WO2024149283A1 - 具有CasRx活性的突变体及其应用 - Google Patents

具有CasRx活性的突变体及其应用 Download PDF

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WO2024149283A1
WO2024149283A1 PCT/CN2024/071517 CN2024071517W WO2024149283A1 WO 2024149283 A1 WO2024149283 A1 WO 2024149283A1 CN 2024071517 W CN2024071517 W CN 2024071517W WO 2024149283 A1 WO2024149283 A1 WO 2024149283A1
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mutant
aav
vector
disease
sequence
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张冰
杨平
娄应梅
吴硕
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Shanghai Jiao Tong University
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Definitions

  • the present invention relates to the field of molecular biology technology, and in particular to mutants with CasRx activity and applications thereof.
  • the system is composed of a single effector protein Cas13 and CRISPR RNA (crRNA) to form an RNA targeting effector complex guided by crRNA.
  • crRNA CRISPR RNA
  • crRNA CRISPR RNA
  • CRISPR/Cas13d Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 13d
  • Cas13d CRISPR/Cas13d nuclease Cas13d
  • 930 aa is a relatively small class II CRISPR effector found in mammalian cells (20% smaller than its family members Cas13a-c and 33% smaller than Cas9), making it easier to package into application vectors with limited capacity such as AAV vectors.
  • Cas13d's cutting of RNA targets does not depend on the PFS sequence (protospacer flanking sequence, equivalent to Cas9's PAM sequence for DNA), which greatly increases the application range of Cas13d and makes it a potential platform for further development of targeted RNA tools, which can achieve effective gene editing in multiple species, different cells and animals.
  • Cas13 proteases require crRNA to ensure their specificity in binding to target ssRNA and to exert their ribonuclease (RNase) activity under the guidance of crRNA.
  • Repeat Direct Repeat, DR
  • spacer spacer sequence that specifically targets the transcript.
  • Cas13d usually requires a 22-30nt guide RNA (guide RNA) to complement the target RNA and activate the nuclease domain for cutting.
  • guide RNA guide RNA
  • Cas13d can usually cut other non-specific RNAs.
  • Cas13d family protein CasRx can target one or two plant RNA viruses alone, show strong specificity for viral nucleic acids, and do not show non-specific RNase activity in plants.
  • RNA editors do not change the genetic material DNA.
  • Cas13d-mediated gene silencing does not change the genomic DNA. This gene silencing is reversible and safer, so it has broad therapeutic prospects in the field of gene therapy.
  • the Cas protease family is generally not used to treat genetic diseases caused by single base mismatches.
  • Single base mutations account for more than 40% of all genetic diseases. Such as hereditary cardiomyopathy, sickle cell anemia, thalassemia, hemophilia, progeria, phenylketonuria, glycogen storage disease, type I diabetes, familial hypercholesterolemia, hypertriglyceridemia, Krabbe disease, Gaucher disease, color blindness, hereditary deafness, progressive muscular dystrophy, and various tumors caused by single base mutations.
  • Inherited cardiomyopathy is a myocardial damage or functional decompensation disease mainly caused by mutations in sarcomere genes and metabolic-related genes.
  • the incidence rate is about 1/200, which is the main cause of sudden death in adolescents.
  • hypertrophic cardiomyopathy dilated cardiomyopathy
  • restrictive cardiomyopathy arrhythmogenic right ventricular cardiomyopathy, etc.
  • MYH7 myosin ⁇ -MHC
  • a mutant having CasRx activity which has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and has one or more mutations located at the following positions with reference to the amino acid position changes of SEQ ID NO: 1:
  • a molecular system which is a complex or composition, comprising (a) the mutant as described above and (b) a collection of guide RNA molecules (gRNA).
  • gRNA guide RNA molecules
  • nucleic acid construct encoding the molecular system as described above.
  • a vector comprising the polynucleotide as described above, or the nucleic acid construct as described above.
  • a delivery system comprising i) the molecular system as described above, and ii) a delivery vehicle.
  • composition comprising the delivery system as described above and a pharmaceutically acceptable excipient.
  • kit which comprises: the mutant as described above, or the molecular system as described above, or the vector as described above, or the delivery system as described above, or the pharmaceutical composition as described above, and optionally instructions for use of the kit.
  • the present invention relates to the use of the mutant as described above, or the molecular system as described above, or the vector as described above, or the delivery system as described above in the preparation of a drug for treating a disease caused by a gene mutation; wherein the disease is caused by a mutation comprising one or more single-base mutations, or can be treated by knocking down or knocking out a gene.
  • a molecular system which is a complex or composition, comprising a collection of mutants and guide RNA molecules (gRNA) as described above, wherein the nucleotide sequence of the gRNA is shown in any one of SEQ ID NO: 44 to 173.
  • gRNA guide RNA molecules
  • the present invention relates to the use of the molecular system as described above in the preparation of a drug for treating cardiomyopathy caused by MYH7 mutation.
  • FIG 1 shows the construction and genotype identification of Myh6-pR872H mice
  • A Schematic diagram of the construction of Myh6-pR872H single-base mutant mice using the CRISPR-Cas9 system
  • B Arms-PCR was used to identify the mouse genotype
  • C-E Sanger sequencing was used to identify the mouse genotype.
  • Figure 2 uses a fluorescent dual reporter system to prove that wild-type Cas13d cannot recognize and cleave single-base mutant RNA;
  • the PEST proline (P), glutamate (E), serine (S), and threonine (T) sequence is used to accelerate the degradation time of EGFP and mCherry; Cas13d plasmid and dual fluorescent reporter plasmid are co-transfected, and the knockdown efficiency of Cas13d on mutant transcripts and wild-type transcripts is analyzed by detecting the expression levels of EGFP and mCherry after 48 hours; BE.
  • Myh6-R872H Multiple mutant Myh6-specific sgRNAs designed by the displacement method cannot distinguish between wild-type and mutant Myh6, and have similar cleavage effects on both wild-type and mutant myh6; Proving that wild-type Cas13d cannot Distinguish Myh6 mRNA with single base mutations; Kd mu represents the knockdown efficiency of mutant Myh6 mRNA, and Kd wt represents the knockdown efficiency of wild-type Myh6 mRNA; Kd mu -Kd wt reflects the ability of Cas13d to distinguish mutant and wild-type mRNA.
  • Figure 3 shows the engineering modification of RfxCas13d to screen single amino acid mutants with single-base recognition ability;
  • A Homology modeling of RfxCas13d to find candidate nucleic acid binding sites near the nucleic acid groove;
  • B Predict the position where the candidate nucleic acid binding site binds to the target RNA and the complementary guide RNA "Spacer"region;
  • the candidate sites are mutated to alanine in turn to destroy the binding force with nucleic acids.
  • Kd mu represents the knockdown efficiency of mutant Myh6 mRNA
  • Kd wt represents the knockdown efficiency of wild-type Myh6 mRNA
  • Kd mu -Kdwt reflects the ability of different Cas13d mutants to distinguish between mutant and wild-type mRNA.
  • Figure 4 shows the optimized combination of mutants, and SuperCas13d that can accurately identify single-base mismatches was screened out; AF.
  • Kd mu represents the knockdown efficiency of mutant Myh6 mRNA, and
  • Kd wt represents the knockdown efficiency of wild-type Myh6 mRNA;
  • Kd mu -Kd wt reflects the ability of different Cas13d mutants to distinguish between mutant and wild-type mRNA; GJ.
  • Figure 5 shows a concentration-dependent and time-dependent in vitro cleavage experiment.
  • SuperCas13d significantly improves the cleavage efficiency and speed of mismatched RNA substrates.
  • Figure 6 is a specific window detection experiment. When there is a mismatch between the 15-21nt position of the gRNA spacer sequence and the substrate, the knockdown efficiency of SuperCas13d on the substrate is reduced.
  • Figure 7 is an intracellular para-cleavage activity detection experiment.
  • A Different Cas13d variants fused with GFP Expression, mutant Myh6 (muMyh6) and mCherry-PEST fusion expression. Different gRNAs were designed to target mCherry, PPIA or RPL4 RNA, and the fluorescence intensity of GFP and mCherry was analyzed by flow cytometry.
  • BD It shows that when targeting mCherry, wild-type Cas13d exhibits high non-specific cleavage of GFP, while SuperCas13d significantly reduces non-specific cleavage while retaining high cleavage activity, which is better than the reported high-fidelity variant hfCas13d.
  • EG qPCR was used to detect the knockout efficiency of the target gene and the side-cutting activity of other non-target genes.
  • Figure 8 is a molecular dynamics simulation experiment proving that SuperCas13d changes the interaction mode between the Cas protein and the RNA substrate;
  • A. GROMACS-2021 was used to perform molecular dynamics simulations on wild-type RfxCas13d, T486A-Cas13d, N641A-Cas13d, R648A-Cas13d and SuperCas13d, indicating that the three point mutations in SuperCas13d all weakened the binding of Cas13d to nucleic acids;
  • B is a molecular dynamics simulation experiment proving that SuperCas13d changes the interaction mode between the Cas protein and the RNA substrate.
  • Figure 9 is a molecular dynamics simulation that proves that SuperCas13d changes the binding mode of RNA and Cas protein to improve base resolution; analysis of the binding dynamics (RMSF) of wild-type RfxCas13d, T486A-Cas13d, N641A-Cas13d, R648A-Cas13d and SuperCas13dCas13d with each base of the guide RNA Spacer region (A) and the target RNA (B) shows that: N641A-Cas13d and R648A-Cas13d significantly increased the binding instability of Cas13d protein with bases 40 to 43 on the guide RNA, while having no obvious effect on the binding of Cas13d to the target RNA; T486A-Cas13d has no effect on the binding of guide RNA and target RNA; while SuperCas13d significantly increased the binding instability of Cas13d protein with bases 39 to 43 on the guide RNA, and significantly increased The binding instability of Cas13d
  • FIG. 10 shows that SuperCas13d can significantly improve the left ventricular hypertrophy phenotype of RH/+ mice in vivo;
  • A Experimental process of using adeno-associated virus to deliver SuperCas13d to treat left ventricular hypertrophy in RH/+ point mutation mice;
  • B-F respectively use HE staining (B), cardiac ultrasound (C-D), and electrocardiogram (E-F) to illustrate that RH/+ point mutation mice show left ventricular hypertrophy phenotypes after cyclosporine A (CsA) induction, including ventricular wall thickening, smaller heart chambers, increased ejection fraction, QTc prolongation and S wave abnormality in ECG; neonatal (P3-P7) injection of SuperCas13d significantly inhibited the development of RH/+ point mutation to left ventricular hypertrophy phenotype; LVPWd, diastolic left ventricular posterior wall thickness; LVIDd, diastolic left ventricular cavity diameter; IVSd
  • Figure 11 proves that SuperCas13d can also improve the ability to distinguish Myh6 R404Q single base mutation;
  • A. sgRNA is designed for the myh6 R404Q point mutation;
  • C is sgRNA is designed for the myh6 R404Q point mutation
  • SuperCas13d and sgRNA15 and sgRNA18 targeting R404Q can significantly reduce the cutting of wild-type Myh6 mRNA, while retaining the cutting efficiency of mutant R404Q mRNA, that is, SuperCas13d significantly improves the ability to distinguish single-base mutation mRNA from wild-type mRNA; Kd R404Q represents the knockdown efficiency of R404Q mRNA, and Kd wt represents the knockdown efficiency of wild-type Myh6 mRNA.
  • Figure 12 demonstrates that the ventricular hypertrophy phenotype of RH/RQ double-point heterozygous mutant mice develops earlier and is more severe;
  • A. HE staining shows that RH/RQ double-point heterozygous mutant mice show severe left ventricular hypertrophy phenotype at 4 months of age compared with RH/+ mice;
  • B. Masson's Trichrome staining shows that RH/RQ mice have significant left ventricular fibrosis at 12 months of age;
  • C. Cardiac ultrasound shows that RH/RQ mice have significantly increased ejection fraction, significantly increased left ventricular wall thickness, and significantly reduced left ventricular cavity diameter at 4 months of age compared with wild-type mice and RH/+ mice;
  • DG. ECG detection shows that at 2 months of age, Compared with wild-type mice (D and E), RH/RQ (F and G) mice had abnormal ECG waveforms, with decreased R wave voltage and increased S wave voltage.
  • Figure 13 proves that SuperCas13d can also significantly improve the ventricular hypertrophy phenotype of RH/RQ mice in vivo;
  • A AAV9-cTnT promoter-SuperCas13d treatment vector diagram;
  • B treatment experiment flow chart;
  • C-D Cardiac ultrasound detection results show that compared with RH/RQ mice injected with PBS, the left ventricular wall thickness, ventricular septum thickness, and ejection fraction of mice injected with AAV-SuperCas13d 4 months ago were significantly reduced, and the diameter of the left ventricular cavity was significantly increased, and there was no statistical difference with RH/+ mice;
  • E-F ECG detection results show that the ECG waveform of mice 4 months after injection of AAV-SuperCas13d is closer to that of RH/+ mice.
  • Figure 14 shows the histological detection of ventricular hypertrophy in RH/RQ mice treated with SuperCas13d.
  • A Overall image of the heart;
  • B Ratio of heart weight (HW) to body weight (BW);
  • C HE staining of the heart cross section shows that AAV-SuperCas13d significantly blocks the development of left ventricular hypertrophy phenotype in RH/RQ mice;
  • D-E WGA staining shows that AAV-SuperCas13d significantly reduces the cross-sectional area of cardiomyocytes;
  • F HE staining shows that AAV-SuperCas13d blocks the disordered arrangement of myocardial fibers;
  • G-H. Masson’s Trichrome staining shows that AAV-SuperCas13d blocks cardiac fibrosis; I-J. TUNEL staining shows that AAV-SuperCas13d blocks cardiac cell apoptosis.
  • Figure 15 shows ddPCR detection to illustrate that SuperCas13d specifically knocks out mutant Myh6 transcripts in mice.
  • A The efficiency of SuperCas13d knocking out wild-type Myh6 (WT) and Myh6 R87H mRNA 2 months after being injected into Myh6 R87H (RH/+)-CsA mice;
  • B The efficiency of SuperCas13d knocking out wild-type Myh6 R87H (RH) mRNA and Myh6 R404Q (RQ) mRNA 2 months after being injected into RH/RQ mice.
  • Figure 16 is an experiment of SuperCas13d targeting 43 pathogenic point mutations on the human MYH7 gene.
  • the technical solution of "A, and/or, B, and/or, C, and/or, D” includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").
  • the terms “increase,” “enhance,” and “enhance” describe an increase of at least about 5%, 10%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more compared to a control.
  • the terms “reduce,” “attenuate,” and “reduce” describe a decrease of at least about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100% compared to a control. In certain embodiments, the decrease may result in no or substantially no (i.e., a negligible amount, such as less than about 10% or even 5%) detectable activity or amount.
  • the present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2%, it can allow fluctuations within ⁇ 0.1%. For values that are large or do not require too fine control, it is also allowed to include greater fluctuations. For example, 100mM, it can allow fluctuations within the range of ⁇ 1%, ⁇ 2%, ⁇ 5%, etc. Involving molecular weight, it is allowed to include fluctuations of ⁇ 10%.
  • the term "about”, when referring to a measurable value such as an amount or concentration, is meant to include variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified value as well as the specified value.
  • “about X”, where X is a measurable value is meant to include X as well as variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of X.
  • the ranges of measurable values provided herein may include any other ranges and/or individual values therein.
  • the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.
  • a “native” or “wild-type” nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence.
  • a “wild-type mRNA” is an mRNA naturally occurring in an organism or endogenous to an organism.
  • a “homologous” nucleic acid sequence is a nucleotide sequence naturally associated with the host cell into which it is introduced.
  • nucleic acid refers to RNA or DNA that is linear or branched, single-stranded or double-stranded or a hybrid thereof.
  • the term also includes RNA/DNA hybrids.
  • less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc. can also be used for synthesis.
  • polynucleotides containing C-5 propyne analogs of uridine and cytidine bind RNA with high affinity.
  • nucleic acid molecules are shown in the 5' to 3' direction from left to right herein and are represented using the standard code for representing nucleotide symbols as specified in the ST.26 standard of the World Intellectual Property Organization (WIPO).
  • guide nucleic acid means a nucleic acid comprising at least one spacer sequence complementary to (and hybridizing with) a target nucleic acid and at least one repeat sequence (such as a direct repeat sequence), wherein the repeat sequence can be connected to the 5' end and/or 3' end of the spacer sequence.
  • the design of the gRNA of the present invention can be based on the Cas13d system.
  • the repeat sequence comprises at least 10 nucleotides, depending on the particular repeat and whether the guide RNA comprising the repeat is processed or unprocessed (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 to 100 or more nucleotides or any range or value therein).
  • the guide RNA comprising the repeat is processed or unprocessed (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 to 100 or more nucleotides or any range or value therein).
  • the repetitive sequence comprises, consists essentially of, or consists of about 10 to about 20, about 10 to about 30, about 10 to about 45, about 10 to about 50, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 40, about 40 to about 80, about 50 to about 100, or more nucleotides.
  • the repeat sequence attached to the 5' end of the spacer sequence can include a portion of the repeat sequence (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more consecutive nucleotides of the wild-type repeat sequence).
  • the portion of the repeat sequence attached to the 5' end of the spacer sequence can be about 5 to about 10 consecutive nucleotides in length (e.g., about 5, 6, 7, 8, 9, 10 nucleotides) and has at least 90% identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the same region (e.g., the 5' end) of the wild-type CRISPR Cas repeat nucleotide sequence.
  • a portion of the repeat sequence may comprise a pseudoknot-like structure (eg, a "handle-like structure") at its 5' end.
  • a "spacer sequence” is a nucleotide sequence that is complementary to a target nucleic acid (such as a target ssRNA).
  • the spacer sequence can be completely complementary or substantially complementary to the target nucleic acid (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more complementary, and any range or value therein)).
  • 70% complementary e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • the spacer sequence can be completely complementary or substantially complementary to the target nucleic acid (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more complementary)).
  • the spacer sequence can have one, two, three, four or five mispairings, which can be continuous or non-continuous.
  • the spacer sequence can have about 70% complementarity with the target nucleic acid. In other embodiments, the spacer nucleotide sequence can have about 80% complementarity with the target nucleic acid. In other embodiments, the spacer nucleotide sequence can have about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% complementarity with the target nucleic acid (pre-spacer sequence). In some embodiments, the spacer sequence is 100% complementary to the target nucleic acid.
  • the length of the spacer sequence can be about 15 nucleotides to about 30 nucleotides (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or any range or value therein).
  • the spacer sequence can have complete complementarity or substantial complementarity on a target nucleic acid region (e.g., a pre-spacer sequence), and the target nucleic acid region length can be at least about 15 nucleotides to about 30 nucleotides.
  • the length of the spacer can be about 20, 21, 22, 23, 24, or 25 nucleotides.
  • the length of the spacer region can be 23 nucleotides.
  • sgRNA is used to refer to a spacer sequence.
  • the direct repeat sequence adopts the sequence shown in SEQ ID NO: 39.
  • mutations can be introduced into the CRISPR system so that the CRISPR system can distinguish between a target sequence having greater than 80%, 85%, 90% or 95% complementarity and an off-target sequence.
  • the degree of complementarity is from 80% to 95%, for example, about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% (for example, distinguishing a target having 18 nucleotides from an off-target of 18 nucleotides having 1, 2 or 3 mismatches).
  • the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 99.9%. In some embodiments, the degree of complementarity is 100%.
