Circular RNA-based prime editor

Abstract

The present invention relates to a circular RNA-based prime editor for realizing site-directed and accurate genome editing. Specifically, in the circular RNA-based prime editor, a nuclease other than Cas9 or a single nickase is used to couple with a reverse transcriptase, and accurate base insertion, deletion and replacement on a target sequence in a biological genome is realized on the basis of the circular RNA. According to the method, genetically modified but not transgenic organisms and the progeny thereof are generated.

Description

Circular RNA-based guide editing system Technical Field

[0001] The present invention relates to the field of genetic engineering. Specifically, the present invention relates to a circular RNA-based guide editing system for achieving site-specific, precise genome editing. More specifically, the circular RNA-based guide editing system of the present invention utilizes nucleases other than Cas9 or single-nickases coupled to reverse transcriptase, and achieves precise base insertion, deletion, and substitution of target sequences in the genome of an organism based on circular RNA, as well as genetically modified but non-transgenic organisms and their offspring produced by the method.

[0002] Background of the Invention

[0003] With the rapid development of biology, targeted, precise editing of organismal genomes has become an urgent need in medicine, agriculture, and scientific research. Precision genome editing technology can treat a large number of genetic diseases in medicine, create excellent agricultural traits in agriculture, and promote the development of biology. Currently, there are three main types of commonly used precision base editing systems: cytosine base editors (CBEs), adenine base editors (ABEs), and prime editors (PEs). Among them, cytosine base editors can convert cytosine (C) to uracil (U) at the target site; adenine base editors can convert adenine (A) to hypoxanthine (I); and prime editors can achieve various single-base conversions and transversions, as well as insertions, deletions, and replacements of fragments. As can be seen, prime editing is more powerful. However, the prime editors currently used are only reverse transcriptases coupled to the Cas9 protein, which is limited by the Cas9 protein in many aspects, such as target sequence selection and biological delivery. The Cas12 series of proteins discovered later: Cas12a (i.e., Cpf1), Cas12b (i.e., C2c1), Cas12c, Cas12d (i.e., CasY), Cas12e (i.e., CasX), Cas12f (i.e., Cas14), Cas12h, Cas12i, Cas12j (i.e., CasΦ), Cas12k, Cas12m, and smaller proteins such as IsrB, IscB, and TnpB, have structural characteristics different from Cas9 and each has unique functional advantages. For example, Cpf1 has the advantages of high efficiency and low off-target rate; Cas12f, IsrB, IscB, and TnpB proteins have the advantages of small sequences and easy delivery. Most of these protein-fused base deaminases have good applications in cytosine base editors and adenine base editors. However, to date, these large numbers of newly discovered proteins have not been applied to the more functional guide editors. Therefore, the development of new guide editing systems that do not depend on the Cas9 protein using proteins such as Cas12a (i.e., Cpf1), Cas12b (i.e., C2c1), Cas12c, Cas12d (i.e., CasY), Cas12e (i.e., CasX), Cas12f (i.e., Cas14), Cas12h, Cas12i, Cas12j (i.e., CasΦ), Cas12k, Cas12m, IsrB, IscB and TnpB will overcome the limitations of the Cas9 protein, effectively utilize the advantages of other proteins, better promote the application of guide editors, and play a very important role in disease treatment, agricultural trait improvement and biological research.

[0004] Summary of the Invention

[0005] The present invention develops a novel guide editing system (CPE, Circular RNA-dependent Prime Editing system, Figure 1) that depends on circular RNA. According to the type of protein used in the guide editing, CPE is divided into a guide editing system (niCPE, Circular RNA-dependent Prime Editing system with nickase, Figure 1a) that utilizes a non-targeted chain single nickase and depends on circular RNA and a guide editing system (nuCPE, Circular RNA-dependent Prime Editing system with nuclease, Figure 1b) that utilizes a nuclease and depends on circular RNA. At the same time, for the convenience of delivery, we have developed detachable sniCPE (split Circular RNA-dependent Prime Editing system with nickase, Figure 1c) and snuCPE (split Circular RNA-dependent Prime Editing system with nuclease) guide editing systems. In addition, we also found that multiple RNAs can be expressed in series, and on this basis, we developed an efficient one-CPE (All RNA under one U6 promoter CPE) guide editing system. More importantly, we have used the Cas12a-CPE system to achieve multi-gene editing in HEK293T human cell lines. The CPE-guided editing system will further enable precise genome editing of single-base transitions and transversions, fragment insertions, deletions, and replacements in animals, bacteria, dicots, and monocots.

[0006] BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1: Schematic diagram of different guide editing systems.

[0008] Figure 2: Editing efficiency of the copGFP reporter system for different guide editing systems.

[0009] Figure 3: Comparison of guide editing efficiency using pegRNA and circular RNA.

[0010] Figure 4: Editing efficiency of the Cas12a-niCPE-guided editing system at various target sites in human 293T cells using different editing methods.

[0011] Figure 5: Editing efficiency of the Cas12a-nuCPE-guided editing system in human 293T cells.

[0012] Figure 6: Editing efficiency of the Cas12a-sniCPE-guided editing system in human 293T cells.

[0013] Figure 7: Editing efficiency of the Cas12a-snuCPE-guided editing system in human 293T cells.

[0014] Figure 8: Comparison of editing efficiency of different guided editing systems.

[0015] Figure 9: One-CPE boot editing system.

[0016] Figure 10: Multi-gene guided editing system.

[0017] Figure 11: Off-Target Detection of CPE Using a CircRNA-Guided Editing System.

[0018] Figure 12: Editing efficiency of Cas12i-nuCPE and Cas12i-niCPE-guided editing systems in human 293T cells.

[0019] Detailed Description of the Invention

[0020] 1. Definition

[0021] In the present invention, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and routine procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following literature: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook"). At the same time, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0022] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0023] When the word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0024] "Genome" as used herein encompasses not only the chromosomal DNA present in the nucleus of a cell, but also the organelle DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0025] As used herein, "organism" includes any organism suitable for genome editing, preferably a eukaryotic organism. Examples of organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, and cats; poultry such as chickens, ducks, and geese; and plants, including monocots and dicots, such as rice, corn, wheat, sorghum, barley, soybeans, peanuts, and Arabidopsis.

[0026] "Genetically modified organisms" refers to organisms or plants that contain exogenous polynucleotides or modified genes or expression control sequences within their genome. For example, the exogenous polynucleotides are capable of stably integrating into the genome of the organism and being inherited through successive generations. The exogenous polynucleotides can be integrated into the genome alone or as part of a recombinant DNA construct. Modified genes or expression control sequences are those that contain one or more nucleotide substitutions, deletions, or additions within the plant genome.

[0027] "Exogenous" with respect to a sequence refers to a sequence that is from a foreign species, or, if from the same species, has been significantly altered from its native form in composition and / or locus through deliberate human intervention.

[0028] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded polymers of RNA or DNA that optionally contain synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single-letter designations as follows: "A" for adenosine or deoxyadenosine (RNA or DNA, respectively), "C" for cytidine or deoxycytidine, "G" for guanosine or deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "D" for A, T, or G, "I" for inosine, and "N" for any nucleotide. Although nucleotide sequences herein may be presented as DNA sequences (including T), when reference is made to RNA, one skilled in the art can readily determine the corresponding RNA sequence (i.e., replacing T with U).

[0029] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" may also include modified forms including, but not limited to, glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0030] As used herein, an "expression construct" refers to a vector, such as a recombinant vector, suitable for expressing a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to produce mRNA or functional RNA) and / or translation of RNA into a precursor or mature protein.

[0031] The "expression construct" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be a translatable RNA (e.g., mRNA), for example, RNA generated by in vitro transcription. For use in mammals such as humans, the expression construct is preferably a viral vector, such as an adeno-associated virus (AAV), a lentivirus, or an adenoviral vector, with an AAV vector being most preferred.

[0032] An "expression construct" of the present invention may comprise regulatory sequences and a nucleotide sequence of interest from different sources, or regulatory sequences and a nucleotide sequence of interest from the same source but arranged in a manner different from that normally found in nature.

[0033] "Promoter" refers to a nucleic acid fragment that is capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the present invention, a promoter is a promoter that is capable of controlling the transcription of a gene in a cell, whether or not derived from the cell. A promoter can be a constitutive promoter, a tissue-specific promoter, a developmentally regulated promoter, or an inducible promoter.

