategy. For polar uncharged residues such as Q / N / S / T, Arg substitution was mainly designed to enhance the local surface positive charge and retain some hydrogen bonding ability; for nonpolar aliphatic residues such as A / V / L / I, Lys substitution was mainly designed to introduce a more flexible positively charged side chain; for E106, Gln substitution was designed to remove the local negative charge and maintain similar side chain properties as much as possible; for F457, Lys substitution was designed to preliminarily evaluate the impact of local positive charge enhancement in the near-terminal region on MRT3 editing activity.

[0161] To evaluate the impact of the second round of candidate sites on MRT3-guided editing activity, this embodiment constructed 23 new mutants based on MRT3 (N63R-N234R) and analyzed their target editing efficiency at the FANCF site in the same detection system. PE4max, MarathonRT-5m, wild-type MRT3, and MRT3 (N63R-N234R) were set as controls. Figure 13 ).

[0162] Among all second-round mutants, MRT3 (N63R-N234R-F457K) performed best, with an average editing efficiency of 24.96%, approximately 2.36 times that of MRT3 (N63R-N234R), significantly higher than MarathonRT-5m's 11.64%, and reaching approximately 84% of PE4max (29.75%). This result indicates that introducing F457K into the MRT3 (N63R-N234R) background can further improve guided editing efficiency, making the editing activity of this non-M-MLV-derived RT variant on the FANCF target closer to that of the optimized M-MLV-derived PE system.

[0163] Besides F457K, T361R, Q59R, and L58K also showed significant efficiency improvements, with average editing efficiencies of 16.23%, 14.25%, and 13.55%, respectively, approximately 1.54 times, 1.35 times, and 1.28 times that of MRT3 (N63R-N234R). Q358R, Q55R, and V105K also showed slightly higher editing efficiencies than MRT3 (N63R-N234R), but the improvement was relatively limited, with average editing efficiencies of 11.98%, 11.88%, and 11.69%, respectively. In contrast, the editing efficiencies of mutants such as V60K, L117K, N132R, Q231R, L235K, and L310K were significantly reduced, suggesting that positive electronegation modification at these sites may be detrimental to the editing activity of MRT3 at this target site. Overall, the second round of structure-guided site optimization further enhanced the editing activity of MRT3 (N63R-N234R) through F457K.

[0164] Example 7: Purity analysis of target product from representative candidate variants To further evaluate whether the second round of candidate variants affects the quality of editing results while improving editing efficiency, this study analyzes the purity of the target product of representative candidate variants. Target product purity is defined as the proportion of target edited product reads to all edited product reads, reflecting the relative proportion of the target product in the editing results. Figure 14 ).

[0165] The results showed that the purity of the target product of wild-type MRT3 at this target site was relatively low, averaging 74.91%; the purity of the target product of MRT3 (N63R-N234R) increased to 95.30%, indicating that the favorable mutation combination in the first round improved the composition of the editing results while increasing editing efficiency. In the second round of candidate variants, the purity of the target product of MRT3 (N63R-N234R-F457K) remained at 100% in all three replicates, indicating that its efficiency improvement was not accompanied by an increase in detectable non-target edited products. Other representative candidate variants also generally maintained high target product purity, indicating that the main high-efficiency variants obtained in the second round did not improve editing efficiency at the expense of significantly reduced product purity.

[0166] In summary, MRT3 (N63R-N234R-F457K) exhibits both high editing efficiency and high target product purity at the current target site, making it the most worthy candidate variant for further validation in this round of screening. Further testing of its efficiency and the stability of editing results is needed at more targets and with different editing types.

[0167] Analysis of the editing efficiency and target product purity of the mutants in the second round of screening shows that F457K is the most outstanding candidate mutation in this round. This mutation significantly improves the target editing efficiency of FANCF in the MRT3 (N63R-N234R) background while maintaining high target product purity.

[0168] Example 8: Validation of MRT3 (N63R-N234R) in a stable rice transformation system To further evaluate the editing performance of the two optimized reverse transcriptases in a stable rice transformation system, this embodiment uses ePPEmax as the backbone to construct guide editing vectors containing MarathonRT-5m and MRT3 (N63R-N234R), respectively, covering the OsEhd1 and OsSPL14 targets. Stable transformation of rice was carried out using Agrobacterium-mediated transformation, and positive transformed plants were identified by PCR.

[0169] After identifying the transgenic plants as positive, Hi-TOM sequencing was used to perform in-depth analysis of the target sites in the positive plants. The results showed that a certain proportion of low-frequency target editing signals were detected at both target sites, but the overall editing efficiency was low, with most T0 plants showing no target editing. To avoid overestimating the overall system efficiency by only counting a few edited plants, this embodiment simultaneously calculated the proportion of plants with target editing and the editing efficiency of individual plants. The proportion of plants with target editing was calculated as the ratio of the number of plants with target editing to the number of plants detected by Hi-TOM; the editing efficiency of individual plants was calculated as the ratio of the number of target-edited reads to the number of valid reads in that sample; plants without detected target editing were included as 0% in the average editing efficiency statistics of all detected plants.

