Nucleic acid for coding CRISPR related protein and application thereof

Abstract

The present invention provides artificial nucleic acids, in particular RNA. Compositions and kits comprising the artificial nucleic acids are also provided.

Description

[0001] This invention claims priority to PCT / CN2023 / 116171 filed on August 31, 2023 and PCT / CN2024 / 074906 filed on January 31, 2024, the contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to artificial nucleic acids, particularly RNA encoding polypeptides or proteins of interest, especially CRISPR-related polypeptides or proteins, as well as (pharmaceutical) compositions comprising these components and kits comprising these components. The artificial nucleic acids, particularly RNA, the (pharmaceutical) compositions, and the kits are intended for use in the medical field, particularly in gene therapy, especially in the treatment and / or prevention of diseases that can be treated by methods such as gene editing, gene knock-in, gene knockout, or regulation of target gene expression using CRISPR-related proteins. Background Technology

[0003] With the development of mRNA drug technology, therapeutic mRNAs have been applied in many fields, such as cancer immunotherapy, infectious disease vaccines, allergy tolerance treatment, protein replacement and supplementation therapy, genome engineering, gene therapy, and gene reprogramming, especially in the field of CRISPR-related gene therapy. In gene therapy, the long-term presence of editing enzymes translated from DNA vectors can lead to off-target effects. However, transient expression of nucleases via mRNA expression can minimize these non-specific effects. Engineered mRNAs encoding ZFNs, TALENs, and Cas9 have been successfully applied to genome editing, enabling in vitro and in vivo editing by disrupting or integrating sequences. (Sahin, U., Karikó, K. & Türeci, Ö. mRNA-basedtherapeutics — developing a new class of drugs. Nat Rev Drug Discov 13, 759–780 (2014). doi.org / 10.1038 / nrd4278).

[0004] However, some unresolved issues remain, such as the control and regulation of mRNA translation, which require further investigation. Addressing these needs is the objective of this invention. Summary of the Invention

[0005] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0006] In one aspect, the present invention provides an mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the open reading frame comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 212, 213, 215, 225, 226, 227, 228, 229, 230, 231, 232 and 247.

[0007] In some embodiments, the open reading frame includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 212, 213, 215, 225, 226, 227, 228, 229, 230, 231, 232, and 247.

[0008] In some embodiments, the mRNA further comprises a 5′ untranslated region (5′ UTR) having at least 90% sequence identity with any one of SEQ ID NOs: 110-171 or 1-62.

[0009] In some embodiments, the mRNA further comprises a 5′ untranslated region (5′ UTR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 110-171 or 1-62.

[0010] In some embodiments, the mRNA further comprises a 3′ untranslated region (3′ UTR) having at least 90% sequence identity with any one of SEQ ID NOs: 172-210 or 63-101.

[0011] In some embodiments, the mRNA further comprises a 3′ untranslated region (3′ UTR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 172-210 or 63-101.

[0012] In some embodiments, the mRNA further comprises a poly-A tail having at least 90% sequence identity with any one of SEQ ID NO: 243, 244, 245 or 246.

[0013] In some embodiments, the mRNA further comprises a poly-A tail having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 243, 244, 245, or 246.

[0014] In another aspect, the present invention provides an mRNA comprising a sequence having at least 98% sequence identity with any one of SEQ ID NOs: 233, 234, 235, 236, 237, 238, 239, 240, 250, 252 and 254.

[0015] In some embodiments, the mRNA comprises a sequence selected from any one of SEQ ID NOs: 233, 234, 235, 236, 237, 238, 239, 240, 250, 252 and 254.

[0016] In some embodiments, at least 10% of the uridine is replaced with modified uridine, wherein the modified uridine is one or more of N1-methylpseudouridine, pseudouridine, 5-methoxyuridine, or 5-iodouridine.

[0017] In another aspect, the present invention provides an expression construct comprising a promoter operatively linked to a sequence encoding the aforementioned mRNA, wherein the expression construct is optionally a plasmid expression construct.

[0018] In another aspect, the present invention provides an isolated host cell comprising the above-described expression construct.

[0019] In one aspect, the present invention provides a composition comprising the above-described mRNA and at least one guide RNA.

[0020] In another aspect, the present invention provides a lipid nanoparticle comprising the above-mentioned mRNA.

[0021] In another aspect, the present invention provides a pharmaceutical composition comprising the above-described mRNA and a pharmaceutically acceptable carrier.

[0022] In another aspect, the present invention provides a method for genome editing or target gene modification, comprising contacting cells with the aforementioned mRNA.

[0023] In one aspect, the present invention provides an artificial nucleic acid molecule comprising: a. At least one coding region encoding at least one CRISPR-related protein; b. At least one 5′ untranslated region (5′ UTR) element; and c. At least one 3′ untranslated region (3′ UTR) element, The coding region therein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 212, 213, 215, 225, 226, 227, 228, 229, 230, 231, 232, and 247.

[0024] In some embodiments, the coding region further includes at least one nuclear positioning signal.

[0025] In some embodiments, the artificial nucleic acid molecule is RNA.

[0026] In some embodiments, the RNA comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 233, 234, 235, 236, 237, 238, 239, 240, 250, 252, and 254.

[0027] In some embodiments, the artificial nucleic acid molecule is DNA.

[0028] In some embodiments, the DNA comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 217-224 and 249-251.

[0029] In another aspect, the present invention provides a recombinant expression vector comprising the aforementioned artificial nucleic acid molecule.

[0030] In some embodiments, the artificial nucleic acid molecule is operatively linked to a promoter.

[0031] In some embodiments, the promoter is an inducible promoter.

[0032] In some embodiments, the artificial nucleic acid molecule further includes a multiple cloning site.

[0033] In another aspect, the present invention provides an in vitro genetically modified host cell comprising any one of the artificial nucleic acid molecules of claims 18 to 23 or the above-described recombinant expression vector.

[0034] In another aspect, the present invention provides a composition comprising the above-described artificial nucleic acid molecule or the above-described recombinant expression vector, and a pharmaceutically acceptable carrier and / or excipient.

[0035] In some embodiments, the composition further comprises guide RNA or nucleic acid encoding thereon, the guide RNA being capable of targeting CRISPR-related proteins to a target DNA sequence.

[0036] In some embodiments, the guide RNA is a single guide RNA (sgRNA).

[0037] In some embodiments, the guide RNA is a dual guide RNA (dgRNA).

[0038] In one aspect, the present invention provides a kit comprising the above-described artificial nucleic acid molecule or the above-described recombinant expression vector, and optionally comprising a liquid carrier and / or comprising technical instructions regarding the administration method and dosage information of the artificial nucleic acid molecule or the composition.

[0039] In some embodiments, the kit further comprises guide RNA (gRNA) or nucleic acid encoding it, the guide RNA being capable of targeting CRISPR-related proteins to a target DNA sequence, or a regulatory element operatively linked thereto.

[0040] In some embodiments, the guide RNA is a single guide RNA (sgRNA).

[0041] In some embodiments, the guide RNA is a dual guide RNA (dgRNA).