[0034] Examples of promoters include, but are not limited to, polymerase (pol) I, pol II, or pol III promoters. Examples of pol I promoters include the chicken RNA pol I promoter. Examples of pol II promoters include, but are not limited to, the cytomegalovirus immediate early (CMV) promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, and the simian virus 40 (SV40) immediate early promoter. Examples of pol III promoters include the U6, U3, and H1 promoters. Inducible promoters such as metallothionein promoters can be used. Other examples of promoters include the T7 phage promoter, the T3 phage promoter, the β-galactosidase promoter, and the Sp6 phage promoter. When used in plants, the promoter can be the cauliflower mosaic virus 35S promoter, the maize Ubi-1 promoter, the wheat U6 promoter, the rice U3 promoter, the maize U3 promoter, or the rice actin promoter.

[0035] "Introducing" a nucleic acid molecule (e.g., a plasmid, a linear nucleic acid fragment, RNA, etc.) or a protein into an organism refers to transforming the cells of the organism with the nucleic acid or protein so that the nucleic acid or protein can function in the cell. "Transformation" as used in the present invention includes stable transformation and transient transformation. "Stable transformation" refers to the introduction of an exogenous nucleotide sequence into the genome, resulting in the stable inheritance of the exogenous gene. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof. "Transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell to perform a function without the stable inheritance of the exogenous gene. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.

[0036] "Trait" refers to a physiological, morphological, biochemical, or physical characteristic of a cell or organism.

[0037] 2. Boot Editing System

[0038] In one aspect, the present invention provides a guide editing system for targeted modification of a genomic DNA sequence in an organism, comprising:

[0039] i) a) a CRISPR nuclease and / or an expression construct comprising a nucleotide sequence encoding said CRISPR nuclease, and a reverse transcriptase and / or an expression construct comprising a nucleotide sequence encoding said reverse transcriptase, or

[0040] b) a guide editing fusion protein and / or an expression construct containing a nucleotide sequence encoding the guide editing fusion protein, wherein the guide editing fusion protein comprises a CRISPR nuclease and a reverse transcriptase;

[0041] ii) an editing guide RNA (gRNA) directed against a target sequence in genomic DNA and / or an expression construct containing a nucleotide sequence encoding the editing gRNA; and

[0042] iii) a circular RNA comprising a reverse transcription template (RTT) sequence and a primer binding site (PBS) sequence and / or an expression construct comprising a nucleotide sequence encoding said circular RNA,

[0043] Preferably, the CRISPR nuclease is not a Cas9 nuclease or a variant thereof.

[0044] As used herein, "guide editing system" refers to a combination of components required for reverse transcription-based genome editing of the genome in a cell. The various components of the system, such as CRISPR nuclease, reverse transcriptase, guide editing fusion protein, gRNA, circular RNA or their expression constructs, etc., can exist independently of each other or can exist in the form of a composition in any combination. The various components of the system can be packaged independently or together into viruses, virus-like particles, virions, liposomes, vesicles, exosomes, liposomal nanoparticles (LNPs), etc.

[0045] The CRISPR nuclease of the present invention may be a CRISPR nuclease other than a Cas9 nuclease or a variant thereof. Examples of CRISPR nucleases suitable for the present invention include, but are not limited to, Cas12a (i.e., Cpf1), Cas12b (i.e., C2c1), Cas12c, Cas12d (i.e., CasY), Cas12e (i.e., CasX), Cas12f (i.e., Cas14), Cas12h, Cas12i, Cas12j (i.e., CasΦ), Cas12k, Cas12m, IsrB, IscB, and TnpB. In some specific embodiments, the CRISPR nuclease is Cas12a. In other specific embodiments, the CRISPR nuclease is Cas12i.

[0046] In some embodiments, the CRISPR nuclease also includes a nickase form thereof. The nickase form of the CRISPR nuclease may also be referred to herein as a CRISPR nickase, which forms a nick in only one strand of a double-stranded nucleic acid molecule, but does not completely cut the double-stranded nucleic acid. In some embodiments, the CRISPR nickase forms a nick only in the complementary strand of the strand where the target sequence is located in the double-stranded nucleic acid molecule (also referred to in the art as the non-targeting strand).

[0047] As used herein, “target sequence” refers to a sequence of about 20 nucleotides in length characterized by a PAM (protospacer adjacent motif) sequence flanking the 5’ or 3’ flank in the genome. Generally speaking, PAM is necessary for the complex formed by the CRISPR nuclease or its variants and the guide RNA to recognize the target sequence. For example, for the Cas12a nuclease and its variants, its target sequence is adjacent to the PAM at the 3’ end, such as 5’-TTTV-3’. Based on the presence of PAM, those skilled in the art can easily determine the target sequence that can be used for targeting in the genome. And depending on the position of the PAM, the target sequence can be located on either strand of the genomic DNA molecule, the strand to which crRNA binds is called the targeting strand, and the strand complementary to the targeting strand is called the non-targeting strand. For Cas12a or its derivatives, such as the Cas12a nickase, the target sequence is preferably 20 to 23 nucleotides. Depending on different CRISPR nucleases or their different variants, the PAM sequence will be different.

[0048] In some specific embodiments, the CRISPR nuclease is Cas12a, which comprises the amino acid sequence of SEQ ID NO: 1 or 2. In some preferred embodiments, the CRISPR nuclease is a Cas12a nickase, which has a substitution at amino acid position 1138 relative to SEQ ID NO: 1 or 2, e.g., amino acid R at position 1138 is substituted with A. In some preferred embodiments, the CRISPR nuclease comprises the amino acid sequence of SEQ ID NO: 3.

[0049] In some specific embodiments, the CRISPR nuclease is Cas12i, which comprises the amino acid sequence shown in SEQ ID NO: 4. In some preferred embodiments, the CRISPR nuclease is a Cas12i nickase, which has a substitution at position 854 relative to SEQ ID NO: 4, for example, the amino acid W at position 854 is substituted with A. In some preferred embodiments, the Cas12i nickase comprises the amino acid sequence shown in SEQ ID NO: 5.

[0050] The reverse transcriptases of the present invention can be derived from various sources. In some embodiments, the reverse transcriptase is derived from a virus. For example, in some embodiments, the reverse transcriptase is M-MLV reverse transcriptase. An exemplary wild-type M-MLV reverse transcriptase sequence is shown as the amino acid sequence in SEQ ID NO:6.

[0051] In some embodiments, the reverse transcriptase, such as M-MLV reverse transcriptase, has its RNase H domain mutated or deleted. In some embodiments, the M-MLV reverse transcriptase with a mutated or deleted RNase H domain comprises the amino acid sequence shown in SEQ ID NO:7.

[0052] In some embodiments, the reverse transcriptase and / or the guide editing fusion protein of the present invention may further comprise one or more RNA aptamer binding protein sequences. In some embodiments, the reverse transcriptase of the present invention further comprises one or more RNA aptamer binding protein sequences. In some embodiments, the guide editing fusion protein of the present invention further comprises one or more RNA aptamer binding protein sequences.

[0053] "RNA aptamer-binding proteins" refer to proteins that can specifically bind to "RNA aptamers" with specific sequences and structures. Proteins containing one or more RNA aptamer-binding protein sequences can thus be recruited intracellularly to nucleic acid molecules containing corresponding RNA aptamers, and vice versa. Many such combinations of "RNA aptamer-binding proteins" and "RNA aptamers" capable of specific binding are known in the art.

[0054] In some embodiments, the RNA aptamer binding protein is an MCP protein, and the corresponding RNA aptamer is MS2. The MCP protein can specifically bind to MS2 or multiple MS2s in tandem. An exemplary MCP comprises the amino acid sequence set forth in SEQ ID NO: 8. An exemplary MS2 comprises the nucleotide sequence set forth in SEQ ID NO: 17.

[0055] As used herein, a "linker" can be a non-functional amino acid sequence of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 20-25, 25-50) or more amino acids in length, without secondary or higher structure. For example, the linker can be a flexible linker. In some specific embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO: 9.

[0056] In some embodiments of the present invention, the CRISPR nuclease, reverse transcriptase, or guide editing fusion protein of the present invention may further comprise one or more nuclear localization sequences (NLS). In general, the one or more NLSs in the CRISPR nuclease, reverse transcriptase, or guide editing fusion protein should have sufficient strength to drive the CRISPR nuclease, reverse transcriptase, or guide editing fusion protein to accumulate in the nucleus of the cell in an amount that can achieve its gene editing function. In general, the intensity of nuclear localization activity is determined by the number, position, one or more specific NLSs used, or a combination of these factors in the CRISPR nuclease, reverse transcriptase, or guide editing fusion protein. An exemplary NLS may comprise the amino acid sequence shown in SEQ ID NO: 10 or 11.