  • target nucleic acid refers to a region in the genome of an organism that is fully complementary (100% complementary) or substantially complementary (e.g., at least 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and any range or value therein)) to the spacer sequence in the gRNA of the invention.
  • 70% complementary e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the target region is at least 15 consecutive nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length, and any range or value therein; for example, about 19 to about 25 nucleotides, about 20 to about 24 nucleotides, etc.).
  • the target RNA can be any suitable form of RNA, including but not limited to mRNA, tRNA, ribosomal RNA (rRNA), microRNA (miRNA), interfering RNA (siRNA), ribozyme, riboswitch, satellite RNA, microswitch, microzyme, or viral RNA.
  • the target nucleic acid is associated with a disorder or disease.
  • mutant refers to point mutations (e.g., missense or nonsense, or insertions or deletions of single base pairs resulting in a frameshift), insertions, deletions and/or truncations.
  • mutation is typically described by determining the original residue, then determining the position of the residue in the sequence, and determining the identity of the newly substituted residue.
  • sequence identity means that two polynucleotide or amino acid sequences are identical over a comparison window (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis).
  • sequence identity percentage is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, U, or I) or residue appears in the two sequences, thereby obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.
  • nucleic acid base e.g., A, T, C, G, U, or I
  • the term "substantial identity” refers to a feature of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises at least A sequence having at least 85% sequence identity, preferably at least 90% to 95% sequence identity, more usually at least 99% sequence identity, compared to a reference sequence over a comparison window of 18 nucleotide (6 amino acid) positions, usually over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that may include deletions or additions that total 20% or less of the reference sequence within the comparison window.
  • the reference sequence may be a subset of a larger sequence.
  • Cas13d is generally used to broadly refer to wild-type and/or mutant Cas13d proteins, and depending on the context, a more common scenario is used to refer to wild-type and mutant CasRx proteins. Mutated CasRx proteins are also referred to herein as “mutants”, “mutants with CasRx activity”, “CasRx mutants”, “CasRx variants”, etc. "SuperCas13d” specifically refers to CasRx mutants with three point mutations of N641, T486, and R648.
  • AAV refers to adeno-associated virus.
  • AAV can be used to refer to the virus itself or its derivatives, such as but not limited to viral capsids, viral genomes, viral particles, viral fragments and combinations thereof.
  • AAV includes all naturally occurring and recombinant subtypes and variants thereof, unless otherwise required.
  • a naturally occurring form of AAV refers to any adeno-associated virus or its derivatives, which comprises a viral capsid composed of naturally occurring viral capsid proteins.
  • Non-limiting examples of naturally occurring AAV include any one or more of AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV2-6), AAV type 7 (AAV1-7), AAV type 8 (AAV6-8), AAV9, AAV10, AAV11, AAV12, AAV13, and rh10.
  • the source of AAV can be avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV.
  • Prime AAV refers to AAV that infects primates
  • non-primate AAV refers to AAV that infects non-primate mammals
  • bovine AAV refers to AAV that infects bovine mammals
  • Recombinant AAV or “rAAV” includes any AAV that contains a heterologous polynucleotide sequence in its viral genome.
  • Other examples of AAV serotypes and variants that can be used as vectors include, but are not limited to, any one or more of AAVDJ, AAV-PHP.S, AAV-PHP.B, AAV-PHP.eB and Anc80.
  • the base editors mentioned in this article are those composed of DNA editing tools (such as Cas9 or Cas12) and The deaminase fused protein can achieve base conversion at the DNA level, such as ABE and CBE.
  • the present invention relates to a mutant having CasRx activity, which has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and has one or more mutations located at the following positions with reference to the amino acid position changes of SEQ ID NO: 1:
  • the mutation at a specific position can be any amino acid other than its wild-type site.
  • the R648 mutation means that the amino acid at position 648 can mutate to any one of G, A, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, and H;
  • the N641 mutation means that the amino acid at position 641 can mutate to any one of G, A, V, L, I, P, F, Y, W, S, T, C, M, Q, D, E, K, R, and H;
  • the T486 mutation means that the amino acid at position 486 can mutate to any one of G, A, V, L, I, P, F, Y, W, S, C, M, N, Q, D, E, K, R, and H.
  • the amino acid after mutation is not a conservative replacement of the amino acid before mutation.
  • Constant replacement means that one amino acid is replaced by another amino acid with common properties.
  • a method for functionally defining the common properties between individual amino acids is to analyze the standardized frequencies of amino acid changes between corresponding proteins in homologous organisms (Schulz (1979) Principles of Protein Structure, Springer-Verlag). Based on such an analysis, families of amino acids can be determined, in which amino acids within the family preferentially replace each other, so that their effects on the overall structure of the protein are most similar (Schulz (1979) supra).
  • Examples of groups of amino acids defined in this way include: “charged/polar family”, including Glu, Asp, Asn, Gln, Lys, Arg and His; “aromatic or cyclic family”, including Pro, Phe, Tyr and Trp; and “aliphatic family”, including Gly, Ala, Val, Leu, Ile, Met, Ser, Thr and Cys.
  • subfamilies can also be determined.
  • the family of charged/polar amino acids can be subdivided into subfamilies, including: the "positive charge subfamilies” including Lys, Arg, and His; the “negative charge subfamilies” including Glu and Asp; and the "polar subfamilies” including Asn and Gln.
  • the aromatic or cyclic families can be subdivided into subfamilies, including: the "nitrogen ring subfamilies”, including Pro, His, and Trp; and the “phenyl subfamilies”, including Phe and Tyr.
  • the aliphatics can be subdivided into subfamilies, including: the “large aliphatic nonpolar subfamilies”, including Val, Leu, and Ile; the “aliphatic slightly polar subfamilies”, including Met, Ser, Thr, and Cys; and the "small residue subfamilies” including Gly and Ala.
  • conservative mutations include amino acid substitutions of amino acids within the above subfamilies, such as, but not limited to: Lys for Arg or vice versa to maintain positive charge; Glu for Asp or vice versa to maintain negative charge; Ser for Thr or vice versa to maintain free -OH; Gln for Asn or vice versa to maintain free -NH2 .
  • the mutant has one or two mutations located at the following positions:
  • the mutant has one or two mutations located at the following positions:
  • the mutant has one or two of the following mutations:
  • the mutant has one or two of the following mutations:
  • the mutant has a mutation at the following position:
  • the mutant has the following mutations N641A, T486A and R648A.
  • the mutant has the following mutations N641A, T486A and R648A; and has one or more mutations located at the following positions:
  • the mutant is shown in SEQ ID NO: 2.
  • the mutant has significantly improved mutant differentiation and in addition to the mutations described above, the mutant may further contain one or more additional substitutions, deletions or insertions, preferably the substitutions, deletions or insertions together result in the mutant: (1) having gRNA-specific nuclease activity that is substantially the same as SEQ ID NO: 1 or 2 (e.g., at least about 80%, 90%, 95%, 99% or more), and/or (2) having substantially no (e.g., at most 20%, 15%, 10%, 5%) side-cutting (non-gRNA-dependent) nuclease activity.
  • the mutant has a significantly improved ability to distinguish mutant and wild-type transcripts associated with single base mutations.
  • the binding instability of the complex composed of the mutant and the guide RNA molecule (gRNA) to the target RNA is increased.
  • the mutant has a significantly reduced negative correlation between the nucleic acid recognition domain and the nuclease domain; wherein the nucleic acid recognition domain includes a Helical1 domain, and the nuclease domain includes a Helical2 and HEPN domains.
  • the mutant substantially lacks flanking endonuclease activity.
  • the mutant of the present invention after the RNP complex of the mutant of the present invention binds to the target RNA, the mutant does not exhibit substantial (or detectable) para-RNase activity.
  • the mutant retains the ability to bind gRNA.
  • the mutant retains gRNA-activated RNase activity.
  • the derivatives retain the ability to bind to and/or cleave a target RNA in the presence of a bound guide/crRNA that is complementary in sequence to at least a portion of the target RNA.
  • the present invention also relates to an isolated polynucleotide encoding a mutant as described above.
  • the polynucleotide comprises a regulatory element, and the regulatory element is operably associated with the portion encoding the mutant.
  • the polynucleotides or nucleic acid constructs of the present invention can be operably associated with a variety of regulatory elements for expression in cells.
  • the regulatory elements include one or more of the following elements: promoters, introns, enhancers, terminators, 5' and 3' untranslated regions, nuclear localization signal (NLS) sequences, or nuclear export signals (NES).
  • NLS nuclear localization signal
  • NES nuclear export signals
  • the number of each can be one or more; preferably at least a promoter is included.
  • the choice of promoter can vary according to the temporal and spatial requirements of expression, and can also be based on the cell to be transformed.
  • the promoter of the host cell of interest can be varied. The promoters used for many different organisms are well known in the art.
  • suitable promoters can be selected for interested specific host organisms. Therefore, for example, a lot of promoters upstream of highly constitutively expressed genes in model organisms (such as Arabidopsis thaliana, nematodes, yeast, fruit flies, mice, rats, etc.) are known, and this knowledge can be easily obtained and implemented in other systems under appropriate circumstances.
  • model organisms such as Arabidopsis thaliana, nematodes, yeast, fruit flies, mice, rats, etc.
  • the promoter comprises a U6 promoter.
  • the promoter is a tissue-specific promoter.
  • tissue-specific promoters include, but are not limited to, B29 promoter (B cell expression), runt transcription factor (CBFa2) promoter (stem cell specific expression), CD14 promoter (monocyte expression), CD43 promoter (leukocyte and platelet expression), CD45 promoter (hematopoietic cell expression), CD68 promoter (macrophage expression), CYP4503A4 or ALB promoter (hepatocyte expression), desmin promoter (muscle cell expression), elastase I promoter (pancreatic acinar cell expression), endoglin promoter (endothelial cell expression), fibroblast-specific protein 1 promoter (FSPl) promoter (fibroblast expression), fibronectin promoter (fibroblast expression), fms-associated tyrosine kinase 1 (FLT1) promoter (endothelial cell expression), glial fibrillary acidic protein (GFAP) promoter (B29 promoter (B cell expression
  • the promoter is a cell-free specific promoter.
  • exemplary cell-free specific promoters include, but are not limited to, the cytomegalovirus (CMV) very early promoter, the viral simian virus 40 (SV40) (e.g., early or late), the Moloney murine leukemia virus (MLV) promoter, the SV40 ...
  • CMV cytomegalovirus
  • SV40 viral simian virus 40
  • MMV Moloney murine leukemia virus
  • MoMLV Rous sarcoma virus
  • RSV Rous sarcoma virus
  • HSV herpes simplex virus
  • vaccinia virus H5 P7.5 and P11 promoters
  • EF1a elongation factor 1-alpha
  • EGR1 early growth response I
  • FerH ferritin H
  • FerL ferritin L
  • GPDH 3-phosphoglyceraldehyde dehydrogenase
  • EIF4A1 heat shock 70kDa protein 5
  • HSPA5 heat shock protein 90kDa-beta member 1
  • HSP70 heat shock protein 70kDa
  • ⁇ -kinesin ⁇ -KIN
  • human R0SA26 locus Irions et al., (2007) Nature Biotechnology 25, 1477-1482
  • UBC ubiquitin C promoter
  • the vector of the present invention may also contain genes used for screening (e.g., antibiotic resistance genes), such as nucleic acid fragments for generating fluorescent proteins.
  • Fluorescent proteins may be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein.
  • Green fluorescent protein may use common GFP, or may use modified GFP gene, such as enhanced GFP gene EGFP, etc.; blue fluorescent protein may be selected from EBFP, Azuritc, TagBFP, etc.; yellow fluorescent protein may be selected from EYFP, Ypct, PhiYFP, etc.; orange fluorescent protein may be selected from mKO, mOrange, mBanana, etc.; red fluorescent protein may be selected from TagRFP, mRuby, mCherry, mKatc, etc.
  • operably linked or “operably associated” when referring to polynucleotides means that the designated elements are functionally related to each other, and usually physically related as well.
  • operably linked or “operably associated,” as used herein, refer to nucleotide sequences that are functionally associated on a single nucleic acid molecule.
  • a first nucleotide sequence that is operably linked to a second nucleotide sequence means when the first nucleotide sequence is in a functional relationship with the second nucleotide sequence.
  • a promoter is operably associated with a nucleotide sequence if it affects the transcription or expression of the nucleotide sequence.
  • a regulatory sequence e.g., a promoter
  • a promoter need not be contiguous with the nucleotide sequence with which it is operably associated, as long as the function of the regulatory sequence is to direct its expression.
  • An intervening untranslated but transcribed nucleic acid sequence may be present between the promoter and the nucleotide sequence, and the promoter can still be considered "operably linked" to the nucleotide sequence.
  • polypeptide refers to the attachment of one polypeptide to another polypeptide.
  • a polypeptide can be linked to another polypeptide directly (e.g., via a peptide bond) or through a linker (at the N-terminus or C-terminus).
  • the polynucleotide is codon optimized for expression in an organism.
  • nucleotide sequence, polynucleotide and/or nucleic acid construct of the present invention can be codon optimized for expression in any organism of interest. Codon optimization is well known in the art and involves modifying nucleotide sequences for codon usage preferences using species-specific codon usage tables. Codon usage tables are generated based on sequence analysis of the highest expressed genes of the organism/species of interest. When the nucleotide sequence is to be expressed in the nucleus, the codon usage table is generated based on sequence analysis of highly expressed nuclear genes of the species of interest. Modification of the nucleotide sequence is determined by comparing the species-specific codon usage table with the codons present in the native polynucleotide sequence.
  • codon optimization of a nucleotide sequence results in a nucleotide sequence that has less than 100% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%, and any range or value therein) with a native nucleotide sequence (or nucleotide sequence before optimization), but it still encodes a polypeptide that has the same function as the polypeptide encoded by the original native nucleotide sequence.
  • polynucleotides, nucleic acid constructs, expression cassettes and/or vectors of the invention are codon-optimized for expression in a particular species of interest, such as a particular plant species, a particular bacterial species, a particular animal species, etc.
  • codon-optimized nucleic acid constructs, polynucleotides, expression cassettes and/or vectors of the invention have about 70% to about 99.9% (e.g., about 70%, 71%, 72%, 73%, 74%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 99%, 90%, 99%, 90%, 99%, 90%, 99%, 90%, 99%, 90%, 91%, 99%, 99%, 99%, 90 ... 97%, 98%, 99%, 99.5%, 99.9% or 100% identity) or more.
  • the mutant with CasRx activity of the present invention and the polynucleotides and nucleic acid constructs encoding it can be used to modify the target nucleic acid in any organism, including but not limited to animals, plants, fungi, archaea or bacteria.
  • Animals may include but are not limited to mammals, insects, fish, birds, etc.
  • Exemplary mammals that the present invention can be used for include but are not limited to primates (humans, and non-humans such as chimpanzees, baboons, monkeys, gorillas, etc.), cats, dogs, mice, rats, ferrets, gerbils, hamsters, cattle, pigs, donkeys, horses, goats, pandas, elephants or sheep.
  • Plants can be angiosperms, gymnosperms, monocots, dicots, C3, C4, CAM plants, bryophytes, ferns and/or pseudoferns, microalgae and/or macroalgae.
  • the present invention also relates to a molecular system, which is a complex or composition, comprising (a) a mutant as described above and (b) a collection of guide RNA molecules (gRNA).
  • a molecular system which is a complex or composition, comprising (a) a mutant as described above and (b) a collection of guide RNA molecules (gRNA).
  • the types of gRNA in the molecular system of the present invention can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.
  • the gRNA may further comprise at least one detectable marker.
  • the detectable marker may be a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags or suitable fluorescent dyes), a detection label (e.g., biotin, digoxigenin, etc.), quantum dots or gold particles.
  • the gRNA may comprise standard ribonucleotides and/or modified ribonucleotides. In some embodiments, the gRNA may comprise standard or modified deoxyribonucleotides. In embodiments where the gRNA is enzymatically synthesized (i.e., in vivo or in vitro), the gRNA generally comprises standard ribonucleotides. In embodiments where the gRNA is chemically synthesized, the gRNA may comprise standard or modified ribonucleotides and/or deoxyribonucleotides.
  • Modified ribonucleotides and/or deoxyribonucleotides include base modifications (e.g., pseudouridine, 2-thiouridine, N6-methyladenosine,
  • the gRNA backbone can also be modified to include phosphorothioate linkages, boranophosphate linkages, or peptide nucleic acids.
  • a composition refers to a composition in which the components (including mutants and gRNA) are loosely combined or mixed together, for example, they are respectively loaded in different carriers, respectively packaged in different containers, or mixed together but not bound by strong intermolecular forces.
  • a complex refers to a composition in which the components (including mutants and gRNA) are combined in the form of ribonucleoprotein (RNP) and have RNA cleavage activity.
  • the present invention also relates to nucleic acid constructs encoding the molecular system as described above.
  • the present invention also relates to a vector comprising the polynucleotide as described above, or the nucleic acid construct as described above.
  • the vector may be a composition, for example, a virus or plasmid containing different polynucleotides (respectively expressing mutants with CasRx activity and other components, such as crRNA),
  • vector refers to a macromolecule or biomacromolecule association complex comprising or associated with a polynucleotide, which can be used to mediate the transfer, delivery or introduction of a polynucleotide to a cell.
  • the vectors used to transform host organisms are well known in the art.
  • Non-limiting examples of general vector categories include viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, bacteriophages, artificial chromosomes, minicircle vectors or Agrobacterium (Agrobacterium) binary vectors, in double-stranded or single-stranded linear or circular forms, which may or may not be self-transmitted or mobile.
  • the vector is a viral vector.
  • the viral vector may include but is not limited to adenoviral vectors, adeno-associated virus (AAV) vectors, lentiviral vectors or retroviral vectors.
  • the vector is an AAV vector.
  • AAV is a small virus that infects humans and some other primate species.
  • AAV is not known to cause disease, and when it does, it has been shown to cause only a mild immune response.
  • AAV can infect both dividing and non-dividing cells. Cells, and can incorporate their genome into the genome of the host cell.
  • adeno-associated viruses mostly remain in a free state (that is, they can replicate in the host without incorporating their payload into the host chromosome); long-term stable expression. These characteristics make adeno-associated viruses a suitable candidate for creating viral vectors for gene therapy. Therefore, in one example, the viral vector is an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • the AAV vector is but is not limited to AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV9, AAV10, AAV11, AAV12, AAV13, rh10, AAVDJ, AAV-PHP.S, AAV-PHP.B, AAV-PHP.eB and Anc80.
  • the AAV vector serotype can be matched to the target cell type.
  • Table 2 of WO 2018002719A1 lists exemplary cell types that can be transduced by a specified AAV serotype (incorporated herein by reference).
  • recombinant AAV can be produced using a triple transfection method (described in detail in U.S. Pat. No. 6,001,650).
  • recombinant AAV is produced by transfecting host cells with a recombinant AAV vector (containing a gene of interest) to be packaged into AAV particles, an AAV helper function vector, and an auxiliary function vector.
  • the AAV helper function vector encodes "AAV helper function" sequences (e.g., rep and cap) that act in trans for productive AAV replication and encapsidation.
  • the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes).
  • the auxiliary function vector encodes nucleotide sequences for non-AAV-derived viral and/or cellular functions (e.g., "auxiliary functions") that AAV relies on for replication.
  • Auxiliary functions include those required for AAV replication, including but not limited to those parts involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and AAV capsid assembly.
  • the viral-based helper function may be derived from any of the known helper viruses, such as adenovirus, herpes virus (except herpes simplex virus-1), and vaccinia virus.
  • the AAV genome comprises a 5'AAV ITR sequence and a 3'AAV ITR sequence.