[0042] In another aspect, the present invention provides a method for inducing double-strand breaks (DSBs) and / or modifying target genes within a target gene, comprising delivering the aforementioned artificial nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned composition, or the aforementioned kit to cells.

[0043] In another aspect, the present invention provides a method for treating, preventing or diagnosing diseases related to a target gene, comprising administering the artificial nucleic acid molecule, the recombinant expression vector, the composition or the kit described above to a subject in need.

[0044] In another aspect, the present invention provides the use of the above-described artificial nucleic acid molecules, the above-described recombinant expression vectors, the above-described compositions, or the above-described kits in the preparation of pharmaceuticals.

[0045] In one aspect, the present invention provides the use of the above-described artificial nucleic acid molecule, the above-described recombinant expression vector, the above-described composition, or the above-described kit in gene therapy.

[0046] In another aspect, the present invention provides the use of the above-described artificial nucleic acid molecules, the above-described recombinant expression vectors, the above-described compositions or the above-described kits in inducing double-strand breaks (DSBs) in target genes, modifying target genes and / or regulating the expression of target genes in cells or subjects.

[0047] In another aspect, the present invention provides the above-mentioned artificial nucleic acid molecule, the above-mentioned recombinant expression vector, the above-mentioned composition or the above-mentioned kit for inducing double-strand breaks (DSBs) in target genes, modifying target genes and / or regulating the expression of target genes in cells or subjects.

[0048] In another aspect, the present invention provides the above-described artificial nucleic acid molecule, the above-described recombinant expression vector, the above-described composition or the above-described kit for the treatment, prevention or diagnosis of diseases related to the target gene.

[0049] In one aspect, the present invention provides an artificial nucleic acid molecule comprising: i. At least one coding region encoding at least one target peptide; ii. At least one 5′ untranslated region (5′ UTR) element comprising a DNA sequence having at least 90% sequence identity with any one of SEQ ID NOs: 110-171, or an RNA sequence having at least 90% sequence identity with any one of SEQ ID NOs: 1-62; iii. At least one 3′ untranslated region (3′ UTR) element comprising a DNA sequence having at least 90% sequence identity with any one of SEQ ID NOs: 172-210, or an RNA sequence having at least 90% sequence identity with any one of SEQ ID NOs: 63-101.

[0050] In some embodiments, the 5′ untranslated region (5′ UTR) element comprises a DNA sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 110-171, or an RNA sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 1-62.

[0051] In some embodiments, the 3′ untranslated region (3′ UTR) element comprises a DNA sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 172-210, or an RNA sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 63-101.

[0052] Sequence List-1

[0053] Sequence List-2

[0054] Sequence List-3

[0055] Table 1. Detailed description of sequence listings 1-3

[0056] Brief description of the attached diagram

[0057] Figure 1 The percentage of editing is shown after transfection of PHH cells with a lipid complex containing SpCas9 sgRNA (sg5815-3, SEQ ID NO:242) and SpCas9 mRNA (m5815-1, SEQ ID NO:233 / CAS001D, SEQ ID NO:236 / CAS001F, SEQ ID NO:238).

[0058] Figure 2 The percentage of editing is shown after transfection of PHH cells with a lipid complex containing SpCas9 sgRNA (sg5815-3, SEQ ID NO:242) and SpCas9 mRNA (m5815-1, SEQ ID NO:233 / CAS001C, SEQ ID NO:235 / CAS001F, SEQ ID NO:238).

[0059] Figure 3 The percentage of editing after transfecting WT mice with LNP (ALC-0315) containing SpCas9 sgRNA (sg5815-1, SEQ ID NO:241) and SpCas9 mRNA (m5815-1, SEQ ID NO:233 / m5815-9, SEQ ID NO:240 / CAS001F, SEQ ID NO:238) is shown.

[0060] Figure 4 The percentage of editing after transfecting WT mice with LNP (ALC-0315) containing SpCas9 sgRNA (sg5815-1, SEQ ID NO:241) and SpCas9 mRNA (m5815-1, SEQ ID NO:233 / m5815-9, SEQ ID NO:240 / CAS001F, SEQ ID NO:238) is shown.

[0061] Figure 5 The activity of different 5'UTR (A) and 5'UTR and 3'UTR combinations (B) on SpCas9 (CAS001F) mRNA in HepG2 cells is shown, along with the corresponding SEQ ID NO names in Table 12 from left to right. Different SpCas9 mRNAs bind to the sgRNA of SEQ ID NO:109 to form different vectors.

[0062] Table 2

[0063] Figure 6A The activity of different 5'UTR (A&B) and 5'UTR / 3'UTR combinations (C) for SpCas9 (CAS001F) mRNA in HepG2 cells is shown, along with the corresponding SEQ ID NO names in Table 14 from left to right. Different SpCas9 mRNAs bind to the sgRNA of SEQ ID NO:109 to form different vectors.

[0064] Table 3

[0065] Figure 6B The activity of different 5'UTR (A&B) and 5'UTR / 3'UTR combinations (C) on SpCas9 (CAS001F) mRNA in HepG2 cells is shown, along with the corresponding SEQ ID NO names in Table 15 from left to right. Different SpCas9 mRNAs bind to the sgRNA of SEQ ID NO:109 to form different vectors.

[0066] Table 4

[0067] Figure 6C The activity of different 5'UTR (A&B) and 5'UTR / 3'UTR combinations (C) for SpCas9 (CAS001F) mRNA in HepG2 cells is shown, along with the corresponding SEQ ID NO names in Table 16 from left to right. Different SpCas9 mRNAs bind to the sgRNA of SEQ ID NO:109 to form different vectors.

[0068] Table 5

[0069] Figure 7A The activity of different 5'UTR and 3'UTR combinations on SpCas9 (CAS001F) mRNA in HepG2 cells was demonstrated. Different SpCas9 mRNAs bound to the sgRNA of SEQ ID NO:109 to form different vectors.

[0070] Table 6

[0071] Figure 7BThe activity of different 5'UTR and 3'UTR combinations on SpCas9 (CAS001F) mRNA in HepG2 cells was demonstrated. Different SpCas9 mRNAs bound to the sgRNA of SEQ ID NO:109 to form different vectors.

[0072] Table 7

[0073] Figure 7C The activity of different 5'UTR and 3'UTR combinations on SpCas9 (CAS001F) mRNA in HepG2 cells was demonstrated. Different SpCas9 mRNAs bound to the sgRNA of SEQ ID NO:109 to form different vectors.

[0074] Table 8

[0075] Figure 8A The activity of different 5'UTR (A, B, C, and D) and 3'UTR (E) combinations on Fluc mRNA in HEK293T cells was shown.

[0076] Table 9

[0077] Figure 8B The activity of different 5'UTR (A, B, C, and D) and 3'UTR (E) combinations on Fluc mRNA in HEK293T cells was shown.

[0078] Table 10

[0079] Figure 8C The activity of different 5'UTR (A, B, C, and D) and 3'UTR (E) combinations on Fluc mRNA in HEK293T cells was shown.