[0057] However, depending on the DNA location to be edited, the CRISPR nuclease, reverse transcriptase, or guide editing fusion protein of the present invention may also include other localization sequences, such as cytoplasmic localization sequences, chloroplast localization sequences, mitochondrial localization sequences, etc.

[0058] In some embodiments, the guide editing fusion protein comprises an amino acid sequence selected from one of SEQ ID NOs: 12-15, preferably comprises the amino acid sequence of SEQ ID NO: 12.

[0059] In some embodiments, the guided editing system includes:

[0060] i) a guide editing fusion protein and / or an expression construct comprising a nucleotide sequence encoding the guide editing fusion protein, wherein the guide editing fusion protein comprises an amino acid sequence selected from one of SEQ ID NOs: 12-15, preferably comprises the amino acid sequence of SEQ ID NO: 12;

[0061] ii) an editing guide RNA (gRNA) and / or an expression construct containing a nucleotide sequence encoding the editing gRNA; and

[0062] iii) a circular RNA comprising a reverse transcription template (RTT) sequence and a primer binding site (PBS) sequence and / or an expression construct comprising a nucleotide sequence encoding the circular RNA.

[0063] In some embodiments, the guided editing system includes:

[0064] i) a CRISPR nuclease and / or an expression construct comprising a nucleotide sequence encoding said CRISPR nuclease, and a reverse transcriptase and / or an expression construct comprising a nucleotide sequence encoding said reverse transcriptase;

[0065] ii) an editing guide RNA (gRNA) and / or an expression construct containing a nucleotide sequence encoding the editing gRNA; and

[0066] iii) a circular RNA comprising a reverse transcription template (RTT) sequence and a primer binding site (PBS) sequence and / or an expression construct comprising a nucleotide sequence encoding said circular RNA,

[0067] wherein the CRISPR nuclease comprises an amino acid sequence selected from one of SEQ ID NOs: 1-5, preferably an amino acid sequence of SEQ ID NOs: 3 or 5,

[0068] The reverse transcriptase comprises the amino acid sequence shown in SEQ ID NO: 16.

[0069] In order to obtain effective expression in different organisms, in the present invention, the nucleotide sequence encoding the CRISPR nuclease, reverse transcriptase, or guide editing fusion protein can be codon-optimized for the organism species whose genome is to be modified.

[0070] Codon optimization refers to a process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with codons that are more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which is believed to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons that are most frequently used for peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp / codon / , and these tables can be adapted in various ways. See, Nakamura et al., 2002, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044 Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0071] The guide RNA (gRNA) described in the present invention refers to a gRNA compatible with the CRISPR nuclease. gRNAs may generally include a scaffold sequence and a guide sequence (also known as a seed sequence or spacer sequence). The guide sequence is configured to have sufficient sequence identity (preferably 100% identity) with the target sequence so that it can bind to the complementary strand of the target sequence through base pairing to achieve sequence-specific targeting. The scaffold sequence generally depends on the CRISPR nuclease used. For example, for Cas12a such as LbCas12a, the scaffold sequence of its gRNA may include the nucleotide sequence shown in SEQ ID NO: 23. And for Cas12i, the scaffold sequence of its gRNA may include the nucleotide sequence shown in SEQ ID NO: 24. CRISPR nuclease can bind to the scaffold sequence of gRNA and form a CRISPR nuclease-gRNA complex, and under the action of the guide sequence, target to the genomic target sequence, and exert its nuclease or nickase function. Those skilled in the art can design suitable gRNAs for different CRISPR nucleases and different target sequences. The guide RNA targeting the target sequence to be edited is referred to as editing guide RNA (editing gRNA) in the present invention.

[0072] The "circular RNA" of the present invention contains a directly connected reverse transcription template (RTT) sequence and a primer binding site (PBS) sequence (RTT-PBS sequence), wherein the RTT sequence is located at the 5' end of the PBS sequence.

[0073] In some embodiments, the circular RNA further comprises one or more (e.g., 1 or 2 or 3 or more) RNA aptamer sequences, such as an MS2 sequence. The one or more RNA aptamer sequences in the circular RNA can be used to recruit a reverse transcriptase containing an RNA aptamer binding protein in a cell, or to recruit the circular RNA to a CRISPR nuclease-gRNA-genomic DNA complex in a cell.

[0074] In some embodiments, the primer binding site sequence is configured to be complementary to at least a portion of the target sequence (preferably completely paired with at least a portion of the target sequence). Preferably, the primer binding site sequence is complementary to at least a portion of the 3' free single strand caused by the nick in the DNA chain where the target sequence is located (preferably completely paired with at least a portion of the 3' free single strand), in particular, complementary to the nucleotide sequence at the 3' end of the 3' free single strand (preferably completely paired). When the 3' free single strand of the chain binds to the primer binding sequence through base pairing, the 3' free single strand can serve as a primer, using the reverse transcription template sequence adjacent to the primer binding site sequence as a template, and reverse transcription is performed under the action of the reverse transcriptase in the guide editing fusion protein or the reverse transcriptase recruited by the RNA aptamer binding protein, thereby extending a DNA sequence corresponding to the reverse transcription template (RTT) sequence.

[0075] The primer binding site sequence depends on the length of the free single strand formed by the CRISPR nuclease used in the target sequence, however, it should have a minimum length to ensure specific binding. In some embodiments, the primer binding site sequence length can be 4-20 nucleotides, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides in length.

[0076] The RTT sequence can be any sequence. Through the above-mentioned reverse transcription, its sequence information can be integrated into the DNA chain where the target sequence is located (that is, the chain containing the target sequence PAM), and then through the DNA repair function of the cell, a DNA double-stranded chain containing the RTT sequence information is formed. In some embodiments, the RTT sequence contains a desired modification. For example, the desired modification includes substitution, deletion and / or addition of one or more nucleotides. In some embodiments, the RTT sequence is set to a sequence corresponding to the downstream of the target sequence nick (for example, complementary to at least a portion of the sequence downstream of the target sequence nick), but contains a desired modification. The desired modification includes substitution, deletion and / or addition of one or more nucleotides.

[0077] In some embodiments, the guide editing system further comprises a nicking gRNA (nicking gRNA, for generating additional nicks) and / or an expression construct containing a nucleotide sequence encoding the nicking gRNA, wherein the nicking gRNA comprises a guide sequence and a scaffold sequence. In some preferred embodiments, the nicking gRNA does not comprise a reverse transcription (RT) template sequence and a primer binding site (PBS) sequence.

[0078] The guide sequence (also known as the seed sequence or spacer sequence) in the nicking gRNA of the present invention is set to have sufficient sequence identity (preferably 100% identity) with the nicking target sequence in the genome, so that the fusion protein of the present invention can be targeted to the nicking target sequence and cause a nick within the nicking target sequence, and the nicking target sequence and the target sequence targeted by the editing gRNA (the target sequence to be edited) are located on opposite strands of the genomic DNA. In some embodiments, the nick formed by the nicking RNA and the nick formed by the editing gRNA are about 1 to about 300 or more nucleotides apart, for example, 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300 nucleotides or more nucleotides apart. In some embodiments, the nick formed by the nicking RNA is located upstream or downstream of the editing gRNA (the upstream or downstream refers to the DNA chain where the editing gRNA target sequence is located). In some embodiments, the leader sequence in the nicking gRNA has sufficient sequence identity (preferably 100% identity) with the opposite chain (modified) of the editing gRNA target sequence after the editing event occurs, so that the nicking gRNA only targets the nicking target sequence generated after the editing gRNA-induced target sequence targeting and modification is completed. In some embodiments, the PAM of the nicking target sequence is located within the complementary sequence of the editing gRNA target sequence.

[0079] In some embodiments, the guide editing system comprises multiple (e.g., 2, 3, 4, 5, or more) editing gRNAs and / or an expression construct containing a nucleotide sequence encoding the multiple editing gRNAs.

[0080] Accordingly, in some embodiments, the circular RNA comprises multiple groups (e.g., 2, 3, 4, 5 or more) of RTT-PBS sequences corresponding to the multiple editing gRNAs in series, each group of RTT-PBS sequences comprising a directly connected reverse transcription template (RTT) sequence and a primer binding site (PBS) sequence, and the RTT sequence is located at the 5' end of the PBS sequence. In some embodiments, the multiple editing gRNAs target different target sequences, and the corresponding multiple groups of RTT-PBS sequences are used to introduce different modifications, thereby enabling the guide editing system to target and modify different target sequences.