  • baculovirus packaged in insect cells is used.
  • the expression system produces the AAV virus particles of the present invention. See, for example, WO 2007046703, WO 2007148971, WO 2009014445, WO 2009104964, WO 2013036118, WO 2011112089, WO 2016083560, WO 2015137802 and WO 2019016349, all of which are incorporated herein by reference.
  • Vector titer is typically expressed as viral genomes/ml (vg/ml). In certain embodiments, the viral titer is greater than 1 ⁇ 10 9 , greater than 5 ⁇ 10 10 , greater than 1 ⁇ 10 11 , greater than 5 ⁇ 10 11 , greater than 1 ⁇ 10 12 , greater than 5 ⁇ 10 12 , or greater than 1 ⁇ 10 13 vg/ml.
  • the present invention also relates to a delivery system comprising i) a molecular system as described above, and ii) a delivery vehicle.
  • the delivery vehicle comprises one or more liposomes, one or more exosomes, one or more microvesicles, one or more dendrimers, one or more inorganic nanoparticles, one or more cell-penetrating peptides, a gene gun, one or more plasmids, one or more viral vectors (in some embodiments, the viral vector is as defined above), and a group consisting of them.
  • Liposomes can be cationic liposomes or neutral liposomes, which can be prepared or modified by known methods, for example, adding polyethylene glycol (PEG) modified liposomes can effectively prevent the aggregation of liposome carriers and increase their stability.
  • Liposomes or lipid transfection preparations can be prepared by methods known to those skilled in the art. Such methods are described in, for example, WO 2016205764 and U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, each of which is incorporated herein by reference in its entirety.
  • Dendrimers are a special family of polymers with a well-defined molecular structure, precisely controllable chemical structure, and unique multivalent properties, and are gradually becoming non-viral vectors for gene delivery.
  • Typical dendrimers are poly(amidoamine) (PAMAM) dendrimers, which can be further modified, such as modifying the nucleobase analog 2-amino-6-chloropurine on the surface of PAMAM to construct a derivative AP-PAMAM, or preparing CS-PAMAM by coupling chondroitin sulfate (CS) with PAMAM, and so on.
  • PAMAM poly(amidoamine)
  • CS-PAMAM chondroitin sulfate
  • Inorganic nanoparticles may include gold nanoparticles (AuNPs), magnetic nanoparticles, mesoporous silica nanoparticles (MSNs), and the like.
  • AuNPs gold nanoparticles
  • MSNs mesoporous silica nanoparticles
  • CPPs Cell-penetrating peptides
  • cationic CPPs such as TAT, Penetratin, Polyarginine, P22N, DPV3 and DPV6, etc.
  • amphiphilic CPPs which can be formed by covalently linking hydrophobic peptide sequences and NLSs, or separated from natural proteins, such as pVEC, ARF (1-22) and BPrPr (1-28)
  • hydrophobic CPPs generally only contain non-polar amino acid residues, and the net charge is about less than 20% of the total charge of the amino acid sequence).
  • the delivery is carried out via a plasmid.
  • the dosage can be a sufficient number of plasmids to elicit a response.
  • the appropriate amount of plasmid DNA in a plasmid composition can be from about 0.1 to about 2 mg.
  • the plasmid will typically include (i) a promoter; (ii) a sequence encoding a CRISPR-associated protein and/or an auxiliary protein targeting a nucleic acid, each sequence being operably connected to a promoter (e.g., the same promoter or a different promoter); (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator located downstream of (ii) and operably connected thereto.
  • a promoter e.g., the same promoter or a different promoter
  • a selectable marker e.g., the same promoter or a different promoter
  • an origin of replication e.g., the same promoter or a different
  • Plasmids can also encode RNA components of CRISPR complexes, but one or more of these components can be encoded alternatively on different vectors.
  • the frequency of administration is within the scope of a medical or veterinary practitioner (e.g., a physician, a veterinarian) or a person skilled in the art.
  • Delivery can be performed by any means known in the art, such as transfection, lipofection, electroporation, gene gun, microinjection, ultrasound, calcium phosphate transfection, cationic transfection, viral vector delivery, and the like.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the delivery system as described above and a pharmaceutically acceptable excipient.
  • the present invention also relates to a kit, which comprises: the mutant as described above, or the molecular system as described above, or the vector as described above, or the delivery system as described above, or the pharmaceutical composition as described above, and optionally instructions for use of the kit.
  • the kit of the present invention may include reagents, buffers and/or devices for mixing, measuring, sorting, labeling, etc., as well as instructions and the like.
  • the kit further comprises one or more buffers that can be used to dissolve any of the components and/or provide suitable reaction conditions for one or more of the components.
  • buffers can include one or more of the following: PBS, TBS, HEPES, Tris, Na 2 CO 3 , NaHCO 3 , acetate, or a combination thereof.
  • the reaction conditions include an appropriate pH, such as an alkaline pH. In some embodiments, the pH is between 7-10.
  • any one or more of the kit components may be stored in a suitable container.
  • the present invention also relates to a method for altering the expression of a gene product, comprising the steps of using a CRISPR-Cas nuclease system to cut a target RNA and introduce it into a cell;
  • the CRISPR-Cas nuclease system comprises the mutant as described above, or the molecular system as described above, or the vector as described above, or the delivery system as described above, or the pharmaceutical composition as described above, or the kit as described above.
  • the cell is a eukaryotic cell, such as a mammalian cell, including a human cell (primary human cell or an established human cell line).
  • the cell is a non-human mammalian cell, such as a cell from a non-human primate (e.g., monkey), a cow/bull/cow, a sheep, a goat, a pig, a horse, a dog, a cat, a rodent (e.g., a rabbit, a mouse, a rat, a hamster, etc.).
  • the cell is from a fish (e.g., salmon), a bird (e.g., a bird, including a chicken, a duck, a goose), a reptile, a shellfish (e.g., an oyster, a clam, a lobster, a prawn), an insect, a worm, a yeast, etc.
  • the cell is from a plant, such as a monocot or a dicot.
  • the plant is a food crop, such as barley, cassava, cotton, groundnut or peanut, maize, millet, oil palm fruit, potato, dry bean, rapeseed or canola, rice, rye, sorghum, soybean, sugarcane, beet, sunflower and wheat.
  • the plant is a cereal (barley, maize, millet,
  • the plant is a tuber (cassava and potato).
  • the plant is a sugar crop (sugar beet and sugar cane).
  • the plant is an oil crop (soybean, groundnut or peanut, rapeseed or canola, sunflower and oil palm fruit).
  • the plant is a fiber crop (cotton).
  • the plant is a tree (such as a peach or nectarine tree, an apple or pear tree, a nut tree (such as an almond or walnut or pistachio tree), or a citrus tree (e.g., an orange, grapefruit or lemon tree)), grass, vegetable, fruit or algae.
  • the plant is a Solanum plant; a Brassica plant; a Lactuca plant; a Spinacia plant; a Capsicum plant; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.
  • the cell is modified in vitro, in vivo or ex vivo. In certain embodiments, the cell is a stem cell.
  • the present invention also relates to the use of the mutant as described above, or the molecular system as described above, or the vector as described above, or the delivery system as described above in the preparation of a drug for treating a disease caused by a gene mutation; wherein the disease is caused by a mutation comprising one or more single-base mutations, or can be treated by knocking down or knocking out a gene.
  • the disease is a genetic disease or an idiopathic disease.
  • the disease includes, but is not limited to, one or more of the following diseases:
  • Hereditary heart disease preferably hereditary cardiomyopathy
  • sickle cell anemia thalassemia, hemophilia, progeria, phenylketonuria
  • glycogen storage disease type I diabetes, familial hypercholesterolemia, hypertriglyceridemia, Krabbe disease, Gaucher disease, color blindness, hereditary deafness, hereditary Alzheimer's disease, familial amyloidosis, progressive muscular dystrophy, and tumors caused by single base mutations.
  • the tumor is a carcinoma, sarcoma, myeloma, leukemia, lymphoma, and mixed tumors.
  • tumors that can be treated by the methods and compositions described herein include cancer cells from the following: bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, or uterus.
  • the cancer can be particularly of the following histological types, but is not limited to these: malignant neoplasms; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelioma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; familial polyposis coli adenocarcinoma; solid carcinoma; malignant carcinoid tumor; bronchioalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma;
  • the disease is a genetic cardiomyopathy.
  • Huntington's disease knockdown of Htt
  • Parkinson's syndrome knockout of ptbp1 promotes the transdifferentiation of astrocytes into neurons to treat Parkinson's disease with neuronal loss
  • Rett syndrome Rett syndrome, Mecp2 gene duplication
  • the spacer sequence of the gRNA in the molecular system, the vector, or the delivery system has at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity with the sequence shown in any one of SEQ ID NOs: 3 to 37.
  • the present invention also claims protection for the spacer sequence of the gRNA as described above (especially having at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity); the present invention also claims protection for a gRNA comprising the spacer sequence; the present invention also claims protection for a polynucleotide, a molecular system, a nucleic acid construct, a vector, a delivery system, and a pharmaceutical composition comprising the gRNA.
  • the description of the above-mentioned polynucleotides, molecular systems, nucleic acid constructs, vectors, delivery systems, and pharmaceutical compositions also applies here.
  • the present invention also requests protection for the mutant shown in SEQ ID NO: 2 as described above (or an amino acid molecule having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% sequence identity with the sequence shown in SEQ ID NO: 2); the present invention also requests protection for the polynucleotides used to express the mutant, and related molecular systems, nucleic acid constructs, vectors, delivery systems and pharmaceutical compositions.
  • the descriptions of the above polynucleotides, molecular systems, nucleic acid constructs, vectors, delivery systems and pharmaceutical compositions also apply here.
  • the direct repeat sequence (or "DR sequence" for short) of the gRNA has at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity with the sequence shown in SEQ ID NO: 38 or SEQ ID NO: 39.
  • the present invention can be used for diseases caused by one or more single base mutations.
  • a disease is caused by two point mutations, and the two point mutations are far enough apart to be recognized by two sets of molecular systems provided by the present invention.
  • it can also be treated by the technical solution provided by the present invention.
  • it relates to a method for treating a disease caused by a single base mutation, comprising the steps of introducing an effective amount of a CRISPR-Cas nuclease system to cut a target RNA into a cell;
  • the CRISPR-Cas nuclease system comprises a mutant as described above, or a molecular system as described above, or a vector as described above, or a delivery system as described above, or a pharmaceutical composition as described above.
  • the term "effective amount" in the present invention refers to the amount of the component corresponding to the term in the subject.
  • the dosage is achieved to treat, prevent, alleviate and/or ameliorate the diseases or conditions described in the present invention.
  • the mutant as described above, or the molecular system as described above, or the vector as described above, or the delivery system as described above, or the pharmaceutical composition as described above is delivered to the tissue of interest by, for example, intramuscular injection, intravenous administration, transdermal administration, intranasal administration, oral administration, or mucosal administration.
  • Delivery can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be delivered herein may vary greatly depending on a variety of factors, such as vector selection, target cells, organisms, tissues, the general condition of the subject to be treated, the degree of transformation/modification sought, route of administration, mode of administration, the type of transformation/modification sought, etc.
  • the delivery is via AAV, which may be a single dose containing at least 1 ⁇ 10 5 particles of AAV.
  • the dose is preferably at least about 1 ⁇ 10 6 particles, at least about 1 ⁇ 10 7 particles, at least about 1 ⁇ 10 8 particles, and at least about 1 ⁇ 10 9 particles of AAV.
  • delivery is performed via a delivery system as described above, which has been described in detail above.
  • the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified.
  • acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.
  • CRISPR/Cas9 technology was used to construct Myh6-pR872H point mutation mice homologous to human R870H ( Figure 1).
  • An sgRNA was designed upstream and downstream of the point mutation site, and the sequences were:
  • sgRNA Transcribe sgRNA in vitro. Microinject spCas9 protein, sgRNA and homologous recombination vector samples into fertilized eggs of mice with C57BL/6JGpt background. Take the surviving fertilized eggs after injection and transplant them into pseudo-pregnant female mice, and wait for them to become pregnant and give birth. Positive F0 generation mice were identified by PCR and Sanger sequencing, and after they reached sexual maturity, they were mated with wild-type background mice to obtain stable genetic positive F1 generation mice. PCR primers: 872-tF1, 5'-GCTACAATGCCCCTTGTCCTTTG-3'; 872-tR1, 5'-GCTCCTCTGCATCATTGAGGTTG-3'.
  • Sequencing primer is 872-tF1. Arms-PCR primers: Mu872-F: 5'-AGAGACTGAGAAGGAGATCGCA-3'; Wt872-F: 5'-AGACGGAGAAGGAGATCGCC-3'; 872-tR1: 5'-GCTCCTCTGCATCATTGAGGTG-3'
  • Myh6-pR404Q point mutation mice (T051403) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. This point mutation is homologous to human MYH7-pR403Q.
  • Myh6 pR872H point mutation homozygotes (RH/RH) were hybridized with Myh6 pR404Q heterozygotes (RQ/+) to obtain RQ/RH double-point heterozygous mutant mice.
  • RQ-positive mice were identified by PCR and Sanger sequencing, PCR primers: T051403-F1 CTTCAGCTAGATCCCAGTCAAGCC, T051403-R1 GGTCTGAGTGGGTAGGTGAGAAACAT. Sequencing was performed using the T051403-F1 primer.
  • Arms-PCR can also be used to identify the mouse genotype, using the following primers:
  • Wt404-F TCAAGGGCCTGTGTCACC A TCG
  • cyclosporine (CsA) diet 1 mg/kg cyclosporine (CsA) diet was fed to mice from 5 to 8 weeks of age for 3 consecutive weeks to accelerate the left ventricular hypertrophy phenotype in Myh6-pR872H point mutation mice.
  • mice were anesthetized with 1-2% isoflurane, and long-axis ultrasound images of the heart were acquired using a Visual Sonics 2100 system and MS-400 probe when the heart rate was 450-500 bpm/min.
  • the wall thickness of the left ventricle, the size of the cavity, the thickness of the ventricular septum, and the ejection fraction were measured.
  • mice were anesthetized with 1-1.5% isoflurane, and the electrocardiogram (two limb leads) of the mice under anesthesia was collected using a rechargeable animal telemetry system (Kexin Medical Biotechnology Co., Ltd.) and the electrocardiogram data were analyzed using Labchart7.
  • the hearts were slowly perfused with 4% paraformaldehyde solution, and the removed hearts were placed in 4% paraformaldehyde solution and fixed at 4°C for 24 hours. After gradient dehydration with 70%-100% ethanol, the hearts were permeabilized with xylene for 2 hours and then embedded in paraffin. Paraffin sections with a thickness of 5 ⁇ m were stained with H&E (hematoxylin and eosin) or Masson's Trichrome kit (Yisheng Biotechnology Co., Ltd.) to detect morphological changes, myocardial fiber arrangement and fibrosis of the heart.
  • H&E hematoxylin and eosin
  • Masson's Trichrome kit Yisheng Biotechnology Co., Ltd.
  • Wheat germ agglutinin (WGA, invitrogen, Cat: W32466) specifically binds to N-acetyl-D-glucosamine and sialic acid residues on the cell membrane surface and is used to detect the size of myocardial cells.
  • SV40 NLS-Cas13d-SV40 NLS-HA was cloned into the XbaI and BamHI restriction sites on the lenti-crispr v2 (addgene: 52961) vector, and Cas13d DR-Bsmbi-EGFP-Bsmbi was connected to the downstream of the U6 promoter to obtain the lenti-U6-DR-Bsmbi-Bsmbi-EF1a-Cas13d expression plasmid, referred to as the lenti-Cas13d-sgRNA plasmid.
  • Overlap PCR was used to perform point mutations on the candidate amino acid sites on Cas13d.
  • the Gibson assembly kit ligated the mutant Cas13d DNA to the XbaI and BamHI restriction sites on the lenti-Cas13d-sgRNA to construct different Cas13d mutants.
  • Lipofectamine 3000 (Invitrogen) was used to co-transfect the dual fluorescent reporter plasmid and the lenti-Cas13d-sgRNA plasmid into 293T cells with a 90% confluence. RNA was extracted 48 hours after transfection, and RT-qPCR was used to detect the knockout efficiency of EGFP and mCherry to reflect the knockout efficiency of wild-type and mutant Myh6 mRNA, respectively. At the same time, flow cytometry was used to detect the fluorescence intensity of EGFP and mCherry to reflect the expression levels of wild-type and mutant Myh6 proteins, respectively.
  • qPCR primers are as follows:
  • hGAPDH-R ACCACCCTGTTGCTGTAGCCAA
  • RNAs (872-sgRNA9 and 404-sgRNA15) specifically targeting Myh6-pR872H and Myh6-pR404Q were ligated to the Sap1-Sap1 site of the above plasmid to obtain AAV-cTnT-Cas13d-U6-R872H sgRNA plasmid and AAV-cTnT-Cas13d-U6-R404Q sgRNA plasmid, respectively.
  • the above expression plasmids were co-transfected with the auxiliary packaging plasmids (rep/cap plasmid and pHGTI/delta plasmid) of AAV9 into 293T cells with a confluency of 70%-80%.
  • the supernatant and cell lysate were collected 60 hours after transfection, and the virus particles were concentrated by iodixanol gradient sedimentation and ultrafiltration membrane.
  • the virus titer was detected by absolute quantitative qPCR.
  • the concentrated virus was resuspended in PBS and stored at -80°C after aliquoting.
  • the SWISS-MODEL software was used to obtain the preliminary structure of the RfxCas13d protein by homology modeling with the EsCas13d-crRNA-target RNA structure (PDB: 6e9f) as a template.
  • PDB EsCas13d-crRNA-target RNA structure
  • AlphaFold2 was used to predict the structure of RfxCas13d, and the region with a higher score (770A-821N) in the AlphaFold2 predicted structure was used to replace the structural region built by SWISS-MODEL, and finally the RfxCas13d protein structure was obtained.
  • the crRNA of EsCas13d was mutated into the crRNA sequence of RfxCas13d: 5'-AACCCCUACCAACUGGUCGGGGUUUGAAACGACCUGAAUAUUUCAGAUCAAA-3', and then the mutated crRNA-target RNA nucleic acid structure was superimposed with the above-mentioned protein conformation to obtain the RfxCas13d-RNA complex structure.
  • RfxCas13d was mutated and other mutant Cas13d-RNA complex structures were constructed.
  • each HoxD9-DNA system was placed in the center of the TIP3P water box, and the minimum boundary of the solute to the water box was set to And add appropriate amounts of Na + and Cl - to ensure that the ion concentration is 0.15 mol/L. In the end, each system contains an average of 249,805 atoms.
  • energy minimization is performed by the following steps: first constrain Cas13d and all RNA atoms, then constrain the main chain C atoms of Cas13d and the main chain phosphorus atoms of RNA, and finally, release all atoms for energy minimization.
  • the last frame of energy minimization of each system was taken as the input structure of molecular dynamics simulation.
  • an initial random seed was set and three parallel molecular dynamics simulations of 200 nanoseconds were performed.
  • the results of molecular dynamics simulation were first grouped according to the topology file to construct an index file for proteins, RNA, and magnesium ions.
  • the subcommand trjconv was used to center the system, and the system was rotated and translated to process periodic boundary conditions to prevent the conformation from being destroyed and abnormal bonds from being generated.
  • the crRNA-targetRNA base complementary regions were grouped and an index file was constructed.
  • the rms subcommand was used to calculate the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of RNA phosphorus atoms in each trajectory.