[0080] Table 11

[0081] Figure 8D The activity of different 5'UTR (A, B, C, and D) and 3'UTR (E) combinations on Fluc mRNA in HEK293T cells was shown.

[0082] Table 12

[0083] Figure 8EThe activity of different 5'UTR (A, B, C, and D) and 3'UTR (E) combinations on Fluc mRNA in HEK293T cells was shown.

[0084] Table 13

[0085] Figure 9A Editing efficiencies of mRNA samples modified with different poly(A) structures, specifically fragmented A60+A60 and A95, were shown in human primary hepatocytes (PHH). mRNA-790 and mRNA-784, derived from the same IVT DNA template and modified with poly(A)A60+A60, were modified with different IVT reactions -A and -B. mRNA-768 and mRNA-791, derived from the same IVT DNA template and modified with poly(A)A95, were modified with different IVT reactions -A and -B. All tested mRNA samples contained the CDS sequence encoding SpCas9, CAS001F. The sgRNA used as the guide RNA was named sg5815-3 (SEQ ID NO:242) and was used in this experiment.

[0086] Table 14

[0087] Figure 9B Editing efficiencies of mRNA samples modified with different poly(A) structures, specifically fragmented A60+A60 and A95, were shown in human primary hepatocytes (PHH). mRNA-788 and mRNA-802 were modified to poly(A)A60+A60 and contained different nuclear localization signals (NLS). mRNA-791 and mRNA-792 were modified to poly(A)A95 and derived from the same IVT DNA template. All tested mRNA samples contained the CDS sequence CAS001F encoding SpCas9. The sgRNA used as the guide RNA was named sg5815-3 (SEQ ID NO:242) and was used in this experiment.

[0088] Table 15

[0089] Figure 9C The editing efficiencies of mRNA samples modified with different poly(A) structures are shown, specifically the editing efficiencies of fragmented A60+A60 and A95 in CD-1 mice. The mRNAs used in this experiment... Figure 9BThe same drugs were used. CD-1 mice received two doses of the drug, 0.03 mpk (light gray bar) and 0.1 mpk (dark gray bar), as shown in the figure. The sgRNA used as the guide RNA was named sg5815-1 (SEQ ID NO:241) and was used in this experiment.

[0090] Table 16

[0091] Figure 10 The percentage of edits after transfecting WT mice with LNP (ALC-0315) containing SpCas9 sgRNA (sg5815-1, SEQ ID NO:241) and SpCas9 mRNA (CAS001F, SEQ ID NO:238, mRNA-788 / SEQ ID NO:252) is shown.

[0092] Detailed description

[0093] definition

[0094] “UTR” refers to the “untranslated regions” flanking the coding sequence of an artificial nucleic acid, as defined herein. In this case, a “UTR element” contains or is composed of a nucleic acid sequence derived from the (naturally occurring, wild-type) UTR of a specific gene, preferably as exemplified herein.

[0095] When referring to a UTR element “derived” from a specific UTR, it means a nucleic acid sequence that corresponds to that UTR (“parent UTR”) or its homologs, variants, or fragments. This term includes sequences that correspond to the full-length (full-length) wild-type sequence of that UTR, its homologs, variants, or fragments, including full-length homologs and variants, as well as fragments of these full-length wild-type sequences, homologs, and variants, and variants of these fragments. The term “corresponds” means that the nucleic acid sequence derived from the “parent UTR” may be an RNA sequence (e.g., an RNA sequence equivalent to the sequence defining the parent UTR) or a DNA sequence (including positive and reverse strands, and mature and immature DNA) that corresponds to that RNA sequence.

[0096] When referring to a UTR element derived from a gene's UTR, "or its homologs, fragments or variants" means that it may refer to the gene itself, the UTR, or both.

[0097] In the context of a gene (or a nucleic acid sequence derived from or containing that gene, such as a UTR), a "homolog" refers to a gene (or nucleic acid sequence) that is related to a second gene (or a nucleic acid sequence derived from or containing that gene) through a common ancestral DNA sequence. The term "homolog" includes genes separated by speciation events ("orthologs") and genes separated by gene duplication events ("paralogs").

[0098] Artificial nucleic acid molecules: Artificial nucleic acid molecules are generally understood as nucleic acid molecules that do not occur naturally, such as DNA or RNA. In other words, artificial nucleic acid molecules can be understood as unnatural nucleic acid molecules. Such nucleic acid molecules may be non-natural due to their unique sequence (not found in nature) and / or due to other modifications (e.g., structural modifications of nucleotides that do not exist in nature). Artificial nucleic acid molecules can be DNA molecules, RNA molecules, or hybrid molecules containing both DNA and RNA portions. Typically, artificial nucleic acid molecules are designed and / or generated using genetic engineering methods to correspond to a desired artificial nucleotide sequence (heterologous sequence). In this context, an artificial sequence usually refers to a sequence that does not exist naturally, i.e., differs from the wild-type sequence by at least one nucleotide. "Wild-type" is generally understood to be a sequence found in nature. Furthermore, the term "artificial nucleic acid molecule" is not limited to "a single molecule" and is generally understood to include a group of identical molecules. Therefore, it may involve multiple identical molecules contained in a certain share.

[0099] Nucleic acid molecules: Nucleic acid molecules are molecules composed of nucleic acid components. Ideally, nucleic acid molecules refer to DNA or RNA molecules. It is often synonymous with the term "polynucleotide." Ideally, a nucleic acid molecule is a polymer containing or formed from nucleotide monomers, with the sugar / phosphate backbone of the nucleotides linked together by phosphodiester bonds. "Nucleic acid molecule" also includes modified nucleic acid molecules, such as DNA or RNA molecules with base modifications, sugar modifications, or backbone modifications.

[0100] DNA: DNA is the common abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, that is, a polymer composed of nucleotides. These nucleotides are typically monomers of deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate, and deoxycytidine monophosphate. They themselves consist of a sugar (deoxyribose), a base, and a phosphate group, and are polymerized through a characteristic backbone structure. The backbone structure is usually formed by a phosphodiester bond between the sugar (deoxyribose) of the first nucleotide and the phosphate group of the second adjacent monomer. The specific sequence of the monomers, that is, the sequence of bases linked to the sugar / phosphate backbone, is called the DNA sequence. DNA can be single-stranded or double-stranded. In the double-stranded form, the nucleotides of the first strand are usually complementary to the nucleotides of the second strand, for example, through A / T pairing and G / C pairing.