[0081] The "expression construct comprising a nucleotide sequence encoding the circular RNA" of the present invention may further comprise elements required for RNA cyclization. Such elements may be described in Jacob L. Litke et al., Nature Biotechnology, Vol 37, June 2019, 667–675, or Wesselhoeft AR et al., (2018) Engineering circular RNA for potent and stable translation in eukaryotic cells. Other elements known in the art that can achieve RNA cyclization may also be used in the present invention.

[0082] In some embodiments, the "expression construct containing a nucleotide sequence encoding the circular RNA" includes the following coding sequence: 5'-first ribozyme-first looping arm-RTT-PBS-second looping arm-second ribozyme-3'. After being transcribed into RNA within the cell, the first and second ribozymes can self-cleave to produce "5'-first looping arm-RTT-PBS-second looping arm-3'," and the first and second looping arms can ligate to form a circular RNA comprising RTT and PBS. In some embodiments, the RTT-PBS can also be flanked by one or more RNA aptamer sequences, such as MS2 sequences.

[0083] In some embodiments, the first ribozyme comprises the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the second ribozyme comprises the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the first loop-forming arm comprises the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the second loop-forming arm comprises the nucleotide sequence of SEQ ID NO: 22.

[0084] In some embodiments, the editing gRNA and the circular RNA and the optional nicking gRNA are encoded by the same expression construct. In some embodiments, the expression construct comprises (for example, from 5' to 3' direction) a nucleotide sequence encoding the circular RNA, a nucleotide sequence encoding the editing gRNA, and an optional nucleotide sequence encoding the nicking gRNA. In some embodiments, the nucleotide sequence encoding the circular RNA, the nucleotide sequence encoding the editing gRNA, and the optional nucleotide sequence encoding the nicking gRNA are driven to express (transcribe) by the same promoter such as the U6 promoter. Due to the RNA cleavage activity of the CRISPR nuclease / nickase for crRNA maturation, the RNA molecules driven to transcribe by the same promoter can produce respective circular RNAs, editing gRNAs, and optional nicking gRNAs.

[0085] On the other hand, the present invention provides a Cas12a nickase and / or an expression construct comprising its coding sequence, wherein relative to SEQ ID NO: 1 or 2, the amino acid at position 1138 is substituted, for example, the amino acid R at position 1138 is substituted by A. In some preferred embodiments, the Cas12a nickase comprises the amino acid sequence shown in SEQ ID NO: 3.

[0086] On the other hand, the present invention provides a Cas12i nickase and / or an expression construct comprising its coding sequence, wherein relative to SEQ ID NO: 4, the amino acid at position 854 is substituted, for example, the amino acid W at position 854 is substituted by A. In some preferred embodiments, the Cas12i nickase comprises the amino acid sequence shown in SEQ ID NO: 5.

[0087] On the other hand, the present invention provides the use of the Cas12a nickase or Cas12i nickase of the present invention, or an expression construct comprising its coding sequence in gene editing, such as guide editing.

[0088] 3. Methods for Modifying Target Sequences in Cell Genomes

[0089] In another aspect, the present invention provides a method for producing a genetically modified cell, comprising introducing a guide editing system of the present invention into at least one cell, thereby causing modification of the genomic sequence of the at least one cell, wherein the modification comprises substitution, deletion, and / or addition of one or more nucleotides. For example, the modification comprises one or more substitutions selected from the group consisting of C to T substitutions, C to G substitutions, C to A substitutions, G to T substitutions, G to C substitutions, G to A substitutions, A to T substitutions, A to G substitutions, A to C substitutions, T to C substitutions, T to G substitutions, T to A substitutions; and / or comprises a deletion of one or more nucleotides, e.g., 1 to about 100 or more, e.g., 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide deletions; and / or comprises an insertion of one or more nucleotides, e.g., 1 to about 100 or more, e.g., 1 to about 100 or more, e.g., 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide insertions. The modification can be located in the target sequence of the gRNA or near the target sequence, for example, downstream of the target sequence.

[0090] In another aspect, the present invention also provides a method for producing a genetically modified cell, comprising introducing the guide editing system of the present invention into the cell.

[0091] In another aspect, the present invention also provides a genetically modified organism comprising a genetically modified cell produced by the method of the present invention or a progeny cell thereof.

[0092] In the present invention, the modification can be located anywhere in the genome, for example, within a functional gene such as a protein-coding gene, or within a gene expression regulatory region such as a promoter region or an enhancer region, thereby modifying the gene function or gene expression. The modification in the cell genome sequence can be detected by T7EI, PCR / RE, or sequencing.

[0093] In the method of the present invention, the guide editing system can be introduced into cells by various methods well known to those skilled in the art.

[0094] Methods that can be used to introduce the guide editing system of the present invention into cells include, but are not limited to, calcium phosphate transfection, protoplast fusion, electroporation, lipofection, microinjection, viral infection (such as baculovirus, vaccinia virus, adenovirus, adeno-associated virus, lentivirus and other viruses), gene guns, PEG-mediated protoplast transformation, and Agrobacterium-mediated transformation.

[0095] Cells that can be gene-edited using the methods of the present invention can be derived from, for example, prokaryotes such as bacteria; eukaryotes including mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, and cats; poultry such as chickens, ducks, and geese; and plants, including monocots and dicots such as rice, corn, wheat, sorghum, barley, soybeans, peanuts, and Arabidopsis. In some preferred embodiments, the cells are derived from humans.

[0096] In some embodiments, the methods of the present invention are performed in vitro. For example, the cells are isolated cells, or cells in isolated tissues or organs.

[0097] In other embodiments, the method of the present invention can also be performed in vivo. For example, the cell is a cell in an organism, and the system of the present invention can be introduced into the cell in vivo by, for example, a virus- or Agrobacterium-mediated method.

[0098] IV. Methods for Producing Genetically Modified Plants

[0099] In another aspect, the present invention provides a method for producing a genetically modified plant, comprising introducing a guide editing system of the present invention into at least one plant, thereby causing a modification in the genome of the at least one plant. The modification comprises a substitution, deletion, and / or addition of one or more nucleotides. For example, the modification comprises one or more substitutions selected from the group consisting of C to T substitutions, C to G substitutions, C to A substitutions, G to T substitutions, G to C substitutions, G to A substitutions, A to T substitutions, A to G substitutions, A to C substitutions, T to C substitutions, T to G substitutions, T to A substitutions; and / or comprises a deletion of one or more nucleotides, e.g., 1 to about 100 or more, e.g., 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide deletions; and / or comprises an insertion of one or more nucleotides, e.g., 1 to about 100 or more, e.g., 1 to about 100 or more, e.g., 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide insertions.

[0100] In some embodiments, the method further comprises screening the at least one plant for plants having the desired modification.

[0101] In the methods of the present invention, the guide editing system can be introduced into plants using various methods well known to those skilled in the art. Methods for introducing the guide editing system of the present invention into plants include, but are not limited to, biolistic methods, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube passage, and ovary injection. Preferably, the guide editing system is introduced into plants via transient transformation.

[0102] In the methods of the present invention, genome modification can be achieved simply by introducing or producing relevant proteins and RNA into plant cells, and the modifications are stably inherited, without the need for stably transforming plants with exogenous polynucleotides encoding the components of the directing editing system. This avoids potential off-target effects of a stably existing (continuously produced) editing system and also prevents integration of exogenous nucleotide sequences into the plant genome, thereby providing greater biosafety.

[0103] In some preferred embodiments, the introduction is performed in the absence of selection pressure, thereby avoiding integration of the exogenous nucleotide sequence into the plant genome.

[0104] In some embodiments, the introduction comprises transforming the guide editing system of the present invention into isolated plant cells or tissues, and then regenerating the transformed plant cells or tissues into whole plants. Preferably, the regeneration is performed in the absence of selective pressure, that is, no selective agent targeting the selection gene carried on the expression vector is used during the tissue culture process. The absence of a selective agent can improve the efficiency of plant regeneration and obtain modified plants that are free of exogenous nucleotide sequences.

[0105] In other embodiments, the guided editing system of the present invention can be transformed into specific parts of intact plants, such as leaves, stem tips, pollen tubes, young ears, or hypocotyls. This is particularly suitable for the transformation of plants that are difficult to regenerate through tissue culture.