  • the conformational correlation network is analyzed according to the following steps: the main chain carbon atoms of Cas13d are grouped, the covariance matrix of the entire trajectory is constructed, and the eigenvector is calculated, the trajectory is projected in the direction of the first principal component, and the Cas13d conformation of the main chain carbon atoms on the first principal component is obtained.
  • the correlation between the motion trajectories of the main chain carbon atoms of Cas13d is calculated, and the correlation greater than 0.6 or less than -0.6 has a motion-directed correlation/negative correlation.
  • the correlation network is aligned to the first principal component conformation to visualize the conformational correlation network.
  • Cas13d was constructed into the pET vector, histone tags were fused to the N-terminus and C-terminus, and transformed into BL21 (DE3) competent cells. Positive clones were picked into LB medium and cultured at 37°C for 12 hours. When OD600 was 0.6-0.8, 0.5mM IPTG was added at 16°C for induction for 16-18 hours. Cell lysate was collected, protein was purified by nickel column, dialyzed overnight, concentrated by 100KDa ultrafiltration tube, and the protein was stored at -80°C.
  • the purified Cas13d protein, in vitro transcribed sgRNA and target RNA were placed in the cleavage reaction solution at a molar ratio of 1:1:1, reacted at 37°C for 15 minutes, proteinase K was added to terminate the reaction, RNA dye (NEB) was added for denaturation at 80°C for 10 minutes, and the cleavage results were analyzed by 10% polyacrylamide gel.
  • ddPCR primers and probes are as follows:
  • Cas13d's recognition of single-base mismatches may be sequence-dependent.
  • wild-type Cas13d can specifically target Myh6 R872H mutant transcripts and thus be used to treat hereditary cardiomyopathy caused by mutations at this site.
  • the knockdown ability of the mutant Myh6 RNA with a perfect match was significantly higher than that of the wild-type Myh6 RNA with a single base mismatch. However, the difference in the knockdown level between the two was only about 10%, as shown in Figure 2 and Table 1.
  • RfxCas13d also known as CasRx
  • EsCas13d both belong to the Cas13d family, and the homology of their protein sequences is 30%. Therefore, we used (PS)2 and SWISS-Model to construct the homologous structure of RfxCas13d based on the three-dimensional structure of EsCas13d (PDB: 6E9F).
  • PDB: 6E9F three-dimensional structure of EsCas13d
  • the crRNA sequence of EsCas13d was mutated into the crRNA sequence of RfxCas13d, and docked with the homologous RfxCas13d structure to generate the three-dimensional structure of the RfxCas13d-sgRNA-target RNA tri-complex, and the results are shown in Figure 3.
  • Example 4 Engineering SuperCas13d improves single-base recognition specificity while reducing side-cutting activity
  • the wild-type Cas13d and SuperCas13d proteins were expressed and purified in bacteria, and 1ug RNA substrate was cut at different concentrations to test the concentration-dependent RNA cutting efficiency.
  • the results showed that at different concentrations, the efficiency of SuperCas13d in cutting a completely matched RNA substrate was much higher than that of cutting a single-base mismatched RNA substrate, and the required concentration to achieve the highest cutting efficiency differed by nearly two times.
  • the Cas13d protein concentration was fixed at 225nM and the time-dependent RNA cutting efficiency was tested, SuperCas13d also showed a lower cutting speed for mismatched RNA substrates, with a 20-fold difference in cutting speed. The results are shown in Figure 5.
  • Example 6 SuperCas13d specifically targets Myh6 R872H point mutation transcripts in vivo to prevent the development of hereditary cardiomyopathy (animal model)
  • Example 8 Engineered SuperCas13d specifically targets Myh6 R404Q point mutation transcripts in vivo to prevent the development of double point mutation hereditary cardiomyopathy (animal model)

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Abstract

提供了具有CasRx活性的突变体及其应用，该突变体与SEQ ID NO：1所示的氨基酸序列具体具有至少70％的序列同一性，并且参照SEQ ID NO：1的氨基酸位置变化，具有位于下列位置处中的一个或多个突变：K323、N324、G326、R333、N348、K350、K411、Y415、T486、R529、K585、R601、Y604、N641、R648以及Y649。

Description

具有CasRx活性的突变体及其应用
相关申请的交叉引用
本申请要求于2023年1月10日提交的申请号为202310031086.3的中国专利申请的优先权，其全部内容通过引用的方式并入本文。
技术领域
本发明涉及分子生物学技术领域，具体而言，涉及具有CasRx活性的突变体及其应用。
背景技术
2016年，科学家发现了可以靶向RNA进行切割的CRISPR/Cas13系统，该系统由单一的效应蛋白Cas13和CRISPR RNA(crRNA)组装形成一个由crRNA引导的RNA靶向效应复合物。目前，Cas13家族共鉴定出四种亚型，包括Cas13a(又名C2c2)、Cas13b、Cas13c和Cas13d。其中，CRISPR/Cas13d(Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 13d)核酸酶在2018年有研究发现Cas13d平均大小约为930aa，是目前发现的哺乳动物细胞中相对较小的Ⅱ类CRISPR效应因子(比其家族成员Cas13a-c小20％，比Cas9小33％)，使它更容易包装到容量有限的应用载体如AAV载体中，Cas13d对RNA靶点的切割不依赖于PFS序列(protospacer flanking sequence，相当于Cas9针对DNA的PAM序列)，这极大的增加了Cas13d的应用范围，使其成为进一步开发靶向RNA工具的潜在平台，可以在多种物种、不同的细胞中以及动物体内实现了有效的基因编辑。
所有的Cas13蛋白酶都需要crRNA来确保其结合靶ssRNA的特异性，并在crRNA的引导下发挥其核糖核酸酶(RNase)活性。crRNA通常由直接 重复(Direct Repeat，DR)序列以及特异性靶向转录本的间隔序列(spacer)组成。Cas13d通常需要22-30nt的向导RNA(guide RNA)与靶RNA互补，激活核酸酶结构域进行切割，并且通常Cas13d除特异性切割靶ssRNA外，还能切割其他非特异性的RNA。2019年12月，Magdy Mahfouz课题组发现Cas13d家族蛋白CasRx可单独针对一种或同时针对两种植物RNA病毒，对病毒核酸表现出很强的特异性，并且在植物中不表现非特异的RNase活性。2020年3月，科研人员通过利用CasRx在小鼠肝脏中实现了pcsk9基因的高效沉默，从而降低了小鼠血液中的胆固醇水平。该项工作证实了Cas13d在成体动物体内也有靶向沉默RNA的活性，使Cas13d系统的应用向前推进了一步。
RNA编辑器和DNA编辑器有着最大不同是RNA编辑器不改变遗传物质DNA，与Cas9介导的基因敲除技术相比，Cas13d介导的基因沉默不会改变基因组DNA，这种基因沉默是可逆的，安全性更好，因此在基因治疗领域具有广阔的治疗前景。
但已有研究表明，单个碱基错配几乎不影响野生型Cas13d酶活。因此Cas蛋白酶家族通常不能用于单碱基错配导致的遗传疾病的治疗。单碱基突变占所有遗传病的40％以上。如遗传性心肌病、镰刀状贫血、地中海贫血、血友病、早衰症、苯丙酮尿症、糖原累积病、I型糖尿病、家族性高胆固醇血症、高甘油三脂血症、克拉伯病、戈谢病、色盲、遗传性耳聋，进行性肌营养不良以及各种由单碱基突变导致的肿瘤等。遗传性心肌病(Inherited Cardiomyopathy)是主要由肌节基因和代谢相关基因突变导致的心肌损伤或功能失代偿性疾病。发病率约为1/200，是青少年猝死的主要原因。按照心肌的形态和功能变化可以粗略分为肥厚性心肌病、扩张性心肌病、限制性心肌病、致心律失常型右室心肌病等。目前已确认的致病突变超过1000种，其中约40％为肌球蛋白β-MHC(MYH7)基因上的错义突变，这些肌球蛋白上的氨基酸突变影响了肌节的收缩和舒张功能。
发明内容
在本发明的一个方面，涉及具有CasRx活性的突变体，其与SEQ ID NO：1所示的氨基酸序列具体至少90％的序列同一性，并且参照SEQ ID NO：1的氨基酸位置变化，具有位于下列位置处中的一个或多个突变：
K323、N324、G326、R333、N348、K350、K411、Y415、T486、R529、K585、R601、Y604、N641、R648以及Y649。
在本发明的另一个方面，涉及分离的多核苷酸，其编码权利要求1～8任一项所述的突变体。
在本发明的又一个方面，涉及一种分子系统，其为复合物或组合物，包含(a)如上所述的突变体和(b)向导RNA分子(gRNA)的集合。
在本发明的又一个方面，涉及编码如上所述的分子系统的核酸构建体。
在本发明的又一个方面，涉及一种载体，其包含如上所述的多核苷酸，或如上所述的核酸构建体。
在本发明的又一个方面，涉及一种递送系统，其包含i)如上所述的分子系统，以及ii)递送媒介物。
在本发明的又一个方面，涉及一种药物组合物，其包含如上所述的递送系统以及药学上可接受的赋形剂。
在本发明的又一个方面，涉及一种试剂盒，其包含：如上所述的突变体、或如上所述的分子系统、或如上所述的载体、或如上所述的递送系统、或如上所述的药物组合物，以及任选的该试剂盒的使用说明书。
在本发明的又一个方面，涉及如上所述的突变体、或如上所述的分子系统、或如上所述的载体、或如上所述的递送系统在制备用于治疗基因变异所引起的疾病的药物中的应用；其中所述疾病由包含一个或多个单碱基突变的变异所引起，或者可通过敲减或敲除某个基因得到治疗。
在本发明的又一个方面，涉及一种分子系统，其为复合物或组合物，包含如上所述的突变体和向导RNA分子(gRNA)的集合，其中所述gRNA的核苷酸序列如SEQ ID NO：44～173任一项所示。
在本发明的又一个方面，涉及如上所述的分子系统在制备用于治疗MYH7突变导致的心肌病的药物中的应用。
本发明的这些和其他方面将在以下本发明的描述中更详细地进行阐述。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为Myh6-pR872H小鼠构建和基因型鉴定；A.采用CRISPR-Cas9系统构建Myh6-pR872H单碱基突变小鼠的示意图；B.采用Arms-PCR鉴定小鼠基因型；C-E.采用Sanger测序鉴定小鼠基因型。
图2为利用荧光双报告系统证明野生型Cas13d无法识别和剪切单碱基突变RNA；A.双荧光报告治疗构建方法；野生型Myh6(wtMyh6)RNA和蛋白表达由EGFP指示；突变型Myh6(wtMyh6)RNA和蛋白表达由mCherry指示。PEST(脯氨酸(P)、谷氨酸(E)、丝氨酸(S)和苏氨酸(T))序列用来加快EGFP和mCherry的降解时间；Cas13d质粒和双荧光报告质粒共转，48小时后通过检测EGFP和mCherry的表达量分析Cas13d对突变型转录本和野生型转录本的敲减效率；B-E.采用位移方法设计的多条突变型Myh6(Myh6-R872H)特异的sgRNA无法区分野生型和突变型Myh6，对野生型和突变型myh6都具有相似的剪切效应；证明野生型Cas13d无法 区分单碱基突变的Myh6 mRNA；Kdmu代表突变型Myh6 mRNA的敲减效率，Kdwt代表野生型Myh6 mRNA的敲减效率；Kdmu-Kdwt反映Cas13d区分突变型和野生型mRNA的能力。
图3为RfxCas13d的工程化改造，筛选具有单碱基识别能力的单氨基酸突变体；A.RfxCas13d的同源建模，寻找核酸凹槽附近候选的核酸结合位点；B.预测候选的核酸结合位点与靶RNA和互补的向导RNA“Spacer”区域结合的位置；C-H.依次对候选位点进行丙氨酸突变、破坏与核酸的结合力，以两个向导RNA为例，在双荧光报告系统中筛选可能提高单碱基错配识别分辨率的位点N641A和R648A；Kdmu代表突变型Myh6mRNA的敲减效率，Kdwt代表野生型Myh6 mRNA的敲减效率；Kdmu-Kdwt反映不同Cas13d突变体区分突变型和野生型mRNA的能力。
图4为突变体的优化组合，筛选出可以准确识别单碱基错配的SuperCas13d；A-F.将具有单碱基识别分辨率的突变位点N641A和R648A，以及其他两个对活性影响较小的突变位点K350A和T486A，进行组合，进一步在双荧光报告系统中筛选到最优的单碱基识别突变体，称为SuperCas13d；Kdmu代表突变型Myh6 mRNA的敲减效率，Kdwt代表野生型Myh6 mRNA的敲减效率；Kdmu-Kdwt反映不同Cas13d突变体区分突变型和野生型mRNA的能力；G-J.体外切割表明在特定浓度如550nM Cas13d蛋白时，相比野生型Cas13d，SuperCas13d切割完全匹配的RNA底物(mu-Myh6)的效率相当，但切割单碱基错配RNA底物(wt-Myh6)的效率降低。
图5为浓度依赖以及时间依赖的体外切割实验，SuperCas13d显著提高了对错配RNA底物的切割效率和切割速度。
图6为特异性窗口检测实验，当gRNA间隔序列15-21nt位置与底物存在错配时，SuperCas13d对底物的敲减效率降低。
图7为细胞内的旁切活性检测实验。A.不同Cas13d变体与GFP融合 表达，突变型的Myh6(muMyh6)与mCherry-PEST融合表达。设计不同的gRNA分别靶向mCherry、PPIA或RPL4RNA，用流式分析GFP和mCherry荧光强度。B-D.表明当靶向mCherry时，野生型Cas13d表现出很高的对GFP的非特异性切割，而SuperCas13d的非特异性切割显著降低，同时还保留了较高的切割活性，切割活性优于已报道的高保真变体hfCas13d。E-G.采用qPCR检测对目标基因的敲除效率以及对其它非目标基因的旁切活性。
图8为分子动态学模拟实验证明SuperCas13d改变了Cas蛋白与RNA底物之间的作用模态；A.采用GROMACS-2021对野生型RfxCas13d、T486A-Cas13d、N641A-Cas13d、R648A-Cas13d和SuperCas13d进行分子动态学模拟，表明SuperCas13d中三个点突变均削弱了Cas13d与核酸的结合；B.分析Cas13d与RNA互补配对区域的RMSD的结果表明：SuperCas13d中的N641A、R648A突变显著提高了Cas13d与RNA的结合不稳定性，而T486A突变对RNA的结合影响较小。