[0101] RNA, mRNA: RNA is the common abbreviation for ribonucleic acid. It is a nucleic acid molecule, a polymer composed of nucleotides. These nucleotides are typically monomers of adenosine monophosphate, uridine monophosphate, guanine monophosphate, and cytidine monophosphate, linked together along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar (ribose) of the first monomer and the phosphate group of the second adjacent monomer. The specific sequence of monomers is called the RNA sequence. RNA is typically obtained by transcribing DNA sequences, for example, within cells. In eukaryotic cells, transcription usually occurs in the nucleus or mitochondria. In vivo, DNA transcription usually produces so-called precursor RNA, which must be processed into so-called messenger RNA, commonly abbreviated as mRNA. The processing of precursor RNA, such as in eukaryotes, involves various post-transcriptional modifications, such as splicing, 5' capping, tailing, and export from the nucleus or mitochondria. The sum of these processes is also called RNA maturation. Mature messenger RNA typically provides a nucleotide sequence that can be translated into a specific peptide or protein amino acid sequence. Typically, mature mRNA contains a 5' cap, a 5' UTR, an open reading frame, a 3' UTR, and a poly(A) sequence. In addition to messenger RNA, there are various non-coding RNA types that may be involved in the regulation of transcription and / or translation.

[0102] Heterologous sequences: These are generally understood as two sequences that are "heterologous" if they are not derived from the same gene. That is, although heterologous sequences may originate from the same organism, they will not appear in the same nucleic acid molecule, such as the same mRNA, under natural conditions (in nature).

[0103] Open Reading Frame (ORF): In the context of this invention, an open reading frame (ORF) is typically a sequence consisting of multiple nucleotide triplets that can be translated into a peptide or protein. An ORF typically contains a start codon, usually consisting of three adjacent nucleotides encoding the amino acid methionine (ATG), located at its 5' end, followed by a region typically a multiple of 3 in length. An ORF typically terminates with a stop codon (e.g., TAA, TAG, TGA). This is usually the only stop codon for the ORF. Therefore, in the context of this invention, an ORF is typically a sequence consisting of several nucleotides divisible by three, beginning with a start codon (e.g., ATG) and typically ending with a stop codon (e.g., TAA, TGA, or TAG). Open reading frames can be isolated or contained within longer nucleic acid sequences, such as in vectors or mRNA. Open reading frames may also be referred to as "(protein) coding sequences" or "coding sequences".

[0104] Codon: In this article, a "codon" refers to a sequence of three nucleotides that together form a unit of genetic code in a DNA or RNA molecule. A codon is defined by the initial nucleotide at which translation begins and sets a frame for a series of consecutive nucleotide triplets, known as an "open reading frame" (ORF). For example, the string GGGAAACCC, read from the first position, contains the codons GGG, AAA, and CCC; from the second position, it contains the codons GGA and AAC; and from the third position, it contains the codons GAA and ACC. Therefore, each nucleotide sequence read in the 5'→3' direction contains three reading frames, each producing a potentially different amino acid sequence (in the given example, Gly-Lys-Pro, Gly-Asn, or Glu-Thr, respectively). DNA is double-stranded, thus defining six possible reading frames, three forward-facing on one strand and three reverse-facing on the opposite side strand. The open reading frame encoding a polypeptide is typically defined by a start codon, usually the first AUG codon in the sequence.

[0105] Start Codon: In this paper, the terms "start codon" and "initiation codon" are used interchangeably. It refers to the first codon of the open reading frame (ORF), translated by the ribosome, and consists of a triplet of adenosine, uridine, and guanine nucleotide bases. The start codon is represented by the letter codes for adenosine (A), uridine (U), and guanine (G), commonly abbreviated as "AUG". Although natural mRNA may use codons other than AUG as the start codon, referred to herein as "alternative start codons," the start codons of the polynucleotides described herein all use the AUG codon. During translation initiation, the sequence containing the start codon is recognized by the ribosome through complementary base pairing with the anticodon on the initiating tRNA (Met-tRNAiMet). The open reading frame may contain multiple AUG start codons, referred to herein as "alternative start codons."

[0106] The role of the start codon: The start codon plays a crucial role in translation initiation. It is the first codon translated by the ribosome within the open reading frame. Typically, the start codon consists of the nucleotide triplet AUG; however, in some cases, translation initiation can occur at other codons composed of different nucleotides. Translation initiation in eukaryotes is a multi-step biochemical process involving multiple protein-protein, protein-RNA, and RNA-RNA interactions, including messenger RNA molecules (mRNA), the 40S ribosomal subunit, and other components of the translation machinery (e.g., eukaryotic initiation factors; eIFs). Current models of mRNA translation initiation assume that the pre-initiation complex (also known as the "43S pre-initiation complex"; abbreviated as "PIC") moves from recruitment sites (usually 5' capped) on the mRNA to the start codon by scanning nucleotides in the 5' to 3' direction until it encounters the first AUG codon located within a specific translation-promoting nucleotide environment (Kozak sequence) (Kozak (1989) J Cell Biol 108:229-241). The PIC scan ends with complementary base pairing between the anticodon on the promoter tRNA (Met-tRNAiMet) and the promoter codon nucleotide on the mRNA. Effective base pairing between the AUG codon and the Met-tRNAiMet anticodon triggers a series of structural and biochemical events that ultimately lead to the binding of the large 60S ribosomal subunit to the PIC, forming an active ribosome capable of translational elongation.

[0107] Sequence identity: Two or more sequences are considered identical if they have the same length and the same sequence of nucleotides or amino acids. Typically, the percentage of identity describes the degree of similarity between two sequences, i.e., the percentage of identical nucleotides at their corresponding positions. When determining the degree of identity (“% identity”), it is generally assumed that the sequences to be compared have the same length, i.e., the length of the longest sequence among the sequences to be compared. This means that if the first sequence consists of 8 nucleotides, it has 80% identity with a second sequence consisting of 10 nucleotides (which contains the first sequence). In other words, in the context of this invention, sequence identity preferably refers to the percentage of nucleotides or amino acids in the sequence that are identical at corresponding positions in two or more sequences of the same length. Specifically, the “% identity” of two amino acid sequences or two nucleic acid sequences can be determined by performing an optimal alignment of the sequences (e.g., introducing gaps in either sequence for optimal alignment) and comparing the corresponding amino acids or nucleotides. Gaps are generally considered to be dissimilar positions, regardless of their actual location in the alignment. “Optimal alignment” generally refers to the alignment result of two sequences having the highest percentage of identity. Percentage identity is determined by comparing the number of identical nucleotides in the sequences (i.e., % identity = number of identical positions / total number of positions × 100). The percentage identity between two sequences can be determined using known mathematical algorithms.

[0108] In the context of a gene's nucleic acid sequence, the term "variant" refers to a nucleic acid sequence variant, i.e., a nucleic acid sequence or gene that differs from a reference (or "parental") nucleic acid sequence or the nucleic acid sequence of a reference (or "parental") gene at least one nucleotide. Therefore, a variant's nucleic acid or gene typically contains at least one mutation, substitution, insertion, or deletion compared to its corresponding reference sequence. The term "variant" includes naturally occurring variants as well as engineered nucleic acid sequence or gene variants. Therefore, a "variant" as defined herein can originate from, be isolated from, be associated with, be based on, or be homologous to a reference nucleic acid sequence / gene. The sequence identity of the variant is typically at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or even 97%, compared to the corresponding naturally occurring (wild-type) nucleic acid sequence or gene, or its homolog, fragment, or derivative.