[0106] In some embodiments of the present invention, in vitro expressed proteins and / or in vitro transcribed RNA molecules (e.g., the expression construct is an in vitro transcribed RNA molecule) are directly transformed into the plant. The protein and / or RNA molecule can be guided in plant cells and subsequently degraded by the cells, thereby avoiding integration of the exogenous nucleotide sequence into the plant genome.

[0107] Therefore, in some embodiments, genetic modification and breeding of plants using the methods of the present invention can obtain plants whose genomes have no exogenous polynucleotide integration, ie, non-transgenic (transgene-free) modified plants.

[0108] In some embodiments of the present invention, the modified genomic region is associated with a plant trait such as an agronomic trait, whereby the modification substitution results in the plant having an altered (preferably improved) trait, such as an agronomic trait, relative to a wild-type plant.

[0109] In some embodiments, the method further comprises the step of screening plants for desired modifications and / or desired traits, such as agronomic traits.

[0110] In some embodiments of the present invention, the method further comprises obtaining offspring of the genetically modified plant. Preferably, the genetically modified plant or its offspring has a desired modification and / or a desired trait such as an agronomic trait.

[0111] In another aspect, the present invention also provides a genetically modified plant, or a progeny thereof, or a part thereof, wherein the plant is obtained by the method of the present invention described above. In some embodiments, the genetically modified plant, or a progeny thereof, or a part thereof is non-transgenic. Preferably, the genetically modified plant, or a progeny thereof, has a desired genetic modification and / or a desired trait, such as an agronomic trait.

[0112] In another aspect, the present invention also provides a plant breeding method comprising crossing a first genetically modified plant obtained by the method of the present invention with a second plant that does not contain the modification, thereby introducing the modification into the second plant. Preferably, the genetically modified first plant has a desired trait, such as an agronomic trait.

[0113] "Agronomic traits" specifically refer to measurable indicator parameters of crop plants, including but not limited to: leaf greenness, grain yield, growth rate, total biomass or accumulation rate, fresh weight at maturity, dry weight at maturity, fruit yield, seed yield, plant total nitrogen content, fruit nitrogen content, seed nitrogen content, plant vegetative tissue nitrogen content, plant total free amino acid content, fruit free amino acid content, seed free amino acid content, plant vegetative tissue free amino acid content, plant total protein content, fruit protein content, seed protein content, plant vegetative tissue protein content, herbicide resistance and drought resistance, nitrogen absorption, root lodging, harvest index, stem lodging, plant height, ear height, ear length, disease resistance, cold resistance, salt resistance and tiller number, etc.

[0114] 5. Therapeutic Applications

[0115] The present invention also encompasses the use of the guided editing system of the present invention in disease treatment.

[0116] By modifying the disease-related genes through the guide editing system of the present invention, it is possible to achieve upregulation, downregulation, inactivation, activation or mutation correction of the disease-related genes, thereby achieving the prevention and / or treatment of the disease. For example, the genomic modification described in the present invention can be located in the protein coding region of the disease-related gene, or, for example, can be located in a gene expression regulatory region such as a promoter region or an enhancer region, so as to achieve functional modification of the disease-related gene or modification of the expression of the disease-related gene. Therefore, the modified disease-related genes described herein include modification of the disease-related gene itself (e.g., protein coding region), and also include modification of its expression regulatory region (e.g., promoter, enhancer, intron, etc.).

[0117] "Disease-associated" gene refers to any gene that produces a transcription or translation product at an abnormal level or in an abnormal form in cells derived from tissues affected by the disease, compared to tissues or cells of non-disease controls. In the case where the altered expression is related to the appearance and / or progression of the disease, it can be a gene expressed at an abnormally high level; it can be a gene expressed at an abnormally low level. Disease-associated genes also refer to genes with one or more mutations or genetic variations that are directly responsible for or disequilibrium with one or more genes responsible for the etiology of the disease. The mutation or genetic variation is, for example, a single nucleotide variation (SNV). The transcribed or translated product can be known or unknown and can be at normal or abnormal levels.

[0118] Therefore, the present invention also provides a method for treating a disease in a subject in need thereof, comprising delivering to the subject an effective amount of a guide editing system of the present invention to modify a gene associated with the disease. The present invention also provides a use of a guide editing system in the preparation of a pharmaceutical composition for treating a disease in a subject in need thereof, wherein the guide editing system is used to modify a gene associated with the disease. The present invention also provides a pharmaceutical composition for treating a disease in a subject in need thereof, comprising a guide editing system of the present invention, and optionally a pharmaceutically acceptable carrier, wherein the guide editing system is used to modify a gene associated with the disease.

[0119] Preferably, the "subject" of the present invention is a mammal, such as a human.

[0120] In some embodiments, the primed editing systems described herein are used to introduce point mutations into nucleic acids.

[0121] In some embodiments, the guide editing system described herein is used to cause the correction of genetic defects, such as in point mutations that cause loss of function in gene products. In some embodiments, the genetic defect is associated with a disease or condition (e.g., lysosomal storage disease or metabolic disease, such as, for example, type I diabetes). In some embodiments, the methods provided herein can be used to introduce inactivating point mutations into genes or alleles encoding gene products associated with a disease or condition.

[0122] In some embodiments, the invention provides a method for treating a disease associated with or caused by a point mutation that can be corrected using the guide editing system provided herein. In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a neoplastic disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease.

[0123] In some embodiments, the methods described herein are intended to be used to treat mitochondrial diseases or disorders. As used herein, "mitochondrial diseases" refer to diseases caused by abnormal mitochondria, such as mutations in mitochondrial genes, enzyme pathways, etc. Examples of diseases include, but are not limited to, neurological diseases, loss of motor control, muscle weakness and pain, gastrointestinal diseases and difficulty swallowing, poor growth, heart disease, liver disease, diabetes, respiratory complications, epilepsy, vision / hearing problems, lactic acidosis, developmental delays, and susceptibility to infection.

[0124] Examples of diseases described herein include, but are not limited to, genetic diseases, circulatory system diseases, muscle diseases, brain, central nervous and immune system diseases, Alzheimer's disease, secretase disorders, amyotrophic lateral sclerosis (ALS), autism, trinucleotide repeat expansion disorders, hearing diseases, gene-targeted therapy of non-dividing cells (neurons, muscles), liver and kidney diseases, epithelial cell and lung diseases, cancer, Usher syndrome or retinitis pigmentosa-39, cystic fibrosis, HIV and AIDS, beta thalassemia, sickle cell disease, herpes simplex virus, autism, drug addiction, age-related macular degeneration, schizophrenia. Other diseases that can be treated by correcting point mutations or introducing inactivating mutations into disease-related genes are known to those skilled in the art, and therefore the present disclosure is not limited in this regard. In addition to the diseases exemplarily described herein, other related diseases can also be treated using the strategies and guide editing systems provided by the present invention, and the application will be apparent to those skilled in the art. The diseases or targets to which the present invention can be applied refer to WO2015089465A1 (PCT / US2014 / 070135), WO2016205711A1 (PCT / US2016 / 038181), WO2018141835A1 (PCT / EP2018 / 052491), WO2020191234A1 (PCT / US2020 / 023713), WO2020191233A1 (PCT / US2020 / 023712), WO2019079347A1 (PCT / US2018 / 056146), and WO2021155065A1 (PCT / US2021 / 015580) for the related diseases to which the base editing systems are applicable.

[0125] Administration of the guided editing system or pharmaceutical composition of the present invention can be adjusted for the patient's or subject's weight and species. The frequency of administration is within the medically or veterinary acceptable range. It depends on conventional factors including the patient's or subject's age, sex, general health, other conditions, and the specific condition or symptom being addressed.

[0126] 6. Test Kit

[0127] The present invention also includes a kit for use in the methods of the present invention, comprising the components of the guide editing system of the present invention. The kit may also include reagents for introducing the guide editing system into an organism or a cell of an organism. Kits generally include a label indicating the intended use and / or instructions for use of the kit contents. The term label includes any written or recorded material provided on or with the kit or otherwise accompanying the kit. Example

[0128] Materials and Methods

[0129] 1. Vector Construction

[0130] The vectors were constructed using the existing guide editor vector backbone in the laboratory. The main vectors constructed are:

[0131] niCPE:

[0132] Cas12a-niCPE

[0133] Cas12a-sniCPE

[0134] Cas12i-niCPE

[0135] nuCPE:

[0136] Cas12a-nuCPE

[0137] Cas12a-snuCPE

[0138] Cas12i-nuCPE

[0139] CirRNA:

[0140] 5'+3'MS2-CirRNA

[0141] guide RNA:

[0142] crRNA(Cas12a)

[0143] crRNA(Cas12i)

[0144] 2. Human HEK293T cell culture and transformation

[0145] 2.1 Thawing cells

[0146] (1) Remove HEK293T cell lines from liquid nitrogen and thaw them in a 37°C water bath for 2 minutes.