图9为分子动力学模拟证明SuperCas13d改变RNA与Cas蛋白结合模态进而提高碱基分辨率；分析野生型RfxCas13d、T486A-Cas13d、N641A-Cas13d、R648A-Cas13d和SuperCas13dCas13d与向导RNA Spacer区(A)和靶RNA(B)各碱基的结合动力学(RMSF)表明：N641A-Cas13d和R648A-Cas13d显著提高了Cas13d蛋白与向导RNA上的40至43号碱基的结合不稳定性，而对Cas13d与靶RNA结合的影响不明显；T486A-Cas13d对向导RNA和靶RNA结合的均无影响；而SuperCas13d非常显著地增加了Cas13d蛋白与向导RNA上的39至43号碱基的结合不稳定性，同时显著增加了Cas13d与靶RNA上的14至18号碱基的结合不稳定性；C.进一步分析野生型RfxCas13d、N641A-Cas13d、R648A-Cas13d和SuperCas13d蛋白各结构域之间的相关性，结果表明相对于野生型Cas13d蛋白，N641A-Cas13d、R648A-Cas13d和SuperCas13d显著减少了核酸识别结构域(Helical1)与核酸酶结构域(Helical2及HEPN结构域)之间的负相关 性(蓝色线条减少)，一方面可能导致靶RNA结合时Cas13d蛋白的适应性构象调节减少、增加错配靶RNA的结合不稳定性，另一方面可能导致靶RNA结合后对核酸酶结构域的构象调节减少、增加了核酸酶活性构象激活的能垒。
图10表明SuperCas13d在体内能显著改善RH/+小鼠的左心室肥厚表型；A.采用腺相关病毒递送SuperCas13d治疗RH/+点突变小鼠左心室肥厚的实验流程；B-F分别采用HE染色(B)、心脏超声(C-D)、心电(E-F)说明RH/+点突变小鼠经环孢素A(CsA)诱导后表现出左心室肥厚表型，包括室壁增厚、心腔变小、射血分数增加、心电的QTc延长和S波异常；新生期(P3-P7)注射SuperCas13d显著抑制RH/+点突变向左心室肥厚表型发展；LVPWd，舒张期左心室后壁厚度；LVIDd，舒张期左心室腔直径；IVSd，舒张期室间隔厚度；EF，射血分数；ns，无统计学差异；*，P<0.05；**，P<0.01；***，P<0.001；****，P<0.0001。
图11证明SuperCas13d也能提高对Myh6 R404Q单碱基突变的区分能力；A.针对myh6 R404Q点突变设计sgRNA；B.在双荧光报告系统中，采用野生型Cas13d同时敲减野生型和突变型myh6 R404Q mRNA，发现野生型Cas13d无法区分野生型和突变型mRNA；C.采用SuperCas13d和靶向R404Q的sgRNA15和sgRNA18连用，可显著减少对野生型Myh6 mRNA的切割，而保留对突变型R404Q mRNA的切割效率，即SuperCas13d显著提高了对单碱基突变mRNA与野生型mRNA的区分能力；KdR404Q代表R404Q mRNA的敲减效率，Kdwt代表野生型Myh6 mRNA的敲减效率。
图12证明RH/RQ双点杂合突变小鼠心室肥厚表型发病早更严重；A.HE染色说明RH/RQ双点杂合突变小鼠相比RH/+小鼠在4月龄表现出严重的左心室肥厚表型；B.Masson’s Trichrome染色表明RH/RQ小鼠在12月时左心室具有显著的纤维化；C.心脏超声表明RH/RQ相比野生型小鼠和RH/+小鼠，在4月开始龄射血分数显著增加、左心室室壁厚度显著增加，舒张期左心室腔直径显著减小；D-G.心电检测表明在2月龄时， RH/RQ(F和G)相比野生型小鼠(D和E)心电波形异常，R波电压减小，S波电压增加。
图13证明SuperCas13d在体内也能显著改善RH/RQ小鼠的心室肥厚表型；A.AAV9-cTnT启动子-SuperCas13d治疗载体图；B治疗实验流程图；C-D.心脏超声检测结果表明相比注射PBS的RH/RQ小鼠，注射AAV-SuperCas13d后4个月的小鼠左心室室壁厚度、室间隔厚度、射血分数显著减小，左心室腔的直径显著增加，并且与RH/+小鼠无统计学差异；E-F.心电检测结果表明，注射AAV-SuperCas13d后4个月的小鼠心电波形更接近RH/+小鼠。
图14为组织学水平检测SuperCas13d治疗RH/RQ小鼠的心室肥厚。A.心脏整体图；B.心脏重量(HW)与体重(BW)的比值；C.心脏横切面的HE染色说明AAV-SuperCas13d显著阻滞RH/RQ小鼠发展左心室肥厚表型；D-E.WGA染色显示AAV-SuperCas13d显著减小心肌细胞横切面面积大小；F.HE染色AAV-SuperCas13d阻滞心肌纤维排列紊乱；G-H.Masson’s Trichrome染色显示AAV-SuperCas13d阻滞心脏成纤维化；I-J.TUNEL染色显示AAV-SuperCas13d阻滞心脏细胞凋亡。
图15为ddPCR检测说明SuperCas13d在小鼠体内特异性敲除突变Myh6转录本。A.SuperCas13d注射至Myh6 R87H(RH/+)-CsA小鼠2个月后，敲除野生型Myh6(WT)以及Myh6 R87H mRNA的效率；B.SuperCas13d注射至RH/RQ小鼠2个月后，敲除野生型Myh6 R87H(RH)mRNA以及Myh6 R404Q(RQ)mRNA的效率。
图16为SuperCas13d靶向人MYH7基因上43个致病点突变的实验。A.MYH7基因上致病点突变的分布图。B-E.SuperCas13d提高了对底物的区分效率，且提高了具备高效区分能力的gRNA的比例。F.SuperCas13d在碱基编辑器不能编辑的位点也展现出很好的区分能力。
具体实施方式
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
除非另有说明，用于披露本发明的所有术语(包括技术和科学术语)的意义与本发明所属领域普通技术人员所通常理解的相同。通过进一步的指导，随后的定义用于更好地理解本发明的教导。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
术语描述
本文所使用的术语“和/或”、“或/和”、“及/或”的选择范围包括两个或两个以上相关所列项目中任一个项目，也包括相关所列项目的任意的和所有的组合，所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。需要说明的是，当用至少两个选自“和/或”、“或/和”、“及/或”的连词组合连接至少三个项目时，应当理解，在本申请中，该技术方案毫无疑问地包括均用“逻辑与”连接的技术方案，还毫无疑问地包括均用“逻辑或”连接的技术方案。比如，“A及/或B”包括A、B和A+B三种并列方案。又比如，“A，及/或，B，及/或，C，及/或，D”的技术方案，包括A、B、C、D中任一项(也即均用“逻辑或”连接的技术方案)，也包括A、B、C、D的任意的和所有的组合，也即包括A、B、C、D中任两项或任三项的组合，还包括A、B、C、D的四项组合(也即均用“逻辑与”连接的技术方案)。
本发明中所使用的术语“含有”、“包含”和“包括”是同义词，其 是包容性或开放式的，不排除额外的、未被引述的成员、元素或方法步骤。
本发明中用端点表示的数值范围包括该范围内所包含的所有数值及分数，以及所引述的端点。
如本文所用的，术语“增加”、“增强”和“提高”描述了与对照相比，升高了至少约5％、10％、20％、25％、50％、75％、100％、150％、200％、300％、400％、500％或更多。
如本文所用的，术语“减少”、“减弱”和“降低”描述了与对照相比，降低了至少约5％、10％、20％、25％、30％、40％、50％、60％、70％、75％、80％、90％或100％。在特定的实施方案中，该降低可能导致没有或基本上没有(即，微不足道的量，例如小于约10％或甚至5％的量)可检测的活性或量。
本发明中涉及浓度数值，其含义包括在一定范围内的波动。比如，可以在相应的精度范围内波动。比如2％，可以允许±0.1％范围内波动。对于数值较大或无需过于精细控制的数值，还允许其含义包括更大波动。比如100mM，可以允许±1％、±2％、±5％等范围内的波动。涉及分子量，允许其含义包括±10％的波动。
如本文所用的术语“约”，在提及可测量值诸如量或浓度等时，意指包括指定值的±10％、±5％、±1％、±0.5％或甚至±0.1％的变化以及指定值。例如，“约X”，其中X是可测量值，意指包括X以及X的±10％、±5％、±1％、±0.5％或甚至±0.1％的变化。本文提供的可测量值的范围可以包括其中的任何其他范围和/或各个值。
本发明中，涉及“多个”、“多种”等描述，如无特别限定，指在数量上指大于等于2。
本发明中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。
本发明中，“优选”、“更好”、“更佳”、“为宜”仅为描述效果更好的实施方式或实施例，应当理解，并不构成对本发明保护范围的限制。本发明中，“可选地”、“可选的”、“可选”，指可有可无，也即指选自“有”或“无”两种并列方案中的任一种。如果一个技术方案中出现多处“可选”，如无特别说明，且无矛盾之处或相互制约关系，则每项“可选”各自独立。
“天然”或“野生型”核酸、核苷酸序列、多肽或氨基酸序列指的是天然存在的或内源性的核酸、核苷酸序列、多肽或氨基酸序列。因此，例如，“野生型mRNA”是生物体中天然存在的mRNA或对生物体而言是内源性的mRNA。“同源”核酸序列是与其所导入的宿主细胞天然关联的核苷酸序列。
如本文所用的，术语“核酸”、“核酸分子”、“核苷酸序列”和“多核苷酸”指的是线性或支化的、单链或双链或者其杂合体的RNA或DNA。该术语还包括RNA/DNA杂合体。当以合成方式产生crRNA或gRNA时，不太常见的碱基，如肌苷、5-甲基胞嘧啶、6-甲基腺嘌呤、次黄嘌呤等也可用于合成。例如，已经证明含有尿苷和胞苷的C-5丙炔类似物的多核苷酸以高亲和力结合RNA。还可以进行其他修饰，例如对磷酸二酯骨架或RNA核糖糖基团中的2'-羟基的修饰。术语“核苷酸序列”、“核酸”、“核酸分子”、“核酸构建体”、“寡核苷酸”和“多核苷酸”在本文中还可互换使用。本文提供的核酸分子和/或核苷酸序列在本文中以从左至右的5'至3'方向显示，并使用如世界知识产权组织(WIPO)ST.26标准中规定的用于表示核苷酸符号的标准代码来表示。
如本文所用的“引导核酸”、“指导RNA”、“引导RNA”、“gRNA”、“CRISPR RNA/DNA”、“crRNA”或“crDNA”，意指一种核酸，其包含至少一个与靶核酸互补(并杂交)的间隔子序列和至少一个重复序列(如直接重复序列)，其中重复序列可以连接到间隔子序列的5'端和/或3'端。本发明的gRNA的设计可以基于Cas13d系统。
在一些实施方案中，重复序列包含至少10个核苷酸，这取决于特定的重复以及包含该重复的引导RNA是经过加工还是未加工(例如，约10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50至100个或更多个核苷酸或者其中的任何范围或值的核苷酸)。在一些实施方案中，重复序列包含约10至约20个、约10至约30个、约10至约45个、约10至约50个、约15至约30个、约15至约40个、约15至约45个、约15至约50个、约20至约30个、约20至约40个、约20至约50个、约30至约40个、约40至约80个、约50至约100个，或更多个核苷酸，基本上由其组成，或者由其组成。
与间隔子序列5'端相连的重复序列可以包含重复序列的一部分(例如，野生型重复序列的5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35个或更多个连续核苷酸)。在一些实施方案中，与间隔子序列5'端相连的重复序列的一部分的长度可以是约5至约10个连续核苷酸(例如，约5、6、7、8、9、10个核苷酸)，并且与野生型CRISPR Cas重复核苷酸序列的相同区域(例如5'端)具有至少90％的同一性(例如，至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)。在一些实施方案中，重复序列的一部分可以在其5'端包含假结样结构(例如“柄状结构”)。
如本文所用的“间隔序列”是与靶核酸(如靶ssRNA)互补的核苷酸序列。间隔序列可以与靶核酸完全互补或基本上互补(例如，至少约70％互补(例如，约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的互补，以及其中的任何范围或值))。因此，在一些实 施方案中，与靶核酸相比，间隔子序列可以具有一个、两个、三个、四个或五个错配，该错配可以是连续的或非连续的。在一些实施方案中，间隔子序列可以与靶核酸具有约70％的互补性。在其它实施方案中，间隔子核苷酸序列可以与靶核酸具有约80％的互补性。在另外的其它实施方案中，间隔子核苷酸序列可以与靶核酸(前间隔子序列)具有约85％、90％、95％、96％、97％、98％、99％或99.5％等的互补性。在一些实施方案中，间隔子序列与靶核酸100％互补。间隔子序列的长度可以是约15个核苷酸至约30个核苷酸(例如，15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸，或者其中的任何范围或值)。因此，在一些实施方案中，间隔子序列可以在靶核酸区域(例如，前间隔子序列)上具有完全互补性或基本互补性，该靶核酸区域长度可以是至少约15个核苷酸至约30个核苷酸。在一些实施方案中，间隔子的长度可以是约20、21、22、23、24或25个核苷酸。在一些实施方案中，间隔子区的长度可以是23个核苷酸。
在本发明中，若未特别描述，sgRNA用于指代间隔序列。
在本发明实施例中，若未特别描述，直接重复序列采用SEQ ID NO：39所示的序列。
为了减少脱靶相互作用，例如，为了减少gRNA与具有低互补性的靶序列相互作用，可以将突变引入所述CRISPR系统中，使得所述CRISPR系统可以区分具有大于80％、85％、90％或95％互补性的靶序列与脱靶序列。在一些实施方式中，所述互补程度为从80％至95％，例如，约83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％或95％(例如，区分具有18个核苷酸的靶标与具有1、2或3个错配的18个核苷酸的脱靶)。相应地，在一些实施方式中，指导序列与其对应的靶序列之间的互补程度大于94.5％、95％、95.5％、96％、96.5％、97％、97.5％、98％、98.5％、99％、99.5％或99.9％。在一些实施方式中，所述互补程度是100％。
如本文所用的，“靶核酸”、“靶RNA”、“靶区域”或“基因组中的靶区域”指的是生物体基因组中与本发明gRNA中的间隔序列完全互补(100％互补)或基本上互补(例如，至少70％互补(例如，约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高，以及其中的任何范围或值))的区域。在一些实施方案中，靶区域长度为至少15个连续核苷酸(例如，长度为15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30个或更多个核苷酸，以及其中的任何范围或值；例如，约19至约25个核苷酸、约20至约24个核苷酸等)。所述靶RNA可以是任何合适形式的RNA，包括但不限于mRNA、tRNA、核糖体RNA(rRNA)、微小RNA(miRNA)、干扰RNA(siRNA)、核酶、核糖开关、卫星RNA、微开关、微酶(microzyme)或病毒RNA。在一些实施方式中，所述靶核酸与病症或疾病相关。
术语“突变”指的是点突变(例如，错义或无义，或者导致移框的单碱基对的插入或缺失)、插入、缺失和/或截短。当突变是氨基酸序列中的一个残基取代为另一个残基，或者是序列中一个或多个残基的缺失或插入时，通常通过确定原始残基，然后是确定残基在序列中的位置，并确定新取代的残基的身份，来描述该突变。
如本文所用，术语“序列同一性”是指两个多核苷酸或氨基酸序列在比较窗口上是相同的(即，在逐个核苷酸或逐个残基的基础上)。术语“序列同一性百分比”是通过在比较窗口上比较两个最佳比对的序列来计算的，确定在两个序列中相同的核酸碱基(例如，A、T、C、G、U或I)或残基出现的位置的数量，从而得到匹配位置的数量，将匹配位置的数量除以比较窗口中的位置总数(即窗口大小)，并将结果乘以100得到序列同一性的百分比。如本文所用，术语“可观同一性(substantial identity)”表示多核苷酸或氨基酸序列的特征，其中所述多核苷酸或氨基酸包含在至少 18个核苷酸(6个氨基酸)位置的比较窗口上，通常在至少24-48个核苷酸(8-16个氨基酸)位置的窗口上，与参考序列相比具有至少85％序列同一性、优选至少90％至95％序列同一性、更通常至少99％序列同一性的序列，其中序列同一性的百分比是通过将参考序列与可能包括缺失或添加的序列(在比较窗口内总计为参考序列的20％或更少)进行比较来计算。参考序列可以是更大序列的子集。
如本文所用，“Cas13d”通常用于宽泛地指代野生型和/或突变的Cas13d蛋白，根据语境，更常见的场景是用于指代野生型和突变的CasRx蛋白。突变的CasRx蛋白在本文中又称“突变体”、“具有CasRx活性的突变体”、“CasRx突变体”、“CasRx变体”等。“SuperCas13d”特指具有N641、T486和R648三位点突变的CasRx突变体。
如本文所用，术语“AAV”是指腺相关病毒。术语AAV可用于指病毒本身或其衍生物，例如但不限于病毒衣壳、病毒基因组、病毒颗粒、病毒片段及其组合。术语“AAV”包括所有天然存在和重组形式的亚型及其变体，另有要求除外。天然存在形式的AAV是指任何腺相关病毒或其衍生物，其包含由天然存在的病毒衣壳蛋白组成的病毒衣壳。天然存在的AAV的非限制性实例包括AAV 1型(AAV-1)、AAV 2型(AAV-2)、AAV 3型(AAV-3)、AAV 4型(AAV-4)、AAV 5型(AAV-5)、AAV 6型(AAV2-6)、AAV 7型(AAV1-7)、AAV 8型(AAV6-8)、AAV9、AAV10、AAV11、AAV12、AAV13、rh10中的任一种或多种。此外，AAV的来源可以是禽AAV、牛AAV、犬AAV、马AAV、灵长类AAV、非灵长类AAV和绵羊AAV。“灵长类AAV”是指感染灵长类动物的AAV，“非灵长类AAV”是指感染非灵长类哺乳动物的AAV，“牛AAV”是指感染牛哺乳动物的AAV等。“重组AAV”或“rAAV”包括在其病毒基因组中包含异源多核苷酸序列的任何AAV。可用作载体的AAV血清型和变体的其他实例包括但不限于AAVDJ、AAV-PHP.S、AAV-PHP.B、AAV-PHP.eB和Anc80中的任一种或多种。
本文提到的碱基编辑器是指由DNA编辑器工具(如Cas9或Cas12)与 脱氨酶融合后的蛋白，可在DNA水平实现碱基转换，如ABE和CBE。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。除非和本申请的发明目的和/或技术方案相冲突，否则，本发明涉及的引用文献以全部内容、全部目的被引用。本发明中涉及引用文献时，相关技术特征、术语、名词、短语等在引用文献中的定义也一并被引用。本发明中涉及引用文献时，被引用的相关技术特征的举例、优选方式也可作为参考纳入本申请中，但以能够实施本发明为限。应当理解，当引用内容与本申请中的描述相冲突时，以本申请为准或者适应性地根据本申请的描述进行修正。
发明详述
本发明涉及具有CasRx活性的突变体，其与SEQ ID NO：1所示的氨基酸序列具体具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、至少99.5％、至少99.9％或100％的序列同一性，并且参照SEQ ID NO：1的氨基酸位置变化，具有位于下列位置处中的一个或多个突变：