[0109] In the context of nucleic acid sequences or genes, the term "fragment" refers to a continuous subsequence of a reference (or "parent") nucleic acid sequence or gene. In other words, a "fragment" is typically a portion of a complete nucleic acid sequence or gene. Therefore, a fragment typically consists of a sequence identical to the corresponding region in the complete nucleic acid sequence or gene. This term includes both naturally occurring fragments and engineered fragments. In the context of this invention, a preferred fragment consists of a continuous nucleic acid sequence representing a continuous region from which it is derived, typically representing at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, and most preferably at least 80% of the complete (i.e., full-length) nucleic acid sequence or gene. Sequence identity associated with the fragment typically refers to the complete nucleic acid sequence or gene. The preferred “fragment” may contain at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or even 97%, compared to the reference nucleic acid sequence or gene from which it is derived.

[0110] The following examples further illustrate the content of the present invention, but the content of the present invention is not limited thereto.

[0111] The following illustrates preferred embodiments of the present invention based on the accompanying drawings, in order to provide a detailed description of the technical solutions of the present invention.

[0112] Example 1: LNP Formulation and Analysis

[0113] Cas9 mRNA and sgRNA were encapsulated in lipid nanoparticles (LNPs) using a Precision MPF-L2™ AITESEN device. Generally, the lipid nanoparticle components were dissolved in 100% ethanol, with the lipid components (LP01, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 50:9:38:3, or ALC-0315, DSPC, cholesterol, and ALC-0159 in a molar ratio of 46.3:42.7:9.4:1.6). The RNA cargo was dissolved in a buffer of 25 mM citrate, 100 mM NaCl, pH 5.0, at a concentration of approximately 0.45 mg / mL. The LNP formulation had a lipid amine to RNA phosphate (N:P) molar ratio of approximately 6 and a mRNA to gRNA mass ratio of 2:1. The RNA was mixed with the ethanol-lipid mixture in an aqueous buffer at a volume ratio of 3:1. After mixing, LNP was collected, and the remaining buffer was exchanged through a 10 kDa dialysis bag for TSS at pH 7.5 (containing 5% w / v (g / L) sucrose, 45 mM NaCl, 50 mM Tris, and a 100-fold excess of the sample volume), and gently stirred overnight at 4°C. The resulting mixture was then filtered through a 0.2 μm sterile filter membrane. The resulting filtrate was stored at 2–8°C.

[0114] Lp01 is (9Z,12Z)-3-((4,4-bis(octyloxy)butyryloxy)-2-((((3-(diethylamino)propoxy)carbonyloxy)methyl)propyloctadecenoic acid-9,12-dienoic acid ester, and the structure of Lp01 is shown below: .

[0115] ALC-0315 is 6-((2-hexyldecanoyloxy)-N-(6-((2-hexyldecanoyloxy)-)hexyl)-N-(4-hydroxybutyl)hexane-1-ammonium salt, and its structure is shown below: .

[0116] Table 17 Prescription for LNP-01

[0117] Table 18 Prescription for LNP-0315

[0118] Dynamic light scattering (“DLS”) was used to determine the mean particle size and polydispersity index (“PDI”) of the LNP samples. The mean particle size and polydispersity were measured using a Malvern Zetasizer DLS instrument. Prior to testing, the LNP samples were diluted in 1x PBS at a volume ratio of 1:99. The mean hydrodynamic diameter of each sample was measured by reporting the mean particle size and PDI. The zeta potential of the LNPs was also measured using a Malvern Zetasizer instrument. The samples were diluted in 10 mM NaCl at a volume ratio of 1:19 prior to measurement.

[0119] Fluorescence-based assays (Ribogreen®, ThermoFisher Scientific) were used to determine encapsulation efficiency, calculated as (Total RNA - Free RNA) / Total RNA. LNP samples were diluted in 1x TE buffer with 1% Triton-X 100 added to achieve an appropriate concentration to determine total RNA, or diluted in 1x TE buffer to determine free RNA. Standard curves were prepared using the starting RNA solution used to prepare the formulation, following the manufacturer's instructions. Ribogreen® dye was then added to each standard and sample, and they were incubated at room temperature in the dark for approximately 5 minutes. Samples were read using a Tecan INFINITE 200 PRO with excitation at 480 nm and emission at 520 nm. Total RNA and free RNA were calculated from the appropriate standard curve by subtracting the fluorescence value of the reagent blank from the fluorescence value of each sample.

[0120] Typically, when preparing LNPs, the encapsulation efficiency is greater than 80%, the particle size is less than 120 nanometers, and the PDI is less than 0.2.

[0121] Example 2: SpCas9 mRNA sequence optimization

[0122] Primary human liver hepatocytes (PHH) were thawed and resuspended in hepatocyte thawing medium supplemented with hepatocyte supplements (Lonza, Cat. MCHT50), and then centrifuged at 100 g for 10 min. The supernatant was discarded, and the precipitated cells were resuspended in hepatocyte seeding medium (Lonza, Cat. MP100) with 10% fetal bovine serum added. After cell counting, cells were seeded into 96-well ultra-low adsorption cell culture plates (Liver Biotech, Cat. LV-ULA002-96W) at a seeding density of 40,000 cells / well. The seeded cells were incubated at 37°C and 5% CO2 for 24 hours to settle and adhere. After incubation, cell monolayer formation was checked, and the medium was replaced with hepatocyte culture medium (Lonza, Cat. CC-3198) with 10% fetal bovine serum. Cells were sequentially transfected with a lipid complex containing SpCas9 mRNA / sgRNA (6.25 / 1.56 nM sgRNAsg5815-3, mRNA / sgRNA = 2:1 w / w), Lipofectamine RNAiMax (0.3 μL / well, Invitrogen™), and OptiMem, according to the manufacturer's instructions. The culture plates were incubated at 37°C and 5% CO2, and cells were harvested on day 3 post-transfection. QuickExtract was used according to the manufacturer's instructions. TM DNA Extraction Solution 1.0 was used to extract total genomic DNA separately. Editing efficiency was measured using next-generation sequencing (NGS).

[0123] Figures 1 and 2 show the editing efficiency of SpCas9 mRNA in PHH cells. The mRNA sequences used were m5815-1, SEQ ID NO:233 / CAS001D(327), SEQ ID NO:236 / CAS001F(331), and SEQ ID NO:238. The results indicate that SpCas9 mRNA CAS001F had the highest editing efficiency.

[0124] Example 3: LNP delivery to mice with WT (mouse) TTR

[0125] The selected guide RNA, mRNA, and LNP formulations were further tested in mice. Mice used in each study involving mice ranged in age from 6 to 12 weeks. LNPs were administered via lateral injection into the tail vein. Animals were observed for adverse reactions after administration. Body weight was measured 24 hours prior to administration, and animals were euthanized by blood loss, anesthesia with isoflurane, or carbon dioxide asphyxiation after administration. Blood was collected from the inner canthal vein and deposited in a serum separator. LNPs were prepared as described in Example 1.