[0147] (2) Remove cells from the cryopreservation tube and slowly add them to a 15 mL centrifuge tube containing 10 mL of culture medium.

[0148] (3) Centrifuge at 500 × g for 3 minutes to pellet the cells and remove the supernatant.

[0149] (4) Add 10 mL of culture medium to the precipitate, transfer to a T175 culture flask, and place in a 37°C incubator containing 5% CO2.

[0150] (5) After 24 hours, the culture medium was replaced and the cells were cultured in a 37°C incubator containing 5% CO2.

[0151] 2.2 Cell culture

[0152] (1) Carefully aspirate the cell culture medium from the T175 culture flask.

[0153] (2) Gently wash the cells with 10 mL of PBS solution.

[0154] (3) Trypsinize the cells with 2 mL of trypsin: Place the T175 cell culture flask in a 37°C incubator for 2 minutes to allow trypsin to fully dissociate the cells.

[0155] (4) Add 10 mL of cell culture medium to the T175 cell culture flask to resuspend the cells and inactivate trypsin.

[0156] (5) Use a 10 mL pipette to pipette up and down the cell suspension to obtain a single cell suspension.

[0157] (6) After diluting the cells at a ratio of 1:10, continue to culture in a 37°C incubator containing 5% CO2.

[0158] 2.3 Cell plating

[0159] (1) Take 15 μL of the single-cell suspension from step 2.1-(5) and mix it with 15 μL of trypan blue. Let it stand at room temperature for 1-2 minutes.

[0160] (2) Open a new cell counter. Add 10 μL of the mixed cell and trypan blue solution to each of the A and B counting chambers. Insert the counting chamber into the cell counter and read the count.

[0161] (3) Calculate the required dilution factor so that the final plate count is 45,000 cells per well in 250 μL of cell culture medium in a 48-well plate. Dilution factor = (A count + B count) / (2 × 4 × 45,000).

[0162] (4) Dilute the cells according to the dilution factor and use an adjustable width multichannel pipette to dispense the cell suspension into a 48-well plate, adding 250uL of cell suspension to each well.

[0163] (5) Place the 48-well plate containing cells in a 37°C incubator and culture them for 16 to 24 hours before use for liposome transformation.

[0164] 2.4 Transfection of cells

[0165] (1) Before transfecting cells, observe the cell growth status under a microscope, preferably at a coverage of 60%.

[0166] (2) Mix 300 ng of protein expression plasmid and 100 ng of RNA expression plasmid in Opti-MEM to a total volume of 12.5 uL.

[0167] (3) Add 1 μL of Lipofectamine 2000 and 10-20 ng of copGFP expression plasmid to Opti-MEM to a total volume of 12.5 μL.

[0168] (4) Mix (2) and (3) to a total volume of 25 μL and incubate for 5-15 minutes. Then add the solution to the cell solution in the 48-well plate and incubate in a 37°C incubator for 48-72 hours.

[0169] 3. Fluorescence observation of reporter system

[0170] After culturing human HEK293T cells in a 37°C incubator for 48 hours, they were observed under a fluorescence microscope or a laser confocal microscope. The efficiency of different guide editors was compared based on fluorescence, and the fluorescence images were saved.

[0171] 4. Human HEK293T cell lysis and amplicon sequencing analysis

[0172] 4.1 Lysis of human HEK293T cells

[0173] (1) After culturing the cells in a 37°C incubator for 72 hours, carefully aspirate the cell culture medium using an adjustable-width multichannel pipette.

[0174] (2) Gently add 300uL PBS buffer, shake gently to wash away dead cells, and then aspirate the PBS buffer.

[0175] (3) Add 200 μL of lysis buffer (with proteinase K added) and treat at 55°C for 30 minutes.

[0176] (4) Pipette 50 μL of cell lysate into a 96-well plate and treat at 95°C for 5 minutes before use.

[0177] 4.2 Amplicon Miseq sequencing analysis

[0178] (1) PCR amplification of human HEK293T cell lysate was performed using Miseq first-round primers.

[0179] The first round 15 μL amplification system: 7.5 μL 2×Phanta Max Master Mix, 4.5 uL ddH2O, 1 μL Forward primer (10 μM), 1 μL Reverse primer (10 μM), 1 μL cell lysate.

[0180] The first round of 15 μL amplification conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 50-60°C for 15 s, and extension at 72°C for 20 s, for 34 cycles; and full extension at 72°C for 5 min.

[0181] (2) The first round amplification product was diluted 5 times, and 1 μL was used as the template for the second round of PCR amplification. The amplification primers were Miseq second round sequencing primers containing barcodes.

[0182] Second-round 25 μL amplification system: 12.5 μL 2×Phanta Max Master Mix, 9.5 μL ddH2O, 1 μL Forward primer (10 μM), 1 μL Reverse primer (10 μM), 1 μL DNA template.

[0183] The second round of amplification conditions were as follows: pre-denaturation at 95°C for 3 min; 10 cycles of denaturation at 95°C for 15 s, annealing at 50-60°C for 15 s, and extension at 72°C for 20 s; and full extension at 72°C for 5 min.

[0184] (3) PCR products were detected by 2% agarose gel electrophoresis, and the target fragments were recovered using the AxyPrep DNA Gel Extraction kit. The recovered products were quantitatively analyzed using a NanoDrop ultra-micro spectrophotometer. 100 ng of the recovered products were mixed and sent to Beijing Qihe Biotechnology Co., Ltd. for amplicon sequencing analysis.

[0185] (4) After sequencing is completed, the editing type and editing efficiency of the products at different gene targeting sites in three repeated experiments are compared and analyzed.

[0186] Example 1. The circular RNA-dependent guide editing system Cas12a-CPE repairs copGFP to produce green fluorescence in human 293T cells.

[0187] The present inventors previously utilized pegRNA to develop Cas12a-PE to guide editor when finding that the efficiency of guiding editing is relatively low (Fig. 2). According to analysis, it is believed that because Cas12a itself has the activity of cutting RNA, even if the domain of its cutting RNA is mutated, pegRNA will still be affected. Moreover, because crRNA is very small, there are only about 20bp, the RTT and PBS sequences of its pegRNA are very easy to complement the Target sequences of crRNA, greatly hindering the function of pegRNA.

[0188] In 2019, Litke and Jaffrey reported the Tornado (Twister-optimized RNA for durable overexpression) human circular RNA expression system (https: / / doi.org / 10.1038 / s41587-019-0090-6). Tornado uses two ribozymes to cut RNA ends, which are then joined to form circular RNAs by the human RNA ligase RtcB. In 2022, Sontheimer (https: / / doi.org / 10.1038 / s41587-022-01255-9) and Hanhui Ma (https: / / doi.org / 10.1093 / procel / pwac014) groups expressed the RTT (reverse transcriptase template) and PBS (primer binding site) sequences of the pegRNA (prime editing guide RNA) in a prime editor into circular RNA, generating a sPE (split prime editor) guide editor.

[0189] The inventors surprisingly found that placing the RTT and PBS sequences in pegRNA in circular RNA for stable expression effectively avoided the above two problems of the Cas12a guide editing system.

[0190] The guide editor is mainly composed of three parts: nCas protein, reverse transcriptase and pegRNA (prime editing guide RNA). When developing the new guide editor Cas12a-CPE, the viral M-MLV reverse transcriptase has high activity and does not need to be completely changed; and LbCas12a, i.e. LbCpf1, does not have the classic nick single incision form. The inventors found that LbCpf1 with R1138A substitution is the Nickase form of LbCpf1, which has nickase activity, and LbCpf1 with R1138A is used to construct Cas12a-CPE. The third part of the guide editor is pegRNA. The inventors separated the PBS (primer binding site) and RT (reverse transcriptase) template sequences from pegRNA and placed them in Circular RNA for stable expression. The inventors also designed a copGFP reporter system in which the GFP coding sequence contains a mutation of three bases. The fluorescence of copGFP can only be restored when precise gene editing occurs in the guide editor.