K323、N324、G326、R333、N348、K350、K411、Y415、T486、R529、K585、R601、Y604、N641、R648以及Y649。
在本发明中，未特别说明书时，特定位置的突变可以为除其野生型位点以外的任何氨基酸。例如R648突变，是指其第648位的氨基酸可以突变为G、A、V、L、I、P、F、Y、W、S、T、C、M、N、Q、D、E、K、H中的任意一种；例如N641突变，是指其第641位的氨基酸可以突变为G、A、V、L、I、P、F、Y、W、S、T、C、M、Q、D、E、K、R、H中的任意一种；例如T486突变，是指其第486位的氨基酸可以突变为G、A、V、L、I、P、F、Y、W、S、C、M、N、Q、D、E、K、R、H中的任意一种。
在较为优选的实施方式中，突变后的氨基酸与突变前的氨基酸并非保守性替换。“保守性替换”是指一种氨基酸被另一种具有共同性质的氨基 酸所取代。从功能上定义单个氨基酸之间的共同性质的方法是分析同源生物中相应蛋白质之间氨基酸变化的标准化频率(Schulz(1979)Principles of Protein Structure，Springer-Verlag)。根据这样的分析，可以确定出氨基酸的族，其中族内的氨基酸优先相互替换，因此它们对蛋白质整体结构的影响最相似(Schulz(1979)同上)。以这种方式定义的氨基酸的组的实例包括:“带电/极性族”，包括Glu、Asp、Asn、Gln、Lys、Arg和His；“芳香族或环族”，包括Pro、Phe、Tyr和Trp；以及“脂肪族”，包括Gly、Ala、Val、Leu、Ile、Met、Ser、Thr和Cys。在每一族中，还可以确定子族。例如，带电荷/极性的氨基酸的族可以再分为亚族，所述亚族包括：“正电荷亚族”包括Lys、Arg和His；“负电荷亚族”包括Glu和Asp；以及“极性亚族”包括Asn和Gln。在另一实例中，芳香或环族可以再分为亚族，包括：“氮环亚族”，包括Pro、His和Trp；和“苯基亚族”，包括Phe和Tyr。在另一个实例中，脂肪族可再分为亚族，包括：“大脂肪族非极性亚族”，包括Val、Leu和Ile；“脂肪族微极性亚族”，包括Met、Ser、Thr和Cys；以及“小残基亚族”包括Gly和Ala。保守性突变的例子包括上述亚族内的氨基酸的氨基酸替换，例如，但不限于：Lys替换Arg或反之，以保持正电荷；Glu替换Asp或反之，这样可以保持负电荷；Ser替换Thr或反之，这样可以保持游离-OH；Gln替换Asn或反之，这样可以保持游离-NH2。
在一些实施方式中，所述的突变体具有位于下列位置处中的一个或两个突变：
R648和N641。
在一些实施方式中，所述的突变体具有位于下列位置处中的一个或两个突变：
R648和N641；并且具有位于下列位置处中的一个或多个突变：
K323、N324、G326、R333、N348、K350、K411、Y415、T486、R529、 K585、R601、Y604以及Y649。
在一些实施方式中，所述的突变体具有下述突变中的一个或两个：
R648A和N641A。
在一些实施方式中，所述的突变体具有下述突变中的一个或两个：
R648A和N641A；并且具有位于下列位置处中的一个或多个突变：
K323A、N324A、G326A、R333A、N348A、K350A、K411A、Y415A、T486A、R529A、K585A、R601A、Y604A以及Y649A。
在一些实施方式中，所述的突变体具有下述位置处的突变：
N641、T486和R648。
在一些实施方式中，所述的突变体具有下述突变N641A、T486A和R648A。
在一些实施方式中，所述的突变体具有下述突变N641A、T486A和R648A；并且具有位于下列位置处中的一个或多个突变：
K323A、N324A、G326A、R333A、N348A、K350A、K411A、Y415A、R529A、K585A、R601A、Y604A以及Y649A。
在一些实施方式中，所述的突变体为SEQ ID NO：2所示。
在一些实施方式中，所述的突变体具有显著提高的区分突变型和除了上述所描述的突变外，所述突变体还可以进一步包含一个或多个额外的取代、缺失或插入，优选所述取代、缺失或插入共同导致所述突变体：(1)具有与SEQ ID NO:1或2基本上相同(例如，至少约80％、90％、95％、99％或更多)的gRNA特异性核酸酶活性，和/或(2)基本上没有(例如，最多20％、15％、10％、5％)旁切(非gRNA依赖性)核酸酶活性。
在一些实施方式中，所述的突变体具有显著提高的区分单碱基突变相关的突变型和野生型转录本的能力。
在一些实施方式中，所述的突变体与向导RNA分子(gRNA)所组成的复合物与靶RNA的结合不稳定性提高。
在一些实施方式中，所述的突变体具有显著减少的核酸识别结构域与核酸酶结构域之间的负相关性；其中所述核酸识别结构域包括Helical1结构域，所述核酸酶结构域包括Helical2和HEPN结构域。
在一些实施方式中，所述的突变体基本上缺乏旁切内切核酸酶活性。
在某些实施方式中，在本发明突变体的RNP复合物与所述靶RNA结合后，所述突变体不会展现出实质性的(或可检测的)旁切RNA酶活性。
在一些实施方式中，所述突变体保留结合gRNA的能力。
在一些实施方式中，所突变体保留gRNA激活的RNA酶活性。
在一些实施方式中，在所结合的在序列方面与至少一部分靶RNA互补的指导/crRNA存在下，所述衍生物保留结合靶RNA和/或切割所述靶RNA的能力。
容易理解，上述的功能描述中所出现的相对趋势变化，如增加或减少，或保留某能力等，根据语境，均是与野生型的CasRx(特别是SEQ ID NO：1所示蛋白)进行比较。
本发明还涉及分离的多核苷酸，其编码如上所述的突变体。
在一些实施方式中，所述的多核苷酸包含调控元件，且所述调控元件与编码所述突变体的部分可操作地关联。
在本文描述的任何实施方案中，本发明的多核苷酸或核酸构建体均可以与多种调控元件可操作性相关联，以便在细胞中表达。因此，在一些实施方案中，所述调控元件包含以下元件中的一种或多种：启动子、内含子、增强子、终止子、5'和3'非翻译区、核定位信号(NLS)序列或核输出信号(NES)。且每种的数量可以为一个或多个；优选至少包含启动子。启动子的选择可以根据表达的时间和空间要求而变化，也可以根据待转化的 宿主细胞而变化。用于许多不同生物体的启动子是本领域众所周知的。基于本领域中存在的已有知识，可以为感兴趣的特定宿主生物体选择合适的启动子。因此，例如，对模式生物(如拟南芥、线虫、酵母、果蝇、小鼠、大鼠等)中高度组成型表达基因上游的启动子了解很多，并且这些知识可以很容易获取并在适当情况下在其他系统中实施。
在一些实施方式中，所述启动子包括U6启动子。
在一些实施方式中，所述启动子是组织特异性启动子。组织特异性启动子的示例性实例包括但不限于：B29启动子(B细胞表达)、runt转录因子(CBFa2)启动子(干细胞特异性表达)、CD14启动子(单核细胞表达)、CD43启动子(白细胞和血小板表达)、CD45启动子(造血细胞表达)、CD68启动子(巨噬细胞表达)、CYP4503A4或ALB启动子(肝细胞表达)、肌间线蛋白启动子(肌肉细胞表达)、弹性蛋白酶I启动子(胰腺泡细胞表达)、内皮糖蛋白启动子(内皮细胞表达)、成纤维细胞特异性蛋白I启动子(FSPl)启动子(成纤维细胞表达)、纤连蛋白启动子(成纤维细胞表达)、fms-相关的酪氨酸激酶I(FLTl)启动子(内皮细胞表达)、胶质纤维酸性蛋白(GFAP)启动子(星形胶质细胞表达)、胰岛素启动子(胰腺细胞表达)、整合蛋白-α-2b(ITGA2B)启动子(巨核细胞)、胞内粘着分子2(ICAM-2)启动子(内皮细胞)、干扰素-β(IFN-β)启动子(造血细胞)、角蛋白5启动子(角化细胞表达)、肌红蛋白(MB)启动子(肌肉细胞表达)、成肌分化I(MYOD1)启动子(肌肉细胞表达)、肾病蛋白启动子(足细胞表达)、骨γ-羧基谷氨酸蛋白2(OG-2)启动子(成骨细胞表达)、3-酮酸CoA转移酶2B(Oxct2B)启动子(单倍体精细胞表达)、表面活化蛋白B(SP-B)启动子(肺细胞表达)、突触蛋白启动子(神经细胞表达)、Wiskott-Aldrich综合征蛋白(WASP)启动子(造血细胞表达)、cTnT启动子(心肌细胞表达)。
在一些实施方式中，所述启动子为无细胞特异性的启动子。示例性的无细胞特异性的启动子包括但不限于巨细胞病毒(CMV)极早期启动子，病毒性猿猴病毒40(SV40)(例如，早期或晚期)、莫罗尼鼠白血病病毒 (MoMLV)LTR启动子，劳氏肉瘤病毒(RSV)LTR、单纯疱疹病毒(HSV)(胸苷激酶)启动子，牛痘病毒的H5、P7.5和Pll启动子，延长因子l-α(EFla)启动子，早期生长应答I(EGRl)、铁蛋白H(FerH)、铁蛋白L(FerL)、3_磷酸甘油醛脱氢酶(GAPDH)、真核生物翻译起始因子4A 1(EIF4A1)、热休克70kDa蛋白5(HSPA5)、热休克蛋白90kDa-β成员1(HSP90B1)、热休克蛋白70kDa(HSP70)、β-驱动蛋白(β-KIN)、人R0SA26基因座(Irions et al.,(2007)Nature Biotechnology25,1477-1482)、泛激素C启动子(UBC)，磷酸甘油酸激酶-1(PGK)启动子，巨细胞病毒增强子/鸡β-肌动蛋白(CAG)启动子，以及β-肌动蛋白启动子。无细胞特异性的启动子能使得所述基因表达盒具有更好的通用性及表达效率。
在一些实施方式中，本发明所述载体中还可以包含筛选所用的基因(例如抗生素抗性基因)，例如用于生成荧光蛋白的核酸等片段。荧光蛋白可以选择绿色荧光蛋白、蓝色荧光蛋白、黄色荧光蛋白、橙色荧光蛋白或红色荧光蛋白。绿色荧光蛋白可以采用常见的GFP，也可以采用经过改造后的GFP基因，例如增强型GFP基因EGFP等；蓝色荧光蛋白可以选自EBFP、Azuritc、TagBFP等；黄色荧光蛋白可以选自EYFP、Ypct、PhiYFP等；橙色荧光蛋白可以选自mKO、mOrange、mBanana等；红色荧光蛋白可以选自TagRFP、mRuby、mCherry、mKatc。
本文中提及多核苷酸时使用的“可操作地连接”或“可操作地相关联”，意指所指定的元件在功能上彼此相关，并且通常在实体上也相关。因此，如本文所用的术语“可操作地连接”或“可操作地相关联”，指的是单一核酸分子上在功能上相关联的核苷酸序列。因此，与第二核苷酸序列可操作地连接的第一核苷酸序列，意指当第一核苷酸序列与第二核苷酸序列处于功能关系时的情况。例如，如果启动子影响核苷酸序列的转录或表达，则该启动子与所述核苷酸序列可操作地相关联。本领域技术人员将会理解，调控序列(例如，启动子)不需要与其可操作地相关联的核苷酸序列相毗连，只要调控序列的功能是指导其表达即可。因此，例如，在启动 子和核苷酸序列之间可以存在间插的未翻译但已转录的核酸序列，并且仍然可以认为该启动子与核苷酸序列“可操作地连接”。
如本文所用的，在提及多肽时的术语“连接”，指的是一个多肽与另一个多肽的附接。一个多肽可以直接(例如，经由肽键)或通过接头与另一个多肽连接(在N-末端或C-末端)。
在一些实施方式中，所述多核苷酸经密码子优化以在生物体中表达。
本发明的任何核苷酸序列、多核苷酸和/或核酸构建体都可以进行密码子优化，以便在任何感兴趣的生物体中表达。密码子优化在本领域中是众所周知的，并且涉及使用物种特异性密码子使用表针对密码子使用偏好性对核苷酸序列进行修饰。密码子使用表是基于对感兴趣的生物体/物种的最高表达基因的序列分析而生成的。当核苷酸序列要在细胞核中表达时，密码子使用表是基于对感兴趣物种的高表达核基因的序列分析而生成的。通过将物种特异性密码子使用表与天然多核苷酸序列中存在的密码子进行比较来确定核苷酸序列的修饰。如本领域所理解的那样，核苷酸序列的密码子优化导致核苷酸序列与天然核苷酸序列(或称优化前的核苷酸序列)具有小于100％的同一性(例如，约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％或99.9％，以及其中的任何范围或值)，但其仍编码与原始天然核苷酸序列编码的多肽具有相同功能的多肽。因此，在本发明的一些实施方案中，本发明的多核苷酸、核酸构建体、表达盒和/或载体(例如，包含/编码本发明的多肽、融合蛋白、复合物，例如修饰的CRISPR-Cas核酸酶)是经密码子优化的，用于在感兴趣的特定物种中表达，例如特定植物物种、特定细菌物种、特定动物物种等。在一些实施方案中，本发明的经密码子优化的核酸构建体、多核苷酸、表达盒和/或载体与未经密码子优化的本发明的多核苷酸、核酸构建体、表达盒和/或载体具有约70％至约99.9％(例如，约70％、71％、 72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％或100％)或更高的同一性。
本发明的具有CasRx活性的突变体以及编码其的多核苷酸和核酸构建体可用于修饰任何生物体中的靶核酸，包括但不限于动物、植物、真菌、古细菌或细菌。动物可以包括但不限于哺乳动物、昆虫、鱼、鸟等。本发明可用于的示例性哺乳动物包括但不限于灵长类动物(人类，和非人类例如黑猩猩、狒狒、猴子、大猩猩等)、猫、狗、小鼠、大鼠、雪貂、沙鼠、仓鼠、牛、猪、驴、马、山羊、熊猫、大象或绵羊。植物可以是被子植物、裸子植物、单子叶植物、双子叶植物、C3、C4、CAM植物、苔藓植物、蕨类植物和/或拟蕨类植物、微藻类和/或大型藻类。
本发明还涉及一种分子系统，其为复合物或组合物，包含(a)如上所述的突变体和(b)向导RNA分子(gRNA)的集合。
本发明的分子系统中gRNA的种类可以为1、2、3、4、5、6、7、8、9或更多种。
在本发明的其他实施方案中，gRNA可进一步包含至少一种可检测标志。可检测标志可为荧光团(例如FAM、TMR、Cy3、Cy5、Texas Red、Oregon Green、Alexa Fluors、Halo标签或适合的荧光染料)、检测标签(例如生物素、地高辛等等)、量子点或者金颗粒。
在本发明的其他实施方案中，gRNA可包含标准核糖核苷酸和/或修饰的核糖核苷酸。在一些实施方案中，gRNA可包含标准或修饰的脱氧核糖核苷酸。在其中gRNA由酶促合成(即体内或体外)的实施方案中，gRNA一般包含标准核糖核苷酸。在其中gRNA由化学合成的实施方案中，gRNA可包含标准或修饰的核糖核苷酸和/或脱氧核糖核苷酸。修饰的核糖核苷酸和/或脱氧核糖核苷酸包括碱基修饰(例如假尿苷、2-硫代尿苷、N6-甲基腺苷 等等)和/或糖修饰(例如2’-O-甲基、2’-氟代、2’-氨基、锁核酸(LNA)等等)。gRNA的主链也可被修饰以包含硫代磷酸酯键合、硼烷磷酸酯键合或肽核酸。
如本文所用，组合物是指其中的组分(包括突变体和gRNA)是松散的组合或混合在一起的，例如分别负载于不同的载体中、分别包装于不同的容器中、或混合在一起但不通过强分子间作用力结合。复合物则是指其中的组分(包括突变体和gRNA)以核糖核蛋白(ribonucleoprotein，RNP)的形式进行结合，并且具有RNA切割活性。
本发明还涉及编码如上所述的分子系统的核酸构建体。
本发明还涉及载体，其包含如上所述的多核苷酸，或如上所述的核酸构建体。
载体可以是组合物，例如其是包含负载不同多核苷酸的病毒或质粒(分别表达具有CasRx活性的突变体和其他成分，如crRNA)，
本文所用的术语“载体”是指包含多核苷酸或与多核苷酸缔合的大分子或生物大分子缔合物，其可用于介导多核苷酸向细胞的转移、递送或导入。用于转化宿主生物体的载体在本领域是众所周知的。通用载体类别的非限制性实例包括病毒载体、质粒载体、噬菌体载体、噬菌粒载体、粘粒载体、fosmid载体、噬细菌体、人工染色体、微环载体(minicircle)或者农杆菌属(Agrobacterium)双元载体，呈双链或单链线性或环状形式，它们可能是或可能不是自我传递或移动的。在一些实施方式中，所述的载体是病毒载体。在一些实施方案中，所述病毒载体可包括但不限于腺病毒载体、腺相关病毒(AAV)载体、慢病毒载体或逆转录病毒载体。在一些实施方式中，所述的载体是AAV载体。
以本公开申请中使用的载体为例，AAV是感染人类和一些其他灵长类物种的小病毒。目前尚不知道腺相关病毒会导致疾病，而且在感染时，已经证明仅会引起轻微免疫反应。腺相关病毒能够感染分裂细胞和非分裂细 胞，并可将其基因组并入宿主细胞的基因组。此外，腺相关病毒大多保持游离状态(也就是说，它可以在宿主体内复制，而不会将其有效载荷并入到宿主染色体中)；进行长时间稳定的表达。这些特征使腺相关病毒成为创建用于基因治疗的病毒载体的合适候选物。因此，在一个实例中，病毒载体是腺相关病毒(AAV)载体。在另一个实例中，AAV载体是但不限于AAV 1型(AAV-1)、AAV 2型(AAV-2)、AAV 3型(AAV-3)、AAV 4型(AAV-4)、AAV 5型(AAV-5)、AAV 6型(AAV-6)、AAV 7型(AAV-7)、AAV 8型(AAV-8)、AAV9、AAV10、AAV11、AAV12、AAV13、rh10、AAVDJ、AAV-PHP.S、AAV-PHP.B、AAV-PHP.eB和Anc80。AAV载体血清型可以与靶细胞类型匹配。例如，WO 2018002719A1的表2列出了可以被指定的AAV血清型转导的示例性细胞类型(通过引用并入本文)。
在一些实施方式中，可以使用三重转染方法(在美国专利号6,001,650中详细描述)产生重组AAV。典型地，通过用有待包装到AAV颗粒中的重组AAV载体(包含感兴趣的基因)、AAV辅助功能载体和辅佐功能载体转染宿主细胞产生重组AAV。AAV辅助功能载体编码“AAV辅助功能”序列(例如，rep和cap)，所述序列以反式起作用，用于生产性AAV复制和衣壳化。优选地，AAV辅助功能载体支持高效的AAV载体生产，而不生成任何可检测的野生型AAV病毒粒子(例如，含有功能性rep和cap基因的AAV病毒粒子)。所述辅佐功能载体编码用于AAV进行复制所依赖的非AAV衍生的病毒和/或细胞功能(例如，“辅佐功能”)的核苷酸序列。辅佐功能包括AAV复制所需的那些功能，包括但不限于参与激活AAV基因转录、阶段特异性AAV mRNA剪接、AAV DNA复制、cap表达产物合成和AAV衣壳组装的那些部分。基于病毒的辅佐功能可衍生自已知的辅助病毒的任一种，如腺病毒、疱疹病毒(除了单纯疱疹病毒-1型)和牛痘病毒。
在某些实施方式中，所述AAV的基因组包含5'AAV ITR序列和3'AAV ITR序列。
在一些实施方式中，使用包装在昆虫细胞(如Sf9细胞)中的杆状病毒 表达系统产生本发明AAV病毒颗粒。参见例如，WO 2007046703、WO 2007148971、WO 2009014445、WO 2009104964、WO 2013036118、WO 2011112089、WO 2016083560、WO 2015137802和WO 2019016349，将所有文献通过引用并入本文。
载体滴度通常表示为病毒基因组/ml(vg/ml)。在某些实施方式中，病毒滴度高于1×109、高于5×1010、高于1×1011、高于5×1011、高于1×1012、高于5×1012、或高于1×1013vg/ml。
本发明还涉及递送系统，其包含i)如上所述的分子系统，以及ii)递送媒介物。
在一些实施方式中，所述递送媒介物包括一种或多种脂质体、一种或多种外泌体、一种或多种微囊泡、一种或多种树状大分子、一种或多种无机纳米粒子、一种或多种细胞穿膜肽、基因枪、一种或多种质粒、一种或多种病毒载体(在一些实施方式中，所述病毒载体如上所定义)，以及它们所组成的组。
脂质体可以为阳离子脂质体或中性脂质体，其可通过公知的方法进行制备或修饰，例如加入聚乙二醇(PEG)修饰的脂质体可以有效防止脂质体载体的聚集并增加其稳定性。脂质体或脂质转染配制品可以通过本领域技术人员已知的方法制备。这样的方法描述于例如WO 2016205764和美国专利号5,593,972、5,589,466、和5,580,859中，将各个文献通过引用以其全文并入本文。