[0126] For studies involving in vivo editing, liver tissue was collected from the left lobe of each animal. Genomic DNA was extracted using a nucleic acid extraction kit (Denogen, DNS033-48). All DNA samples were subjected to Sanger sequencing and NGS analysis as described in Example 2.

[0127] The data in Figures 3 and 4 are from WT mice (n=5 per group) that were administered LNP0315 containing SpCas9 mRNA m5815-1, SEQ ID NO: 233 / m5815-9, SEQ ID NO: 240 / CAS001F, SEQ ID NO: 238, and sgRNA sg5815-1 (SEQ ID NO: 241) at a dose of 0.3 MPK (total RNA content, mRNA / sgRNA = 2:1 w / w) and were euthanized 6 days after administration. Samples were processed as described above.

[0128] Example 4

[0129] method

[0130] In vitro transcription (IVT)

[0131] 1. Template DNA Preparation

[0132] 1.1 Plasmid Construction

[0133] The mRNA was synthesized via IVT from a linearized plasmid DNA template. The plasmid template was constructed using pVAX1 as a backbone. Within the plasmid, the T7 promoter was linked to the 5' untranslated region (UTR), a Kozak sequence, SpCas9 CDS or SaCas9CDS or a firefly luciferase reporter gene, a 3' UTR, and a poly(A) sequence of less than A100 or a separated poly(A)A60+6dNTP+A60. Plasmid construction was performed using Gibson splicing or Golden Gate splicing.

[0134] 1.2 Plasmid Extraction

[0135] P inoculated E. coli strains containing plasmid DNA templates in glycerol onto fresh LB agar plates containing the appropriate antibiotics (ampicillin 100 µg / mL or kanamycin 100 µg / mL) and incubated overnight at 37°C.

[0136] Take a single colony from the plate and inoculate it with 20 / 200 mL of LB liquid medium. Incubate overnight at 37°C and 220 rpm.

[0137] Plasmids were extracted using commercial plasmid extraction kits (TIANGEN Endotoxin Removal Plasmid Medium Extraction Kit DP108 or Macherey-Nagel NucleoBond Xtra Midi EF Medium Endotoxin Removal Plasmid DNA Kit 740420.10).

[0138] 1.3 Plasmid linearization

[0139] According to the manufacturer's instructions, use BsaI, BspQI, Esp3I or NdeI (NEB) to linearize the plasmid template overnight at 37°C.

[0140] 1.4 Proteinase K Treatment

[0141] The linearized plasmid DNA template was treated with proteinase K (100–200 µg / mL) and 0.5% SDS at 50°C for 30 minutes to remove transcriptional repressors (such as RNase) that may have been introduced during plasmid extraction. The enzyme was then inactivated by heating at 65°C for 10 minutes.

[0142] 1.5 Linear plasmid purification

[0143] Subsequently, the restriction endonuclease digestion products were purified by phenol / chloroform extraction (equal volumes) and ethanol precipitation.

[0144] 1) Add 1 / 10 volume of 3 M sodium acetate and mix thoroughly.

[0145] 2) Add an equal volume of DNA extraction reagent (phenol:chloroform:isoamyl alcohol = 25:24:1), mix thoroughly, and let stand at room temperature for 3 minutes.

[0146] 3) Centrifuge at 12000 rpm for 15 minutes.

[0147] 4) Carefully transfer the supernatant into a new EP tube.

[0148] 5) Add an equal volume of isopropanol and precipitate at -20°C for at least 30 minutes.

[0149] 6) Centrifuge at 12000 rpm for 15 minutes to precipitate the linear plasmid DNA, and carefully discard the supernatant.

[0150] 7) Wash the DNA precipitate with 1 mL of 70% pre-cooled ethanol and centrifuge at 12000 rpm for 1 minute.

[0151] 8) Repeat the washing step once.

[0152] 9) Carefully remove any remaining liquid using a fine suction tip.

[0153] 10) Allow the sediment to settle and air dry for about 10 minutes, or longer if necessary.

[0154] 11) Resuspend the precipitate in 30–50 µL of nucleic acid-free water (if 20 µg of plasmid has been digested).

[0155] 12) Use Qubit or NanoDrop to determine the concentration of linear plasmids.

[0156] 1.6 In vitro transcription (IVT)

[0157] Co-transcriptional capping was performed using IVT reagents and a GAG cap provided by Hongene. A 20 µL IVT reaction contained ATP (10 mM), UTP (10 mM), GTP (10 mM), VTP (10 mM), GAG or GAG 3'oMe cap (10 mM), DNA template (1 µg), 5X reaction buffer (1X), and T7 enzyme mixture (1.5 µL). The reaction was assembled at room temperature, mixed by gentle tapping, and incubated at 37°C for 4 hours.

[0158] If using an ARCA cap for co-transcription capping, add an ARCA cap (final concentration 8 mM) to the IVT reaction, and add a final GTP concentration of 2 mM. The remaining components are the same as when using a GAG cap.

[0159] 1.7 DNase Digestion

[0160] After incubating at 37°C for 4 hours, DNase I (2 U for 20 µL of reaction) was added to the IVT reaction and incubated at 37°C for 15 minutes.

[0161] 1.8 mRNA purification

[0162] mRNA was purified by lithium chloride precipitation. The procedure was briefly described as follows: The reaction was terminated (20 ± 1 µL), and 30 µL of nucleic acid-free water and 30 µL of LiCl precipitation solution (7.5 M lithium chloride, 50 mM EDTA, final concentration 2.778 M (2.5–2.8 M)) were added to precipitate the RNA.

[0163] Mix thoroughly and refrigerate at -20°C for ≥30 minutes.

[0164] Centrifuge at 4°C for 15 minutes to precipitate RNA.

[0165] Carefully remove the supernatant, wash once with about 1 mL of pre-cooled 70% ethanol, and centrifuge again to remove as many unincorporated nucleotides as possible.

[0166] Carefully remove 70% ethanol and resuspend the RNA in a suitable solution or buffer†. After determining the RNA concentration, freeze at –20°C or –70°C.

[0167] 1.9 mRNA quantification

[0168] The final mRNA product can be quantified using fluorescent dyes (Qubit) or ultraviolet absorbance (NanoDrop). mRNA purity is assessed by agarose gel electrophoresis and capillary electrophoresis (using a 5300 fragment analyzer, Agilent).

[0169] 1.10 Assay of mRNA activity in HepG2 cells

[0170] HepG2 cells were cultured in Advanced DMEM (Gibco) containing 2 mM L-glutamine (GlutaMAX™-1, Gibco) and supplemented with 10% fetal bovine serum (GEMINI) at 37°C and 5% C. Cultured under the specified conditions. When transfecting mRNA, seed 2.0 × 10⁻⁶ mcg / well of a 24-well plate. HepG2 cells were transfected with 12.5 nM, 3.125 nM, or 1.56 nM gRNA and corresponding mRNA at a 1:1 (w / w) ratio using Lipofectamine RNAiMAX (Life Technologies). Cells were harvested after 72 hours, gDNA was extracted, and the editing efficiency at the target sites was analyzed by NGS or Sanger sequencing.