[0191] Using the above-mentioned copGFP reporter system, Cas12a-niCPE, Cas12a-nuCPE, Cas12a-sniCPE, Cas12a-snuCPE guide editing system and mutated copGFP plasmid were co-transferred into human HEK293T cells. After about 48 hours of culture, obvious copGFP fluorescence was detected in the Cas12a-niCPE (Figure 2i), Cas12a-nuCPE (Figure 2h) and Cas12a-sniCPE (Figure 2o) reporter systems, and the fluorescence of the Cas12a-snuCPE (Figure 2l) guide editing system was weaker. Figure 2a is a positive control with copGFP added, the untreated negative control (Figure 2b) and the control group (Figure 2c) with only the mutated copGFP system added were non-fluorescent, and the Cas12a guide editing system constructed using 3'pegRNA (Figures 2d-e) and 5'pegRNA (Figures 2f-g) was also almost non-fluorescent. The guide editing system without reverse transcriptase (Figure 3j,m) also had no fluorescence, and the guide editing system with free reverse transcriptase (Figure 3k,n) expressed weak fluorescence.

[0192] Further use of the human HEK293T cell line to conduct guide editing reporter system and various target editing efficiency tests revealed that the Cas12a guide editing system constructed using 5'pegRNA and 3'pegRNA had extremely low editing efficiency, while the guide editing system constructed using circular RNA produced precise gene editing (Figure 3).

[0193] Example 2. The circular RNA-dependent guide editing system Cas12a-niCPE achieves precise base editing at the target site in human 293T cells.

[0194] The inventors found in the reporter system that the Cas12a-niCPE guide editing system based on circular RNA and non-targeted chain single nickase nCas12a has a strong gene editing efficiency, so that the copGFP reporter system emits strong green fluorescence. The inventors further verified the guide editing efficiency of Cas12a-niCPE in the DNMT1-1 target of human 293T cells and found that the deletion (DEL, deletion), insertion (INS, insertion) and substitution (SUB, substitution) of bases had higher guide editing efficiency, among which the highest guide editing efficiency can reach 24.06% (Fig. 4).

[0195] Example 3. The circular RNA-dependent guide editing system Cas12a-nuCPE achieves precise base editing at the target site in human 293T cells.

[0196] Although the inventors have found the non-targeted chain single nickase (the chain combined with crRNA is called targeting chain, and the chain complementary to the targeting chain is called non-targeting chain) form Cas12a-R1138A of Cas12a, but the newly discovered nuclease may not also have the non-targeted chain single nickase form, which greatly limits the development of Cas12b (i.e. C2c1), Cas12c, Cas12d (i.e. CasY), Cas12e (i.e. CasX), Cas12f (i.e. Cas14), Cas12h, Cas12i, Cas12j (i.e. CasΦ), Cas12k, Cas12m, IscB and TnpB etc. albumen for guiding editing system.So, the inventors first developed the Cas12a-nuCPE guiding editing system based on circular RNA with wild nuclease Cas12a as a breakthrough. The inventors selected three gene targets in human 293T cells, namely: SITE7, SITE8 and SITE10, to further verify the guide editing efficiency of the Cas12a-nuCPE guide editing system in human cells. After testing, it was found that the Cas12a-nuCPE editing system had guide editing efficiency at all three targets (Figure 5).

[0197] Example 4. The detachable circular RNA-dependent guide editing system Cas12a-sniCPE achieves precise base editing at the target site in human 293T cells.

[0198] The base editing system has gradually shown a surprising prospect in the treatment of human diseases. However, due to the large size of the guide editing system, it is not convenient for the delivery of viral systems such as AAV. Therefore, the inventors further freed the MCP-MMLV protein and expressed it independently of the nCas12a non-targeted chain single nickase. A detachable guide editing system Cas12a-sniCPE that utilizes a non-targeted chain single nickase nCas12a and depends on circular RNA was developed. The Cas12a-sniCPE gene editing efficiency was verified using three gene targets SITE7, SITE8, and SITE10 in human 293T cells, and it was found that the Cas12a-sniCPE editing system had guide editing efficiency at all three targets (Figure 6).

[0199] Example 5. The detachable circular RNA-dependent guide editing system Cas12a-snuCPE achieves precise base editing at the target site in human 293T cells.

[0200] Similarly, since the newly discovered nuclease may not have a non-targeted chain single nickase form, this greatly limits the development of proteins such as Cas12b (i.e., C2c1), Cas12c, Cas12d (i.e., CasY), Cas12e (i.e., CasX), Cas12f (i.e., Cas14), Cas12h, Cas12i, Cas12j (i.e., CasΦ), Cas12k, Cas12m, IscB, and TnpB for guiding editing systems. Therefore, the inventors further developed a detachable guide editing system Cas12a-snuCPE that utilizes nucleases and depends on circular RNA. The Cas12a-snuCPE gene editing efficiency was verified using three gene target sites SITE7, SITE8, and SITE10 in human 293T cells, and it was found that the Cas12a-snuCPE editing system had guide editing efficiency at all three targets (Figure 7). Further research and comparison revealed that among the niCPE, nuCPE, sniCPE and snuCPE systems, the niCPE and sniCPE guided editing systems both had good editing efficiency (Figure 8). At the same time, verification of off-target sites revealed that the niCPE and sniCPE guided editing systems had no off-target efficiency in HEK293T (Figure 11a).

[0201] Example 6. One-CPE-guided editing system for simultaneous expression of all RNAs.

[0202] Since Cas12a itself has the function of cutting RNA, the same human U6 promoter can be used to drive the simultaneous expression of multiple tandem RNAs, including crRNA, circular RNA containing RTT and PBS, and nicking RNA. Under the action of Cas12a, multiple RNAs are expressed simultaneously, crRNA is used to bind to the Cas12a protein to find the target site, and circular RNA expresses RTT and PBS to provide the starting primer and template for the reverse transcriptase. In CPE3, after nicking RNA and Cas12a are combined, a nick is produced in the distal targeting chain, further increasing the efficiency of guide editing (Figure 9a). Further verification in the HEK293T cell line found that one-CPE has an efficient guide editing efficiency (Figure 9b). Verification of the off-target site found that the one-niCPE and one-sniCPE guide editing systems did not show off-target efficiency in HEK293T (Figure 11b).

[0203] Example 7. Human HEK293T multi-gene Cas12a-CPE guided editing system.

[0204] Cas12a protein has been widely used in multi-gene editing in organisms, but guide editing has not yet been performed. We took advantage of Cas12a’s own ability to cut RNA to express multiple crRNAs at the same time. More importantly, we simultaneously connected multiple RTT and PBS sequences in circular RNA (Figure 10a). Experiments in HEK293T human cell lines have shown that multiple RTTs and PBSs expressed in series in circular RNAs produce efficient guide editing for different editing sites (Figures 10b-d). Since many diseases are caused by mutations in multiple targets, we further tested the Cas12a-CPE guide editing system for simultaneous guide editing of two genes, three genes, and even four genes. The results showed that the Cas12a-CPE guide editing system can perform efficient multi-gene guide editing in human HEK293T cells (Figure 10e).

[0205] Example 8. The circular RNA-dependent guide editing system Cas12i-niCPE achieves precise base editing at the target site in human 293T cells.

[0206] The four kinds of guide editing systems niCPE, nuCPE, sniCPE and snuCPE developed above all have good guide editing efficiency on Cas12a, and the gene editing efficiency of the particularly Cas12a-niCPE guide editing system is very high. Therefore, the inventors speculate that most of the newly developed Cas12b (i.e. C2c1), Cas12c, Cas12d (i.e. CasY), Cas12e (i.e. CasX), Cas12f (i.e. Cas14), Cas12h, Cas12i, Cas12j (i.e. CasΦ), Cas12k, Cas12m, IsrB, IscB and TnpB proteins can be applied to the CPE guide editing system. The inventors then selected the CPE guide editing system developed by further detecting Cas12i2 protein. The inventors first found that two amino acid mutations W954A and T882A may make Cas12i2 form a non-targeted chain single nickase form. The inventors utilize the non-targeted chain single nickase form formed by the wild type and these two amino acid point mutations of Cas12i2, and develop Cas12i-nuCPE and Cas12i-niCPE-W854A, Cas12i-niCPE-T882A guide editing system. By verifying the CD34 (Figure 12 a) and RNF2 (Figure 12 b) targets of human 293T cells, it is found that Cas12i-niCPE-W854A has a preferred guide editing efficiency, Cas12i-nuCPE is second, and Cas12i-niCPE-T882A guide editing efficiency is relatively low. Explanation: W854A is the preferred non-targeted chain nickase form of Cas12i, and Cas12i-niCPE-W854A will be named as Cas12i-niCPE later.