树枝状大分子是一种具有明确的分子结构、可精确控制的化学结构和独特的多价性质的特殊聚合物家族，逐渐成为基因传递的非病毒载体。典型的树枝状大分子例如聚(酰氨基胺)(PAMAM)树枝状聚合物，其可做进一步修饰，例如在PAMAM表面修饰核碱基类似物2-氨基-6-氯嘌呤构建衍生物AP-PAMAM，或者通过硫酸软骨素(CS)与PAMAM偶联制备CS-PAMAM等等。
无机纳米粒子可选择金纳米粒子(AuNPs)、磁性纳米粒子、介孔二氧化硅纳米粒子(MSNs)等。
细胞穿膜肽(cell-penetrating peptides，CPPs)是一类具有较强跨膜转运能力的小分子肽，可携带多肽、蛋白质和核酸等多种大分子物质进入细胞。其可以为阳离子型CPPs(如TAT，Penetratin，Polyarginine，P22N，DPV3和DPV6等)、两亲型CPPs(可以由疏水性肽序列和NLSs共价连接而成，或者从天然蛋白质中分离获得，如pVEC，ARF(1-22)和BPrPr(1-28))、疏水型CPPs(一般只含有非极性氨基酸残基，净电荷量约低于氨基酸序列总电荷量的20％)。
在一些实施方式中，所述递送经由质粒进行。所述剂量可以是足够数量的质粒以引发响应。在一些情况下，质粒组合物中质粒DNA的合适量可以是从约0.1至约2mg。质粒将通常包括(i)启动子；(ii)编码靶向核酸的CRISPR相关蛋白和/或辅助蛋白的序列，每个序列与启动子(例如，相同的启动子或不同的启动子)可操作地连接；(iii)可选择标志物；(iv)复制起点；以及(v)位于(ii)的下游并与其可操作地连接的转录终止子。质粒还可以编码CRISPR复合物的RNA组分，但这些组分中的一种或多种可以替代地在不同的载体上编码。施用频率在医学或兽医学从业者(例如，医师、兽医师)或本领域技术人员的范围内。
递送可以通过本领域已知的任何一种方式，如转染、脂质转染、电穿孔、基因枪、显微注射、超声、磷酸钙转染、阳离子转染、病毒载体递送等来进行。
本发明还涉及药物组合物，其包含如上所述的递送系统以及药学上可接受的赋形剂。
本发明还涉及试剂盒，其包含：如上所述的突变体、或如上所述的分子系统、或如上所述的载体、或如上所述的递送系统、或如上所述的药物组合物，以及任选的该试剂盒的使用说明书。
本发明的试剂盒可以包含用于混合、测量、分选、标记等的试剂、缓冲液和/或装置，以及说明书等。
在某些实施方式中，所述试剂盒进一步包含一种或多种缓冲液，所述缓冲液可用于溶解任一所述组分和/或为一种或多种所述组分提供合适的反应条件。这样的缓冲液可以包括以下中的一种或多种：PBS、TBS、HEPES、Tris、Na2CO3、NaHCO3、醋酸盐或其组合。在某些实施方式中，所述反应条件包括适当的pH，如碱性pH。在某些实施方式中，所述pH在7-10之间。
在某些实施方式中，任一种或多种所述试剂盒组分可以储存在合适的容器中。
本发明还涉及一种改变基因产物表达的方法，包括使用CRISPR-Cas核酸酶体系切割靶RNA引入细胞的步骤；
所述CRISPR-Cas核酸酶体系包含如上所述的突变体、或如上所述的分子系统、或如上所述的载体、或如上所述的递送系统、或如上所述的药物组合物、或如上所述的试剂盒。
在某些实施方式中，所述细胞是真核细胞，如哺乳动物细胞，包括人细胞(原代人细胞或已建立的人细胞系)。在某些实施方式中，所述细胞是非人哺乳动物细胞，如来自非人灵长类动物(例如，猴)、母牛/公牛/牛、绵羊、山羊、猪、马、狗、猫、啮齿动物(如兔子、小鼠、大鼠、仓鼠等)的细胞。在某些实施方式中，所述细胞来自鱼(如鲑鱼)、鸟(如禽鸟，包括鸡、鸭、鹅)、爬行动物、贝类(例如，牡蛎、蛤蜊、龙虾、对虾)、昆虫、蠕虫、酵母等在某些实施方式中，所述细胞来自植物，如单子叶植物或双子叶植物。在某些实施方式中，所述植物是粮食作物，如大麦、木薯、棉花、落花生或花生、玉蜀黍、小米、油棕果、马铃薯、干豆、油菜籽或低芥酸菜籽(canola)、稻、黑麦、高粱、大豆、甘蔗、甜菜、向日葵和小麦。在某些实施方式中，所述植物是谷类(大麦、玉蜀黍、小米、 稻、黑麦、高粱和小麦)。在某些实施方式中，所述植物是块茎(木薯和马铃薯)。在某些实施方式中，所述植物是糖料作物(甜菜和甘蔗)。在某些实施方式中，所述植物是含油作物(大豆、落花生或花生、油菜籽或低芥酸菜籽、向日葵和油棕果)。在某些实施方式中，所述植物是纤维作物(棉花)。在某些实施方式中，所述植物是树木(如桃树或油桃树、苹果树或梨树、坚果树(如杏仁树或核桃树或开心果树)、或柑橘树(例如，橙树、葡萄柚树或柠檬树))、草、蔬菜、水果或藻类。在某些实施方式中，所述植物是茄属植物；芸苔属(Brassica)植物；莴苣属(Lactuca)植物；菠菜属(Spinacia)植物；辣椒属(Capsicum)植物；棉花、烟草、芦笋、胡萝卜、卷心菜、西兰花、花椰菜、番茄、茄子、胡椒、生菜、菠菜、草莓、蓝莓、覆盆子、黑莓、葡萄、咖啡、可可等。对于特定国家，可能需要专门排除某些主题，例如人的全能干细胞，受精超过14天以上的受精卵等。
相关方面提供使用本文描述的CRISPR系统通过本发明的方法修饰的细胞或其后代。
在某些实施方式中，所述细胞在体外、在体内或离体进行修饰。在某些实施方式中，所述细胞是干细胞。
本发明还涉及如上所述的突变体、或如上所述的分子系统、或如上所述的载体、或如上所述的递送系统在制备用于治疗基因变异所引起的疾病的药物中的应用；其中所述疾病由包含一个或多个单碱基突变的变异所引起，或者可通过敲减或敲除某个基因得到治疗。
在一些实施方式中，疾病为遗传性疾病或原发性疾病。
在一些示例性的方案中，疾病非限制性地包括如下疾病中的一种或多种：
遗传性心脏病(优选遗传性心肌病)、镰刀状贫血、地中海贫血、血友病、早衰症、苯丙酮尿症、糖原累积病、I型糖尿病、家族性高胆固醇血症、高甘油三脂血症、克拉伯病、戈谢病、色盲、遗传性耳聋、遗传性 老年痴呆、家族性淀粉样病变、进行性肌营养不良以及由单碱基突变导致的肿瘤。
在某些实施方式中，所述肿瘤是癌、肉瘤、骨髓瘤、白血病、淋巴瘤和混合型肿瘤。可以通过本文描述的方法和组合物治疗的肿瘤的非限制性实例包括来自以下的癌细胞：膀胱、血液、骨、骨髓、脑、乳腺、结肠、食道、胃肠道、牙龈、头、肾、肝、肺、鼻咽、颈、卵巢、前列腺、皮肤、胃、睾丸、舌、或子宫。此外，所述癌症可以特别地属于以下组织学类型，但不限于这些：恶性赘生物；癌；未分化的癌；巨细胞癌和梭形细胞癌；小细胞癌；乳头状癌；鳞状细胞癌；淋巴上皮癌；基底细胞癌；毛母质癌；移行细胞癌；乳头状移行细胞癌；腺癌；恶性胃泌素瘤；胆管癌；肝细胞癌；混合型肝细胞癌和胆管癌；小梁腺癌；腺样囊性癌；腺瘤性息肉中的腺癌；家族性结肠息肉病性腺癌；实体癌；恶性类癌肿瘤；细支气管-肺泡腺癌；乳头状腺癌；嫌色细胞癌；嗜酸细胞癌；嗜酸性腺癌；嗜碱细胞癌；透明细胞腺癌；颗粒细胞癌；滤泡状腺癌；乳头状和滤泡状腺癌；非包裹性硬化型癌；肾上腺皮质癌；子宫内膜样癌；皮肤附件癌；大汗腺癌；皮脂腺癌；耵聍腺癌；粘液表皮样癌；囊腺癌；乳头状囊腺癌；乳头状浆液性囊腺癌；粘液性囊腺癌；粘液性腺癌；印戒细胞癌；浸润性导管癌；髓样癌；小叶癌；炎性癌；乳腺佩吉特病；腺泡细胞癌；腺鳞癌；腺癌伴鳞状化生；恶性胸腺瘤；恶性卵巢间质瘤；恶性卵泡膜细胞瘤；恶性颗粒细胞瘤；和恶性成纤维细胞瘤；支持细胞癌；恶性睾丸间质细胞瘤；恶性脂质细胞瘤；恶性副神经节瘤；恶性乳腺外副神经节瘤；嗜铬细胞瘤；血管球肉瘤；恶性黑素瘤；无黑素性黑素瘤；浅表扩散性黑素瘤；巨大色素痣中的恶性黑素瘤；上皮样细胞黑素瘤；恶性蓝痣；肉瘤；纤维肉瘤；恶性纤维组织细胞瘤；粘液肉瘤；脂质肉瘤；平滑肌肉瘤；横纹肌肉瘤；胚胎性横纹肌肉瘤；肺泡横纹肌肉瘤；间质肉瘤；恶性混合瘤；苗勒管混合瘤；肾母细胞瘤；肝母细胞瘤；癌肉瘤；恶性间叶瘤；恶性布伦纳瘤；恶性叶状瘤；滑膜肉瘤；恶性间皮瘤；无性细胞瘤； 胚胎癌；恶性畸胎瘤；恶性卵巢甲状腺肿；绒毛膜癌；恶性中肾瘤；血管肉瘤；恶性血管内皮瘤；卡波西肉瘤；恶性血管外皮细胞瘤；淋巴管肉瘤；骨肉瘤；骨旁骨肉瘤；软骨肉瘤；恶性软骨母细胞瘤；间叶性软骨肉瘤；骨巨细胞瘤；尤因肉瘤；恶性牙源性肿瘤；成釉细胞牙肉瘤；恶性成釉细胞瘤；成釉细胞纤维肉瘤；恶性松果体瘤；脊索瘤；恶性胶质瘤；室管膜瘤；星形细胞瘤；原生质星形细胞瘤；纤维型星形细胞瘤；星形母细胞瘤；胶质母细胞瘤；少突神经胶质瘤；成少突神经胶质细胞瘤；原始神经外胚瘤；小脑肉瘤；神经节神经母细胞瘤；成神经细胞瘤；视网膜母细胞瘤；嗅觉神经源性肿瘤；恶性脑膜瘤；神经纤维肉瘤；恶性神经鞘瘤；恶性颗粒细胞瘤；恶性淋巴瘤；霍奇金病；霍奇金淋巴瘤；副肉芽肿；小淋巴细胞性恶性淋巴瘤；弥漫性大细胞性恶性淋巴瘤；滤泡性恶性淋巴瘤；蕈样肉芽肿；其他指定的非霍奇金淋巴瘤；恶性组织细胞增生症；多发性骨髓瘤；肥大细胞肉瘤；免疫增殖性小肠疾病；白血病；淋巴样白血病；浆细胞白血病；红白血病；淋巴肉瘤细胞白血病；髓性白血病；嗜碱性粒细胞白血病；嗜酸性粒细胞白血病；单核细胞白血病；肥大细胞白血病；巨核细胞白血病；髓性肉瘤；浆细胞瘤、结直肠癌、直肠癌和毛细胞白血病。
在一些实施方式中，所述疾病为遗传性心肌病。
可通过敲减或敲除某个基因得到治疗的疾病是本领域技术人员所熟知的，例如，亨廷顿舞蹈症(敲减Htt)、帕金森综合症(敲除ptbp1促进星形胶质细胞转分化为神经元治疗神经元丢失的帕金森疾病)、Rett综合征(Rett syndrome,Mecp2基因重复)。
在一些实施方式中，所述分子系统、或所述的载体、或所述的递送系统中gRNA的间隔序列与SEQ ID NO：3～37任一项所示序列具有至少70％、至少80％、至少90％、至少95％或100％序列同一性。
本发明同时请求保护如上所述的gRNA的间隔序列(特别是与SEQ ID NO：3～37任一项所示序列具有至少70％、至少80％、至少90％、至少 95％或100％序列同一性的核酸分子)；本发明同时请求保护包含所述间隔序列的gRNA；本发明同时请求保护包含该gRNA的多核苷酸、分子系统、核酸构建体、载体、递送系统以及药物组合物。上述多核苷酸、分子系统、核酸构建体、载体、递送系统以及药物组合物的描述也适用于此处。
本发明同时请求保护如上所述的SEQ ID NO：2所示突变体(或者是与SEQ ID NO：2所示序列具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、至少99.5％、至少99.9％或100％序列同一性的氨基酸分子)；本发明同时请求保护用于表达该突变体的多核苷酸，以及相关的分子系统、核酸构建体、载体、递送系统以及药物组合物。上述多核苷酸、分子系统、核酸构建体、载体、递送系统以及药物组合物的描述也适用于此处。
在一些实施方式中，gRNA的直接重复序列(或简称为“DR序列”)与SEQ ID NO：38或SEQ ID NO：39所示序列具有至少70％、至少80％、至少90％、至少95％或100％序列同一性。
容易理解，本发明可用于一个或多个单碱基突变所引起的疾病，例如某疾病由两个点突变所引发，且两个点突变相距较远，足以被两套本发明所提供的分子系统所识别，显然也可以被本发明所提供的技术方案所治疗。
在本发明的又一个方面，涉及一种治疗单碱基突变所引起的疾病的方法，包括将如使用有效量的CRISPR-Cas核酸酶体系切割靶RNA引入细胞的步骤；
其中所述CRISPR-Cas核酸酶体系包含如上所述的突变体、或如上所述的分子系统、或如上所述的载体、或如上所述的递送系统、或如上所述的药物组合物。
本发明中所述的术语“有效量”是指该术语所对应的组分在受试者中 实现治疗、预防、减轻和/或缓解本发明中所述疾病或病症的剂量。
在一些实施方式中，通过例如肌内注射、静脉内施用、经皮施用、鼻内施用、口服施用或粘膜施用将如上所述的突变体、或如上所述的分子系统、或如上所述的载体、或如上所述的递送系统、或如上所述的药物组合物递送至感兴趣的组织。
递送可以经由单剂量或多剂量进行。本领域技术人员应理解，本文待递送的实际剂量可取决于多种因素而大幅变化，如载体选择、靶细胞、生物、组织、待治疗受试者的一般状况、所寻求的转化/修饰的程度、施用途径、施用模式、所寻求的转化/修饰的类型等。
在某些实施方式中，所述递送经由AAV进行，其可以是含有至少1×105个颗粒的AAV的单剂量。在一些实施方式中，所述剂量优选地是至少约1×106个颗粒、至少约1×107个颗粒、至少约1×108个颗粒、和至少约1×109个颗粒的AAV。
在一些实施方式中，递送通过如上所描述的递送系统进行，在如上内容中已经过详尽描述。
下面将结合实施例对本发明的实施方案进行详细描述。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，优先参考本发明中给出的指引，还可以按照本领域的实验手册或常规条件，还可以参考本领域已知的其它实验方法，或者按照制造厂商所建议的条件。
下述的具体实施例中，涉及原料组分的量度参数，如无特别说明，可能存在称量精度范围内的细微偏差。涉及温度和时间参数，允许仪器测试精度或操作精度导致的可接受的偏差。
实施例1材料和方法
1.1实验动物
采用CRISPR/Cas9技术构建与人R870H同源的Myh6-pR872H点突变小鼠(图1)。在点突变位点上下游分别设计一条sgRNA，序列分别为：
sgRNA1 5'-GCTGCTGAAGAGCGCAGAGA-3'；
sgRNA2 5'-CCCAAACTCCTCCTTCATGT-3'；
体外转录sgRNA。将spCas9蛋白、sgRNA和同源重组载体样品显微注射到C57BL/6JGpt背景的小鼠受精卵中。取注射后存活的受精卵移植到假孕雌鼠体内，待其怀孕产仔。通过PCR和Sanger测序鉴定出阳性的F0代小鼠，待其性成熟后与野生型背景小鼠交配，得到稳定遗传的阳性F1代小鼠。PCR引物：872-tF1,5'-GCTACAATGCCCCTTGTCCTTTG-3'；872-tR1,5'-GCTCCTCTGCATCATTGAGGTTG-3'。测序引物为872-tF1。Arms-PCR引物：Mu872-F:5'-AGAGACTGAGAAGGAGATCGCA-3'；Wt872-F:5'-AGACGGAGAAGGAGATCGCC-3'；872-tR1:5'-GCTCCTCTGCATCATTGAGGTTG-3'
从江苏集萃药康生物科技有限公司购买Myh6-pR404Q点突变小鼠(T051403)，该点突变与人MYH7-pR403Q同源。将Myh6 pR872H点突变纯合子(RH/RH)与Myh6 pR404Q杂合子(RQ/+)杂交，得到RQ/RH双点杂合突变小鼠。采用PCR和Sanger测序鉴定RQ阳性小鼠，PCR引物：T051403-F1 CTTCAGCTAGATCCCAGTCAAGCC，T051403-R1 GGTCTGAGTGGGTAGGTGAGAAACAT。采用T051403-F1引物测序。同时，也可采用Arms-PCR鉴定小鼠基因型，使用引物如下：
Mu404-F:TCAAGGGCCTGTGTCACCGTCA；
Wt404-F:TCAAGGGCCTGTGTCACCATCG；
404-tR1:GGGTCCCCACTACTATCCTGT；
1mg/kg环孢素(CsA)饲料在小鼠5周龄至8周龄连续喂食3周，用于加速Myh6-pR872H点突变小鼠的左心室肥厚表型。
1.2心脏超声
采用1-2％异氟烷麻醉小鼠，当心率为450-500bpm/min时采用Visual Sonics 2100系统和MS-400探头采集心脏长轴超声图像。测量左心室的室壁厚度、腔的大小和室间隔厚度、射血分数等数据。
1.3心电监测
采用1-1.5％异氟烷麻醉小鼠，采用可充电动物遥测系统(科心医学生物技术公司)采集麻醉状态下小鼠的心电(肢体二导联)。采用Labchart7分析心电数据。
1.4组织学染色
动物经CO2处死后立即采用4％多聚甲醛溶液缓慢灌注心脏，摘取的心脏置于4％多聚甲醛溶液中4℃固定24小时。70％-100％乙醇梯度脱水后，二甲苯通透2小时，再进行石蜡包埋。5μm厚度的石蜡切片样品采用H&E(苏木素伊红)或者Masson’s Trichrome试剂盒染色(翌圣生物科技有限公司)检测心脏的形态变化、心肌纤维排列和成纤维情况。小麦胚芽凝集素(WGA，invitrogen，Cat:W32466)特异性结合细胞膜表面的N-乙酰-D-葡萄糖胺和唾液酸残基，用于检测心肌细胞大小。
1.5筛选系统的构建
为了更好地筛选能在体内特异性敲除单点突变转录本的CasRx变体，我们首先构建了野生型myh6融合快速降解型EGFP绿色荧光蛋白的质粒(pCMV-wtMyh6-dEGFP)和单点突变型myh6融合快速降解型mCherry红色荧光蛋白的质粒(pCMV-muMyh6-dmCherry)。我们针对Myh6 pR404Q和Myh6 pR872H分别构建了两套筛选质粒，针对人MYH7基因设计了一套筛选质粒。同时，将SV40 NLS-Cas13d-SV40 NLS-HA克隆至lenti-crispr v2(addgene:52961)载体上的XbaI和BamHI酶切位点，将Cas13d DR-Bsmbi-EGFP-Bsmbi连接至U6启动子下游，得到lenti-U6-DR-Bsmbi-Bsmbi-EF1a-Cas13d表达质粒，简称lenti-Cas13d-sgRNA质粒。采用Overlap PCR的形式对Cas13d上的候选氨基酸位点进行点突变，采用 Gibson assembly试剂盒将突变的Cas13d DNA连接到lenti-Cas13d-sgRNA上的XbaI和BamHI酶切位点，构建不同的Cas13d突变体。
采用Lipofectamine 3000(Invitrogen)将双荧光报告质粒与lenti-Cas13d-sgRNA质粒共转至90％融合度的293T细胞中，转染后48小时提取RNA，采用RT-qPCR分别检测EGFP和mCherry的敲除效率来分别反映野生型和突变型Myh6 mRNA的敲除效率。同时，采用流式分析检测EGFP和mCherry的荧光强度来分别反映野生型和突变型Myh6蛋白的表达水平，通过与转染无特异性靶向的NT sgRNA组对比，计算不同样品中野生型和突变型Myh6蛋白的敲除效率。采用这种方式，我们筛选出靶向Myh6pR872H位点的高活性sgRNA，用于后续Cas13d突变体的筛选。
qPCR引物如下：
EGFP-F:CACCCTGACCTATGGAGTGC
EGFP-R:GGTCTTGTAGTTGCCGTCGT
mCherry-F:TTCATGTACGGCTCCAAGGC
mCherry-R:TGTAGATGAACTCGCCGTCC
hGAPDH-F：GTCTCCTCTGACTTCAACAGCG
hGAPDH-R：ACCACCCTGTTGCTGTAGCCAA
1.6 AAV-cTnT-Cas13d-U6-sgRNA表达质粒构建和AAV9的包装
采用同源重组将U6-DR-Sap1-Sap1序列连接到px601质粒(addgene,61591)上的KpnI-NotI酶切位点，将SV40 NLS-Cas13d-SV40 NLS-HA序列连接到NheI-EcoRI酶切位点(为SEQ ID NO：42和SEQ ID NO：43所示，分别插入wtCas13d和superCas13d)，最后将心肌细胞特异性启动子cTnT序列(cTnT启动子的核苷酸序列如SEQ ID NO：40所示)连接到XbaI-AgeI酶切位点，得到AAV-cTnT-Cas13d-bGH polyA-Sap1-Sap1-DR-U6表达质粒，简称AAV-cTnT-Cas13d-U6-sgRNA质粒(U6 启动子的核苷酸序列如SEQ ID NO：41所示)。特异性靶向Myh6-pR872H和Myh6-pR404Q的向导RNA(872-sgRNA9和404-sgRNA15)连接至上述质粒的Sap1-Sap1位点，分别得到AAV-cTnT-Cas13d-U6-R872H sgRNA质粒和AAV-cTnT-Cas13d-U6-R404Q sgRNA质粒。