[0171] 1.11 Assay of Fluc mRNA activity in HEK293T cells

[0172] HEK293T cells were cultured in DMEM (corning) containing 2 mM L-glutamine (GlutaMAX™-1, Gibco) and supplemented with 10% fetal bovine serum (GEMINI) at 37°C and 5% C. Cultured under the specified conditions. When transfecting Fluc mRNA, seed 3.0 × 10⁻⁶ mcg per well of a 96-well plate. HEK293T cells were transfected with 50 ng of Fluc mRNA using Lipofectamine RNAiMAX (LifeBiotechnologies). At different time points post-transfection (e.g., 4 h, 6 h, 9 h, 21 h, and / or 24 h), each well was treated with the Bright-Lumi™ Firefly Luciferase Reporter Gene Detection Kit (Beyotime, China). Briefly: An equal volume of luciferase reagent was added to 100 µL of cell culture medium, mixed, and incubated at room temperature (approximately 25°C) for 2 minutes. The luminescence signal was then detected using a multi-mode microplate reader (INFINITE 200 PRO, TECAN).

[0173] Example 5

[0174] The 5' capping used in the mRNA sequences named GEB_mRNA_p318, GEB_mRNA_p917, and GEB_mRNA_p676 is from SyngeneBio. The product name is CAP 5 m7G(5')vppp(5')(2'OMeA)pG, and the catalog number is CAP5011. This capping has the following structure: .

[0175] Primary human hepatocytes (PHH) were thawed and resuspended in hepatocyte thawing medium supplemented with hepatocyte supplements (Lonza, catalog number MCHT50), followed by centrifugation at 100 g for 10 minutes. The supernatant was discarded, and the precipitated cells were resuspended in hepatocyte plating medium (Lonza, catalog number MP100) with 10% fetal bovine serum added. After cell counting, cells were seeded at a density of 40,000 cells / well in ultra-low adsorption 96-well cell culture plates (Liver Biotech, catalog number LV-ULA002-96W). After seeding, the plates were incubated at 37°C and 5% C. Cells were cultured in an incubator for 24 hours to allow them to settle and adhere. After culture, the cell monolayer was checked, and the culture medium was replaced with hepatocyte culture medium (Lonza, catalog number CC-3198) supplemented with 10% fetal bovine serum. Cells were then transfected sequentially with a lipid complex containing SpCas9 mRNA / sgRNA (0.12 nM sgRNA sg5815-3, mRNA / sgRNA = 1:1 w / w), Lipofectamine RNAiMax (0.3 μL / well, Invitrogen™), and OptiMem according to the manufacturer's instructions. The plates were incubated at 37°C, 5% C Cells were cultured under controlled conditions and harvested three days after transfection. Total genomic DNA was extracted using QuickExtract™ DNA Extraction Soln 1.0 according to the manufacturer's instructions. Editing efficiency was determined by next-generation sequencing (NGS). Figures 9A and 9B show the editing efficiency of mRNAs modified with different poly(A) structures in PHH cells, specifically A60+A60 and A95 segments, respectively.

[0176] The selected guide RNA, mRNA, and LNP formulations were further tested in mice. Mice aged 6–12 weeks were used in each mouse-involved experiment. LNP was administered via caudal vein injection. Animals were observed for adverse reactions after administration. Body weight was measured 24 hours prior to administration, and bloodletting or C was performed under isoflurane anesthesia after administration. Animals were euthanized by asphyxiation. Blood was collected from the internal canthal vein into serum separation tubes. LNPs were prepared as described in Example 1. For in vivo editing experiments, liver tissue was collected from the left lobe of each animal. Genomic DNA was isolated using a nucleic acid extraction kit (Denogen, catalog number DNS033-48). All DNA samples were prepared as described in Example 2 for Sanger sequencing and NGS analysis. Figure 9C shows the editing efficiency of different poly(A) structure modified mRNAs in CD-1 mice, specifically A60+A60 and A95 segments. The data shown in Figure 9C were obtained from wild-type mice (n=5 per group), which were administered LNP0315 containing SpCas9 mRNA and sgRNA sg5815-1 (SEQ ID NO: 241) at doses of 0.03 MPK (total RNA content, mRNA / sgRNA=2:1 w / w) or 0.1 MPK (total RNA content, mRNA / sgRNA=2:1 w / w) and sacrificed 6 days after administration. Sample processing was performed as described above.

[0177] Example 6

[0178] The selected guide RNA, mRNA, and LNP formulations were further tested in mice. Mice used in each study involving mice ranged in age from 6 to 12 weeks. LNPs were administered via lateral injection into the tail vein. Animals were observed for adverse reactions after administration. Body weight was measured 24 hours prior to administration, and animals were euthanized by blood loss, anesthesia with isoflurane, or carbon dioxide asphyxiation after administration. Blood was collected from the inner canthal vein and deposited in a serum separation tube. LNPs were prepared as described in Example 1. For studies involving in vivo editing, liver tissue was collected from the left lobe of each animal. Genomic DNA was extracted using a nucleic acid extraction kit (Denogen, DNS033-48). All DNA samples were prepared as described in Example 2 and subjected to Sanger sequencing and NGS analysis. Figure 10 shows the editing efficiency of mRNAs with different UTRs in CD-1 mice. The data in Figure 10 are from WT mice (n=5 per group) that were administered LNP0315, which contains SpCas9 mRNA and sgRNA sg5815-1 (SEQ ID NO: 241), at a dose of 0.1 MPK (total RNA content, mRNA / sgRNA = 2:1 w / w), and were euthanized 6 days after administration. Samples were processed as described above.

Claims

1. An mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the open reading frame comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 212, 213, 215, 225, 226, 227, 228, 229, 230, 231, 232 and 247.

2. The mRNA of claim 1, wherein the open reading frame comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 212, 213, 215, 225, 226, 227, 228, 229, 230, 231, 232, and 247.

3. The mRNA according to claim 1 or 2, wherein the mRNA further comprises a 5′ untranslated region (5′ UTR) having at least 90% sequence identity with any one of SEQ ID NOs: 110-171 or 1-62.

4. The mRNA according to any one of the preceding claims, wherein the mRNA further comprises a 5′ untranslated region (5′ UTR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs:110-171 or 1-62.

5. The mRNA according to any one of the preceding claims, wherein the mRNA further comprises a 3′ untranslated region (3′ UTR) having at least 90% sequence identity with any one of SEQ ID NOs:172-210 or 63-101.

6. The mRNA according to any one of the preceding claims, wherein the mRNA further comprises a 3′ untranslated region (3′ UTR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs:172-210 or 63-101.

7. The mRNA according to any one of the preceding claims, wherein the mRNA further comprises a poly-A tail having at least 90% sequence identity with any one of SEQ ID NO:243, 244, 245 or 246.

8. The mRNA according to any one of the preceding claims, wherein the mRNA further comprises a poly-A tail having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO:243, 244, 245, or 246.