[0170] In the OsEhd1 target group, a total of 56 transgenic positive plants were detected in the MarathonRT-5m group, of which 12 plants showed targeted editing, representing 21.43% of the total. The average editing efficiency of the plants with targeted editing was 0.59%, and the average editing efficiency of all detected plants was 0.13%. In the MRT3 (N63R-N234R) group, a total of 80 transgenic positive plants were detected, of which 9 plants showed targeted editing, representing 11.25% of the total. The average editing efficiency of the plants with targeted editing was 0.61%, and the average editing efficiency of all detected plants was 0.07% (Table 4).

[0171] In the OsSPL14 target site, the MarathonRT-5m group detected 44 transgenic positive plants, of which 11 showed targeted editing, representing 25.00% of the plants with targeted editing. The average editing efficiency of the plants with targeted editing was 12.91%, and the average editing efficiency of all detected plants was 3.23%. The MRT3 (N63R-N234R) group detected 79 transgenic positive plants, of which 34 showed targeted editing, representing 43.04% of the plants with targeted editing. The average editing efficiency of the plants with targeted editing was 6.70%, and the average editing efficiency of all detected plants was 2.88%. Compared with MarathonRT-5m, MRT3 (N63R-N234R) increased the proportion of plants with targeted editing in the OsSPL14 target site, but did not increase the average editing efficiency of the plants with targeted editing or the average editing efficiency of all detected plants (Table 4).

[0172] Table 4. Positive identification of OsEhd1 and OsSPL14 targets and Hi-TOM editing results in the stable transformation system of rice. Note: The number of positive plants refers to the number of T0 generation plants confirmed by PCR to carry PE editing elements; the number of plants containing target edits refers to the number of plants in which target edit reads were detected in Hi-TOM sequencing. The average editing efficiency of plants containing target edits only includes plants in which target edits were detected; the average editing efficiency of all tested plants includes plants in which no target edits were detected as 0%. The editing efficiency of a single plant is calculated as the proportion of target edit reads to the total number of valid reads in that sample.

[0173] sequence All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A reverse transcriptase, characterized in that, The amino acid sequence of the reverse transcriptase is based on SEQ ID NO:1, but contains mutations selected from the group consisting of only those shown in SEQ ID NO:1: F457K, N234R, and N63R; T361R, N234R and N63R; Q59R, N234R, and N63R; L58K, N234R, and N63R; Q358R, N234R and N63R; Q55R, N234R, and N63R; V105K, N234R, and N63R; N234R and N63R; or N234R.

2. The reverse transcriptase as described in claim 1, characterized in that, The amino acid sequence of the reverse transcriptase is based on SEQ ID NO:1, but contains mutations selected from the group consisting of only those shown in SEQ ID NO:1: L103K, N234R, and N63R; N365R, N234R and N63R; E106Q, N234R and N63R; V61K, N234R, and N63R; I107K, N234R, and N63R; Q129R, N234R, and N63R; A66K, N234R, and N63R; L359K, N234R, and N63R; S155R, N234R and N63R; N111R, N234R, and N63R; or N111R and N234R.

3. A fusion protein, characterized in that, The fusion protein comprises the reverse transcriptase and Cas protein as described in claim 1; the Cas protein is Cas9 or a variant thereof; the amino acid sequence of the Cas9 is the sequence shown in SEQ ID NO:4; the Cas9 variant has only the following mutations relative to the amino acid sequence shown in SEQ ID NO:4: R221K, N394K and H840A.

4. A CRISPR-Cas composition, characterized in that, The CRISPR-Cas composition comprises: (1) A guide RNA, or one or more DNA molecules encoding the guide RNA; the guide RNA is an epegRNA; and (2) The fusion protein of claim 3, or the nucleic acid molecule encoding the fusion protein of claim 3.

5. A polynucleotide, characterized in that, The polynucleotide encodes the reverse transcriptase of claim 1 or the fusion protein of claim 3.

6. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 5.

7. A delivery system, characterized in that, The delivery system includes the reverse transcriptase of claim 1, or the fusion protein of claim 3, or the CRISPR-Cas composition of claim 4, or the polynucleotide of claim 5, or the vector of claim 6, and a delivery medium.

8. A host cell, characterized in that, The host cell expresses the fusion protein of claim 3, or has its genome integrated with the polynucleotide of claim 5, or contains the vector of claim 6.

9. A reagent kit, characterized in that, The kit comprises the reverse transcriptase of claim 1, or the fusion protein of claim 3, or the CRISPR-Cas composition of claim 4, or the host cell of claim 8.

10. Use of the reverse transcriptase of claim 1, or the fusion protein of claim 3, or the CRISPR-Cas composition of claim 4, or the polynucleotide of claim 5, or the vector of claim 6, or the delivery system of claim 7, or the host cell of claim 8, characterized in that, Prepare formulations for use in guiding editing.

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