将该上述表达质粒分别与AAV9的辅助包装质粒(rep/cap质粒和pHGTI/delta质粒)一起共转至融合度70％-80％的293T细胞中，转染后60小时收集上清和细胞裂解液，再依次采用碘克沙醇梯度沉降和超滤膜浓缩病毒颗粒。采用绝对定量qPCR检测病毒滴度。浓缩的病毒采用PBS重悬，分装后保存在-80℃。
1.7分子动态学模拟
首先采用SWISS-MODEL软件以EsCas13d-crRNA-靶RNA结构(PDB:6e9f)为模板同源建模获得RfxCas13d蛋白初步结构。同时采用AlphaFold2对RfxCas13d的结构进行预测，使用AlphaFold2预测结构中打分较高的区域(770A-821N)替换SWISS-MODEL搭建的结构区域，最终获得RfxCas13d蛋白结构。接着在Pymol中将EsCas13d的crRNA突变成RfxCas13d的crRNA序列：5'-AACCCCUACCAACUGGUCGGGGUUUGAAACGACCUGAAUAUUUCAGAUCAAA-3'，再将突变后的crRNA-靶RNA核酸结构与上述搭建的的蛋白构象进行叠合得到了RfxCas13d-RNA复合物结构。以此为模板，对RfxCas13d进行突变，搭建其他突变体的Cas13d-RNA复合物结构。
接下来，使用GROMACS-2021软件对Cas13d-RNA复合物模型进行能量最小化。带有核酸校正的f14SB力场用于描述Cas13d-RNA复合物体系。每个HoxD9-DNA体系均置于TIP3P水盒子中央，溶质距水盒子的最小边界设置为并添加适当数量的Na+和Cl-以确保离子浓度为0.15摩尔/升。最终每个体系平均含249805个原子。对于每个体系，通过以下步骤进行能量最小化：首先约束Cas13d以及RNA所有原子，接着约束Cas13d的主链C原子和RNA主链磷原子，最后，放开所有原子进行能量最小化。提 取上述每个体系能量最小化的最后一帧，作为分子动力学模拟的输入结构。对于每个体系设定初始随机种子，进行三次平行的200纳秒的分子动力学模拟。
对分子动力学模拟的结果首先根据拓扑文件对蛋白、RNA、镁离子进行编组构建索引文件，使用子命令trjconv，将体系进行中心化，同时对体系进行旋转和平移处理周期性边界条件，防止构像发生破坏产生异常键。对crRNA-targetRNA碱基互补区域进行编组，构建索引文件，使用rms子命令计算各个轨迹中RNA磷原子的均方根偏差(RMSD)和均方根涨落(RMSF)。
同时根据以下步骤分析构象相关性网络：对Cas13d的主链碳原子进行编组，构建整个轨迹的协方差矩阵，并计算特征向量，将轨迹在第一主成分的方向上进行投影，获得主链碳原子在第一主成分上的Cas13d构象。计算Cas13d主链碳原子之间运动轨迹相关性，相关性大于0.6或者小于-0.6具有运动通向相关性/负相关性。将相关性网络比对到第一主成分构象上进行构像相关性网络的可视化。
1.8 Cas13d蛋白纯化和体外切割
将Cas13d构建到pET载体，N端和C端分别融合组蛋白标签，并转化到BL21(DE3)感受态细胞中。挑取阳性克隆至LB培养基中，37℃培养12小时，当OD600为0.6-0.8时，加入0.5mM IPTG 16℃诱导16-18小时。收集细胞裂解液，镍柱纯化蛋白，透析过夜后，100KDa超滤管浓缩，蛋白保存于-80℃。纯化的Cas13d蛋白与体外转录的sgRNA及靶RNA，按1:1：1摩尔比置于切割反应液中，37℃反应15分钟，加入蛋白酶K终止反应，加入RNA染料(NEB)80℃变性10分钟，10％聚丙烯酰胺凝胶分析切割结果。
1.9数字滴度PCR(ddPCR)
采用RNA提取试剂盒(天跟)抽提心室RNA，并稀释至2.5ng/ul。根 据ddPCR试剂盒说明书，将2ul的RNA加入至25ul的ddPCR反应液中，其中包含12.5ul 2 X qScript XLT one-step RT-qPCR Though mix(Quanta bio,cat NO.95132-100),100nM荧光素(Pexbio,China),250nM探针and 1μM引物。然后将配置好的反应液转移至芯片中(NaicaTM Sapphire Chips，Stilla Technologies)，置于ddPCR反应仪(NaicaTM Digital PCR System，Stilla Technologies)，启动反应程序。最终用Crystal Reader和Crystal Miner分别读取和分析阳性油滴数。
ddPCR引物和探针如下：
ddPCR引物和探针
实施例2野生型Cas13d-sgRNA无法特异性靶向Myh6 R872H突变转录本(细胞层面)
Cas13d对单碱基错配的识别可能具有序列依赖性，为了研究野生型Cas13d是否能特异性靶向Myh6 R872H突变转录本，从而用于治疗由该位点突变引起的遗传性心肌病。我们在该点突变对应的双荧光报告细胞系中检测12条sgRNA(SEQ ID NO：3-14)的单碱基识别能力。结果表明，只有sgRNA2、sgRNA7、sgRNA8、sgRNA9、sgRNA10、sgRNA11具有一定的区 分能力，即敲减完全匹配的突变型Myh6 RNA的水平显著高于敲减单碱基错配的野生型Myh6 RNA的水平。但是，针对两者的敲减水平差异只有10％左右，结果示于图2和表1中。
表1
实施例3工程化Cas13d显著提高靶向Myh6 R872H点突变转录本的特异性(细胞层面)
RfxCas13d(又称CasRx)与EsCas13d均属于Cas13d家族，蛋白序列的同源性为30％。因此，我们采用(PS)2和SWISS-Model以EsCas13d的三维结构(PDB:6E9F)构建了RfxCas13d的同源结构。并在Pymol中对EsCas13d的crRNA序列突变成RfxCas13d的crRNA序列，与同源构建的RfxCas13d结构对接，产生RfxCas13d-sgRNA-target RNA三复合物的三维结构，结果示于图3中。
我们根据该结构找到16个RfxCas13d与sgRNA-target RNA相互作用的氨基酸，结合EsCas13d与sgRNA-target RNA相互作用的保守性氨基酸，将这些位点分别突变为丙氨酸，在Myh6 R872H双荧光细胞系中筛选能提高单碱基识别特异性的突变体。结果表明，R648A、N641A突变体分别 与两条sgRNA组合均能显著提高区分突变型和野生型转录本的能力，结果示于图3、表2和表3中。
表2
表3

进一步对剪切活性相对高的突变位点进行两两组合、三个组合，发现N641A/T486A/R648A(SuperCas13d)这个突变体单碱基错配识别能力最强，结果示于图4、表4和表5中。
表4
表5
实施例4工程化SuperCas13d提高单碱基识别特异性的同时降低了旁切活性
将野生型Cas13d和SuperCas13d蛋白在细菌中表达纯化出来，以不同的浓度切割1ug RNA底物，测试浓度依赖的RNA切割效率。结果表明在不同浓度下SuperCas13d切割完全匹配RNA底物的效率远高于切割单碱基错配RNA底物的效率，且达到最高切割效率的所需浓度相差近两倍。而将Cas13d蛋白浓度固定为225nM，检测时间依赖的RNA切割效率时，SuperCas13d也展现对错配RNA底物更低的切割速度，切割速度差20倍。结果示于图5。
进一步在gRNA上引入错配碱基，在HEK293细胞中检测SuperCas13d的特异性识别窗口。结果表明相比野生型Cas13d，SuperCas13d在gRNA的15-21nt位置容错率显著降低。结果示于图6。
由于Cas13d还存在靶RNA激活后的非特异性旁切活性，该活性与靶向底物的切割活性偶联。我们进一步检测了工程化的SuperCas13d的旁切活性，结果表明SuperCas13d对非目标RNA的旁切活性显著降低，与已报道的hfCas13d相当，但对目标RNA的切割活性高于hfCas13d。结果示于图7。
实施例5分子动态学模拟阐明SuperCas13d提高单碱基特异性的机制
我们采用GROMACS软件对野生型Cas13d、Cas13d-N641A、Cas13d-R648A、Cas13d-T486A和SuperCas13d的三复合物结构分别开展分子动态学模拟实验。结果发现，R648A、T486A突变分别减少了自身与周围靶RNA底物的结合，而N641A点突变则改变了K636与周围靶RNA底物的结合位点，三者联合突变整体减少了Cas13d与靶RNA底物的结合力，使得SuperCas13d与靶RNA作用时柔性更大、更不稳定，结果示于图8中。
进一步分析Cas13d分别与sgRNA和靶RNA上每个碱基的动态距离，与野生型Cas13d和其他单点突变体对比，SuperCas13d显著提高了与 sgRNA上的39-43号碱基、靶RNA上的14-18号碱基的结合不稳定性，而靶RNA上的14-18号碱基靠近N641A、R648A点突变。进一步分析不同Cas13d的构象变化发现，相比野生型Cas13d，N641A、R648A突变体和SuperCas13d均显著减少了Helical1与Helical2、HEPN结构域之间的负相关性，这一构象变化可能会导致结合的靶RNA需要更多的能量来激活HEPN结构域的活性构象，结果示于图9中。这些结果提示，SuperCas13d通过减少与靶RNA上的14-18号碱基区域的结合力，从而整体增加SuperCas13d与sgRNA-靶RNA复合物的结合不稳定性，同时减少了核酸识别结构域(Helical1)与核酸酶结构域(Helical2、HEPN)的偶联，从而使得错配的靶RNA底物与Cas13d的结合减弱、激活核酸酶活性构象的能垒更高，最终导致SuperCas13d显著减少对单碱基错配RNA底物的切割。
实施例6 SuperCas13d在体内特异性靶向Myh6 R872H点突变转录本，阻止遗传性心肌病的发展(动物模型)
我们将SuperCas13d构建于AAV9载体中，并在心肌细胞特异性表达启动子cTnT启动子的驱动下实现心脏特异性表达，同时将U6-sgRNA序列(sgRNA采用872-gRNA9)置于该载体中。我们在P3小鼠中皮下注射1×1011vg/mice AAV9-SuperCas13d病毒，对照组注射等体积PBS。在5周龄时，连续3周喂食1mg/kg CsA加速心肌肥厚表型，发现SuperCas13d治疗组相比PBS对照组，心肌肥厚表型显著减轻，表现在左心室壁厚度、射血分数的显著降低。而SuperCas13d治疗组的左心室壁厚度、射血分数与野生型小鼠无差异，结果示于图10中。
实施例7 SuperCas13d显著提高靶向Myh6 R404Q点突变转录本的特异性
为了研究SuperCas13d对其他Myh6点突变如R404Q是否也具有特异 性识别能力，我们先采用野生型Cas13d在Myh6 R404Q双荧光报告细胞系中检测23条sgRNA(SEQ ID NO：15-37)的剪切活性。结果表明sgRNA均具有较高的剪切活性，但是均没有单碱基特异性识别能力。进一步将sgRNA15，sgRNA18与SuperCas13d组合，转染到Myh6 R404Q双荧光报告细胞中，发现SuperCas13d显著提高了单碱基识别特异性，结果示于图11中。
实施例8.工程化SuperCas13d在体内特异性靶向Myh6 R404Q点突变转录本，阻止双点突变遗传性心肌病的发展(动物模型)
多重杂合点突变在儿童遗传性心肌病中比较常见，这类病人相对于单点突变患者发病早、病情进展快、致死率更高。我们推测特异性敲除致病性强的点突变有望治疗这类疾病。Myh6 R872H突变表型较R404Q突变表型轻，两者的单点杂合突变均在成年晚期发病，在小鼠中约6月龄以上发病。而两者的双杂合突变小鼠4月龄异常肥厚，1月龄的心电就存在显著异常，结果示于图12中。为了探究SuperCas13d在治疗双重杂合点突变遗传性心肌病中的应用潜能，我们在P3RH/RQ双点杂合突变小鼠皮下注射1×1011vg AAV9-SuperCas13d-U6-R404Q 404-gRNA15腺相关病毒，对照组注射PBS，同时设置单点突变小鼠作为疾病表型对照。结果发现，SuperCas13d治疗组小鼠的左心室厚度和射血分数相对PBS组显著降低，异常的心电表型也显著改善，结果示于图13中。组织学检测也表明，SuperCas13d治疗显著阻滞了心脏肥厚、心肌肥大、心脏纤维排列紊乱、心脏纤维化、细胞凋亡等异常表型，结果示于图14中。
采用数字滴度PCR技术检测SuperCas13d在动物体内对Myh6不同突变转录本的敲除效率，结果表明在两种不同治疗小鼠模型中，SuperCas13d可靶向特异靶向与gRNA完全匹配的目标RNA，结果示于图15中。
实施例9.工程化SuperCas13d可特异性靶向人MYH7致病点突变(人类细胞)
为了进一步检测SuperCas13d的特异性和普适性，我们设计了人MYH7报告基因，其中野生型MYH7基因融合EGFP-PEST，包含43个致病突变的突变型MYH7基因融合mCherry-PEST。根据SuperCas13d的特异性识别窗口共设计了130条gRNA可特异性靶向这43个致病突变，将这些gRNA分别连接至野生型Cas13d质粒或SuperCas13d质粒，再与MYH7表达质粒共转至人HEK293T细胞中。48小时后，通过检测EGFP-PEST或mCherry-PEST的表达量来反映Cas13d对野生型MYH7或突变MYH7的敲除效率，同时用两者的差值反映底物区分效率(底物区分效率＝突变MYH7敲减效率-野生型MYH7敲减效率)。结果表明，相比野生型Cas13d，SuperCas13d显著提高了对不同MYH7单碱基突变的区分能力，且在一些传统碱基编辑器无法编辑位点展现出高效的敲减和区分能力，结果示于图16中。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准，说明书及附图可以用于解释权利要求的内容。
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Claims (30)

  1. 具有CasRx活性的突变体，其与SEQ ID NO：1所示的氨基酸序列具体具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、至少99.5％、至少99.9％或100％的序列同一性，并且参照SEQ ID NO：1的氨基酸位置变化，具有位于下列位置处中的一个或多个突变：
    K323、N324、G326、R333、N348、K350、K411、Y415、T486、R529、
    K585、R601、Y604、N641、R648以及Y649。
  2. 根据权利要求1所述的突变体，其具有位于下列位置处中的一个或两个突变：
    R648和N641。
  3. 根据权利要求2所述的突变体，其具有下述突变中的一个或两个：
    R648A和N641A。
  4. 根据权利要求1所述的突变体，其具有下述位置处的突变：
    N641、T486和R648。
  5. 根据权利要求4所述的突变体，其具有下述突变N641A、T486A和R648A，优选其为SEQ ID NO：2所示。
  6. 根据权利要求1～5任一项所述的突变体，其具有显著提高的区分单碱基突变相关的突变型和野生型转录本的能力。
  7. 根据权利要求6任一项所述的突变体，其与向导RNA分子(gRNA)所组成的复合物与靶RNA的结合不稳定性提高。
  8. 根据权利要求6所述的突变体，其具有显著减少的核酸识别结构域与核酸酶结构域之间的负相关性；其中所述核酸识别结构域包括Helical1结构域，所述核酸酶结构域包括Helical2和HEPN结构域。
  9. 分离的多核苷酸，其编码权利要求1～8任一项所述的突变体。
  10. 权利要求9所述的多核苷酸，其包含调控元件，且所述调控元件与编码所述突变体的部分可操作地关联。
  11. 权利要求9或权利要求10所述的多核苷酸，其中所述多核苷酸经密码子优化以在生物体中表达。
  12. 权利要求11所述的多核苷酸，其中所述生物体是动物、植物、真菌、古细菌或细菌。
  13. 一种分子系统，其为复合物或组合物，包含(a)权利要求1～8任一项所述的突变体和(b)向导RNA分子(gRNA)的集合。
  14. 编码权利要求13所述的分子系统的核酸构建体。
  15. 载体，其包含权利要求9～12任一项所述的多核苷酸，或权利要求14所述的核酸构建体。
  16. 根据权利要求15所述的载体，其是病毒载体。
  17. 根据权利要求16所述的载体，其是腺病毒载体、腺相关病毒(AAV)载体、慢病毒载体或逆转录病毒载体。
  18. 根据权利要求17所述的载体，所述AAV载体选自由以下组成的组：AAV 1型、AAV 2型、AAV 3型、AAV 4型、AAV 5型、AAV 6型、AAV 7型、AAV 8型、AAV9、AAV10、AAV11、AAV12、AAV13、rh10、AAVDJ、AAV-PHP.S、AAV-PHP.B、AAV-PHP.eB和Anc80。
  19. 递送系统，其包含i)权利要求13所述的分子系统，以及ii)递送媒介物。
  20. 根据权利要求19所述的递送系统，所述递送媒介物包括一种或多种脂质体、一种或多种外泌体、一种或多种微囊泡、一种或多种树状大分子、一种或多种无机纳米粒子、一种或多种细胞穿膜肽、基因枪、一种或多种质粒、一种或多种病毒载体，以及它们所组成的组。
  21. 根据权利要求20所述的递送系统，所述病毒载体如权利要求17或18所定义。
  22. 药物组合物，其包含权利要求19～21任一项所述的递送系统以及药学上可接受的赋形剂。
  23. 试剂盒，其包含：权利要求1～8任一项所述的突变体、或权利要求13所述的分子系统、或权利要求15～18任一项所述的载体、或权利要求19～21任一项所述的递送系统、或权利要求22所述的药物组合物，以及任选的该试剂盒的使用说明书。
  24. 一种改变基因产物表达的方法，包括使用CRISPR-Cas核酸酶体系切割靶RNA引入细胞的步骤；
    所述CRISPR-Cas核酸酶体系包含权利要求1～8任一项所述的突变体、或权利要求13所述的分子系统、或权利要求15～18任一项所述的载体、或权利要求19～21任一项所述的递送系统、或权利要求22所述的药物组合物、或权利要求23所述的试剂盒。
  25. 权利要求1～8任一项所述的突变体、或权利要求13所述的分子系统、或权利要求15～18任一项所述的载体、或权利要求19～21任一项所述的递送系统在制备用于治疗基因变异所引起的疾病的药物中的应用；其中所述疾病由包含一个或多个单碱基突变的变异所引起，或者可通过敲减或敲除某个基因得到治疗。
  26. 根据权利要求25所述的应用，所述疾病选自如下疾病中的一种或多种：
    遗传性心脏病(优选遗传性心肌病)、镰刀状贫血、地中海贫血、血友病、早衰症、苯丙酮尿症、糖原累积病、I型糖尿病、家族性高胆固醇血症、高甘油三脂血症、克拉伯病、戈谢病、色盲、遗传性耳聋、遗传性老年痴呆、家族性淀粉样病变、进行性肌营养不良以及由单碱基突变导致的肿瘤。
  27. 根据权利要求25所述的应用，所述疾病选自如下疾病中的一种或多种：亨廷顿舞蹈症、帕金森综合症和Rett综合征。
  28. 根据权利要求26所述的应用，所述疾病为遗传性心肌病，且所述分子系统、或所述的载体、或所述的递送系统中gRNA的间隔序列与SEQ ID NO：3～37任一项所示序列具有至少70％、至少80％、至少90％、至少95％或100％序列同一性。
  29. 一种分子系统，其为复合物或组合物，包含(a)权利要求1～8任一项所述的突变体和(b)向导RNA分子(gRNA)的集合，其中所述gRNA的核苷酸序列如SEQ ID NO：44～173任一项所示。
  30. 根据权利要求29所述的分子系统在制备用于治疗MYH7突变导致的心肌病的药物中的应用。
PCT/CN2024/071517 2023-01-10 2024-01-10 具有CasRx活性的突变体及其应用 Ceased WO2024149283A1 (zh)

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