9. An mRNA comprising a sequence having at least 98% sequence identity with any one of SEQ ID NOs: 233, 234, 235, 236, 237, 238, 239, 240, 250, 252 and 254.

10. The mRNA of claim 9, wherein the mRNA comprises a sequence selected from any one of SEQ ID NOs: 233, 234, 235, 236, 237, 238, 239, 240, 250, 252 and 254.

11. The mRNA according to any one of claims 1 to 10, wherein at least 10% of the uridine is replaced with a modified uridine, said modified uridine being selected from one or more of N1-methylpseudouridine, pseudouridine, 5-methoxyuridine, or 5-iodouridine.

12. An expression construct comprising a promoter operatively linked to a sequence encoding the mRNA of any one of claims 1-11, wherein the expression construct is optionally a plasmid expression construct.

13. An isolated host cell comprising the expression construct of claim 12.

14. A composition comprising the mRNA of any one of claims 1-11 and at least one guide RNA.

15. A lipid nanoparticle comprising the mRNA of any one of claims 1-11.

16. A pharmaceutical composition comprising the mRNA of any one of claims 1-13 and a pharmaceutically acceptable carrier.

17. A method for genome editing or target gene modification, comprising contacting cells with the mRNA of any one of claims 1-13; preferably, the method is an in vitro or ex vivo method, and / or the method does not include diagnostic, therapeutic, and surgical methods for treating humans or animals.

18. An artificial nucleic acid molecule comprising: a. At least one coding region encoding at least one CRISPR-related protein; b. At least one 5′ untranslated region (5′ UTR) element; and c. At least one 3′ untranslated region (3′ UTR) element, The coding region therein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 212, 213, 215, 225, 226, 227, 228, 229, 230, 231, 232, and 247.

19. The artificial nucleic acid molecule of claim 18, wherein the coding region further comprises at least one nuclear localization signal.

20. The artificial nucleic acid molecule according to claim 18 or 19, wherein the artificial nucleic acid molecule is RNA.

21. The artificial nucleic acid molecule of claim 20, wherein the RNA comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs:233, 234, 235, 236, 237, 238, 239, 240, 250, 252, and 254.

22. The artificial nucleic acid molecule according to claim 18 or 19, wherein the artificial nucleic acid molecule is DNA.

23. The artificial nucleic acid molecule of claim 22, wherein the DNA comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs:217-224 and 249-251.

24. A recombinant expression vector comprising the artificial nucleic acid molecule of any one of claims 18-23.

25. The recombinant expression vector of claim 24, wherein the artificial nucleic acid molecule is operatively linked to a promoter.

26. The recombinant expression vector according to claim 25, wherein the promoter is an inducible promoter.

27. The recombinant expression vector according to any of the preceding claims, wherein the artificial nucleic acid molecule further comprises a multiple cloning site.

28. An in vitro genetically modified host cell comprising the artificial nucleic acid molecule of any one of claims 18-23 or the recombinant expression vector of any one of claims 24-27.

29. A composition comprising an artificial nucleic acid molecule of any one of claims 18-23 or a recombinant expression vector of any one of claims 24-27, and a pharmaceutically acceptable vector and / or excipient.

30. The composition of claim 29, wherein the composition further comprises guide RNA or nucleic acid encoding thereon, the guide RNA being capable of targeting CRISPR-related proteins to a target DNA sequence.

31. The composition according to claim 30, wherein the guide RNA is a single-stranded guide RNA (sgRNA).

32. The composition according to claim 30, wherein the guide RNA is a double-stranded guide RNA (dgRNA).

33. A kit comprising an artificial nucleic acid molecule of any one of claims 18-23 or a recombinant expression vector of any one of claims 24-27, and optionally comprising a liquid carrier and / or comprising technical instructions regarding the administration and dosage information of the artificial nucleic acid molecule or the composition.

34. The kit of claim 33, further comprising guide RNA or nucleic acid encoding thereon, said guide RNA being capable of targeting a CRISPR-related protein to a target DNA sequence, or a regulatory element operatively linked thereto.

35. The kit according to claim 34, wherein the guide RNA is a single-stranded guide RNA (sgRNA).

36. The kit according to claim 34, wherein the guide RNA is a double-stranded guide RNA (dgRNA).

37. A method for inducing double-strand breaks (DSBs) and / or modifying a target gene, comprising delivering to cells an artificial nucleic acid molecule of any one of claims 18-23, a recombinant expression vector of any one of claims 24-27, a composition of any one of claims 29-32, or a kit of any one of claims 33-36.

38. A method for treating, preventing, or diagnosing a disease related to a target gene, comprising administering to a subject in need an artificial nucleic acid molecule of any one of claims 18-23, a recombinant expression vector of any one of claims 24-27, a composition of any one of claims 29-32, or a kit of any one of claims 33-36.

39. Use of the artificial nucleic acid molecule of any one of claims 18-23, the recombinant expression vector of any one of claims 24-27, the composition of any one of claims 29-32, or the kit of any one of claims 33-36 in the preparation of a medicament.

40. Use of the artificial nucleic acid molecule of any one of claims 18-23, the recombinant expression vector of any one of claims 24-27, the composition of any one of claims 29-32, or the kit of any one of claims 33-36 in gene therapy.

41. The use of the artificial nucleic acid molecule of any one of claims 18-23, the recombinant expression vector of any one of claims 24-27, the composition of any one of claims 29-32, or the kit of any one of claims 33-36 in inducing double-strand breaks (DSBs) in a target gene, modifying a target gene, and / or regulating the expression of a target gene in cells or subjects.

42. The artificial nucleic acid molecule of any one of claims 18-23, the recombinant expression vector of any one of claims 24-27, the composition of any one of claims 29-32, or the kit of any one of claims 33-36, for inducing double-strand breaks (DSBs) in a target gene, modifying a target gene, and / or regulating the expression of a target gene in cells or subjects.

43. The artificial nucleic acid molecule of any one of claims 18-23, the recombinant expression vector of any one of claims 24-27, the composition of any one of claims 29-32, or the kit of any one of claims 33-36, for the treatment, prevention, or diagnosis of diseases related to the target gene.

44. An artificial nucleic acid molecule, comprising: i. At least one coding region encoding at least one target peptide; ii. At least one 5′ untranslated region (5′ UTR) element comprising a DNA sequence having at least 90% sequence identity with any one of SEQ ID NOs: 110-171, or an RNA sequence having at least 90% sequence identity with any one of SEQ ID NOs: 1-62; iii. At least one 3′ untranslated region (3′ UTR) element comprising a DNA sequence having at least 90% sequence identity with any one of SEQ ID NOs: 172-210, or an RNA sequence having at least 90% sequence identity with any one of SEQ ID NOs: 63-101.

45. The artificial nucleic acid molecule of claim 44, wherein the 5′ untranslated region (5′ UTR) element comprises a DNA sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 110-171, or an RNA sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 1-62.

46. ​​The artificial nucleic acid molecule of claim 44, wherein the 3′ untranslated region (3′ UTR) element comprises a DNA sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 172-210, or an RNA sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NOs: 63-101.

Images