[0207] sequence:

Claims

1. A guide editing system for targeted modification of an organism's genomic DNA sequence, comprising: i) a) a CRISPR nuclease and / or an expression construct comprising a nucleotide sequence encoding said CRISPR nuclease, and a reverse transcriptase and / or an expression construct comprising a nucleotide sequence encoding said reverse transcriptase, or b) a guide editing fusion protein and / or an expression construct containing a nucleotide sequence encoding the guide editing fusion protein, wherein the guide editing fusion protein comprises a CRISPR nuclease and a reverse transcriptase; ii) an editing guide RNA (gRNA) directed against a target sequence in genomic DNA and / or an expression construct containing a nucleotide sequence encoding the editing gRNA; and iii) a circular RNA containing a reverse transcription template (RTT) sequence and a primer binding site (PBS) sequence and / or an expression construct containing a nucleotide sequence encoding the circular RNA, Preferably, the CRISPR nuclease is not a Cas9 nuclease or a variant thereof.

2. The guide editing system of claim 1, wherein the CRISPR nuclease is selected from Cas12a (i.e., Cpf1), Cas12b (i.e., C2c1), Cas12c, Cas12d (i.e., CasY), Cas12e (i.e., CasX), Cas12f (i.e., Cas14), Cas12h, Cas12i, Cas12j (i.e., CasΦ), Cas12k, Cas12m, IsrB, IscB, and TnpB.

3. The guide editing system of claim 1 or 2, wherein the CRISPR nuclease is a nickase form thereof.

4. The guide editing system of claim 1 or 2, wherein the CRISPR nuclease is Cas12a, which comprises the amino acid sequence shown in SEQ ID NO: 1 or 2.

5. The guide editing system of claim 1 or 2, wherein the CRISPR nuclease is a Cas12a nickase, which is substituted at the amino acid position 1138 relative to SEQ ID NO: 1 or 2, for example, the amino acid R at position 1138 is substituted by A, preferably, the CRISPR nuclease comprises the amino acid sequence shown in SEQ ID NO:

3.

6. The guide editing system of claim 1 or 2, wherein the CRISPR nuclease is Cas12i, which comprises the amino acid sequence shown in SEQ ID NO:

4.

7. The guide editing system of claim 1 or 2, wherein the CRISPR nuclease is a Cas12i nickase, which is substituted at the amino acid position 854 relative to SEQ ID NO: 4, for example, the amino acid W at the 854th position is substituted by A, preferably, the Cas12i nickase comprises the amino acid sequence shown in SEQ ID NO:

5.

8. The guide editing system of any one of claims 1 to 7, wherein the reverse transcriptase is M-MLV reverse transcriptase, such as an M-MLV reverse transcriptase comprising the amino acid sequence shown in SEQ ID NO:

6.

9. The guide editing system of claim 8, wherein the RNase H domain of the M-MLV reverse transcriptase is mutated or deleted.

10. The guide editing system of claim 9, wherein the M-MLV reverse transcriptase with a mutated or deleted RNase H domain comprises the amino acid sequence shown in SEQ ID NO:

7.

11. The guide editing system of any one of claims 1 to 10, wherein the reverse transcriptase and / or the guide editing fusion protein may further comprise one or more RNA aptamer binding protein sequences.

12. The guide editing system of claim 11, wherein the RNA aptamer binding protein is an MCP protein, for example, the MCP comprises the amino acid sequence shown in SEQ ID NO:

8.

13. The guide editing system of claim 1, wherein the guide editing fusion protein of i)-b) comprises an amino acid sequence selected from one of SEQ ID NOs: 12-15, preferably comprises the amino acid sequence of SEQ ID NO:

12.

14. The guide editing system of claim 1, wherein the CRISPR nuclease in i)-a) comprises an amino acid sequence selected from one of SEQ ID NOs: 1-5, preferably an amino acid sequence of SEQ ID NOs: 3 or 5; and the reverse transcriptase comprises the amino acid sequence shown in SEQ ID NO:

16.

15. The guide editing system of any one of claims 1-14, wherein the guide RNA (gRNA) comprises a scaffold sequence shown in SEQ ID NO: 23 or 24.

16. The guide editing system of any one of claims 1 to 15, wherein in the circular RNA, a reverse transcription template (RTT) sequence and a primer binding site (PBS) sequence are directly linked, and the RTT sequence is located at the 5' end of the PBS sequence.

17. The guide editing system of any one of claims 1 to 16, wherein the circular RNA further comprises one or more RNA aptamer sequences, such as the MS2 sequence shown in SEQ ID NO:

17.

18. The guide editing system of any one of claims 1 to 17, wherein the primer binding site sequence is set to be complementary to at least a portion of the target sequence, preferably, the primer binding site sequence is complementary to at least a portion of the 3' free single strand caused by the nick in the DNA chain where the target sequence is located, in particular, is complementary to the nucleotide sequence at the 3' end of the 3' free single strand.

19. The guide editing system of any one of claims 1 to 18, wherein the RTT sequence is set to correspond to a sequence downstream of the target sequence nick, but contains a desired modification, wherein the desired modification comprises a substitution, deletion and / or addition of one or more nucleotides.

20. The guide editing system of any one of claims 1-19, further comprising a nicking gRNA and / or an expression construct containing a nucleotide sequence encoding the nicking gRNA, wherein the nicking gRNA comprises a leader sequence and a scaffold sequence, wherein the leader sequence is configured to have sufficient sequence identity with a nicking target sequence in the genome so as to be able to target the CRISPR nuclease or the fusion protein to the nicking target sequence and cause a nick within the nicking target sequence, wherein the nicking target sequence and the target sequence of the editing gRNA are located on opposite strands of the genomic DNA, and the nick induced by the nicking gRNA and the nick induced by the editing gRNA are about 1 to about 300 nucleotides apart.

21. The guide editing system of any one of claims 1 to 20, wherein the guide editing system comprises a plurality of editing gRNAs and / or an expression construct containing a nucleotide sequence encoding the plurality of editing gRNAs, and accordingly, the circular RNA comprises a plurality of sets of RTT-PBS sequences corresponding to the plurality of editing gRNAs in series.

22. The guide editing system of claim 21, wherein the multiple editing gRNAs target different target sequences, and the corresponding multiple sets of RTT-PBS sequences are used to introduce different modifications into the different target sequences.

23. The guide editing system of any one of claims 1 to 22, wherein the expression construct containing the nucleotide sequence encoding the circular RNA further contains elements required for achieving RNA circularization.

24. The guide editing system of any one of claims 1 to 23, wherein the "expression construct containing a nucleotide sequence encoding the circular RNA" comprises the following coding sequence: 5'-first ribozyme-first looping arm-RTT-PBS-second looping arm-second ribozyme-3', and when transcribed into RNA in the cell, the first ribozyme and the second ribozyme can self-cleave to produce "5'-first looping arm-RTT-PBS-second looping arm-3'", and the first looping arm and the second looping arm can be connected to each other to form a circular RNA containing RTT and PBS.

25. The guide editing system of claim 24, wherein the first ribozyme comprises the nucleotide sequence shown in SEQ ID NO: 19, the second ribozyme comprises the nucleotide sequence shown in SEQ ID NO: 20, the first loop-forming arm comprises the nucleotide sequence shown in SEQ ID NO: 21, and the second loop-forming arm comprises the nucleotide sequence shown in SEQ ID NO:

22.

26. The guide editing system of any one of claims 1-25, wherein the editing gRNA and the circular RNA and optionally the nicking gRNA are encoded by the same expression construct.

27. The guide editing system of claim 26, wherein the expression construct comprises (eg, from 5' to 3' direction) a nucleotide sequence encoding the circular RNA, a nucleotide sequence encoding the editing gRNA, and optionally a nucleotide sequence encoding the nicking gRNA.

28. The guide editing system of claim 27, wherein the nucleotide sequence encoding the circular RNA, the nucleotide sequence encoding the editing gRNA, and optionally the nucleotide sequence encoding the nicking gRNA are driven to express by the same promoter, such as the U6 promoter.

29. A method for producing genetically modified cells, comprising introducing the guide editing system of any one of claims 1-28 into at least one of the cells, thereby resulting in modification of the genomic sequence of the at least one cell.

30. The method of claim 29, wherein the cell is from a mammal such as a human, mouse, rat, monkey, dog, pig, sheep, cow, cat; poultry such as chicken, duck, goose; a plant, including monocots and dicots, for example, rice, corn, wheat, sorghum, barley, soybean, peanut, Arabidopsis.

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