WO2020087631A1 - 基于C2c1核酸酶的基因组编辑系统和方法 - Google Patents

基于C2c1核酸酶的基因组编辑系统和方法 Download PDF

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WO2020087631A1
WO2020087631A1 PCT/CN2018/118458 CN2018118458W WO2020087631A1 WO 2020087631 A1 WO2020087631 A1 WO 2020087631A1 CN 2018118458 W CN2018118458 W CN 2018118458W WO 2020087631 A1 WO2020087631 A1 WO 2020087631A1
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protein
disease
variant
nucleotide sequence
genome editing
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李伟
周琪
滕飞
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中国科学院动物研究所
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Priority to JP2021523583A priority patent/JP7361109B2/ja
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Definitions

  • CRISPR-Cas C lustered R egularly I nterspaced S hort P alindromic R epeats- C RISPR- as sociated protein
  • CRISPR-Cas C lustered R egularly I nterspaced S hort P alindromic R epeats- C RISPR- as sociated protein
  • type II Cas9 Cong, L. et al. Science 339, 819-823 (2013); Mali, P. et al .Science 339,823-826 (2013)
  • VA type Cpfl Zetsche, B. et al. Cell 163,759-771 (2015)
  • RNA and tracRRNA double RNA and tracRRNA
  • protein components are interchangeable and can be optimized initially (Nishimasu, H. et al. Cell 156, 935-949 (2014); Zalatan, JGet al. Cell 160, 339-350 (2015)).
  • CRISPR-Cas systems and research promote the widespread application of CRISPR-Cas systems (Wright, AV, Nunez, JK & Doudna, JACell 164, 29-44 (2016); Shmakov, S. et al. Nat Rev Microbiol 15, 169 -182 (2017)), but little is known about how to redesign or even synthesize the enzymatic genome editing system de novo.
  • the present invention provides a genome editing system for targeted modification of a target sequence in a cell genome, which includes at least one of the following i) to v):
  • An expression construct comprising a nucleotide sequence encoding C2c1 protein or a variant thereof, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
  • the guide RNA can form a complex with the C2c1 protein or its variant, and target the C2c1 protein ortholog or its variant to the target sequence in the cell genome.
  • the C2c1 protein is the AaC2c1 protein from Alicyclobacillus acidiphilus, the AkC2c1 protein from Alicyclobacillus kakegawensis, the AmC2c1 protein from Alicyclobacillus macrosporangiidus, the BhC2c1 protein from Bacillus hissashii, the BhC2c1 protein from Bacillus hissashii, Bs3C2c1 protein, DiC2c1 protein from Desulfovibrio inopinatus, LsC2c1 protein from Laceyella sediminis, SbC2c1 protein from Spirochaetes bacteria, TcC2c1 protein from Tuberibacillus calidus.
  • the C2c1 protein is the AaC2c1 protein from Alicyclobacillus acidiphilus NBRC 100859, the AkC2c1 protein from Alicyclobacillus kakegawensis NBRC 103103, the BaCporus protein from Alicyclobacillus SP2, the C1c1 protein from the Bacillus cirrhosis, the MSC2C1 protein from the Bacillus cirrhica, the MSC2C1 protein
  • the present invention provides a method for targeted modification of a target sequence in the genome of a cell, including introducing the genome editing system of the present invention into the cell.
  • the present invention provides a method of treating a disease in a subject in need thereof, comprising delivering an effective amount of the genome editing system of the present invention to the subject to modify genes in the subject related to the disease .
  • the present invention provides the use of the genome editing system of the present invention in the preparation of a pharmaceutical composition for treating a disease in a subject in need thereof, wherein the genome editing system is used to modify the Describe disease-related genes.
  • the present invention provides a kit for use in the method of the present invention, the kit comprising the genome editing system of the present invention, and instructions for use.
  • the present invention provides a pharmaceutical composition for treating a disease in a subject in need thereof, which comprises the genome editing system of the present invention and a pharmaceutically acceptable carrier, wherein the genome editing system is used for modification Genes associated with the disease in the subject.
  • Figure 1 Phylogenetic trees and their loci for selecting non-redundant C2c1 orthologs for genome editing testing.
  • FIG. 1 Protein alignment of C2c1 orthologs: multiple sequence alignments of the amino acid sequences of the 10 C2c1 orthologs tested. conserveed residues are highlighted with a red background, and conservative mutations are highlighted with outlines and red font.
  • T7EI measurement results indicate the genomic targeting activity of eight C2c1 proteins that bind to their homologous sgRNA in the human genome. The triangle indicates the cut strip.
  • T7EI measurement results indicate that simultaneous multiple genome targeting is mediated by Bs3C2c1 bound to its homologous sgRNA (Bs3sgRNA) in human 293T cells.
  • Sanger sequencing shows representative indels induced by Bs3C2c1 bound to Bs3sgRNA. The PAM and original spacer sequences are colored in red and blue, respectively. Insertion and deletion are indicated by purple dashes and green lowercase characters, respectively.
  • T7EI measurement results indicate the genome targeting activity of eight C2c1 orthologs guided by their homologous sgRNA in human 293T cells. The triangle indicates the cut strip.
  • T7EI assay results indicate the genomic targeting activity of eight C2c1 orthologs directed by AasgRNA (c) and AksgRNA (d) in human 293T cells. The triangle indicates the cut strip.
  • T7EI measurement showed the genome targeting activity of eight C2c1 orthologs guided by AasgRNA (a), AksgRNA (b), AmsgRNA (c), Bs3sgRNA (d) and LssgRNA (e) in human 293T cells.
  • the red triangle indicates the cut band.
  • T7EI assay results indicate the genome targeting activity of AaC2c1, DiC2c1, and TcC2c1 guided by AasgRNA (b) and AksgRNA (c) in human 293T cells. The triangle indicates the cut strip.
  • T7EI assay results indicate that simultaneous multiple genome targeting is mediated by TcC2c1 bound to AksgRNA in human 293T cells.
  • T7EI assay results indicate that simultaneous multiple genome targeting is mediated by TcC2c1 bound to artgRNA13 in human 293T cells.
  • the T7EI measurement results showed that the genome targeting activity of AaC2c1, DiC2c1 and TcC2c1 guided by AasgRNA (a), AksgRNA (b), AmsgRNA (c), Bs3sgRNA (d) and LssgRNA (e) in human 293T cells.
  • the triangle indicates the cut strip.
  • T7EI assay results indicate that simultaneous multiple genome targeting is mediated by TcC2c1 bound to AmsgRNA in human 293T cells.
  • (b-c) Sanger sequencing showed representative insertion deletions induced by TcC2c1 bound to AksgRNA (b) and AmsgRNA (c). The PAM and original spacer sequences are colored in red and blue, respectively. Insertion and deletion are indicated by purple dashes and green lowercase characters, respectively.
  • T7EI assay results indicate the artsgRNA-directed genome targeting activity of TcC2c1 in human 293T cells. The triangle indicates the cut strip.
  • T7EI measurement results indicate that simultaneous multiple genome targeting is mediated by AaC2c1 bound to artgRNA13 in human 293T cells.
  • the present invention provides a genome editing system for targeted modification of a target sequence in a cell genome, which includes at least one of the following i) to v):
  • C2c1 protein or its variant and an expression construct containing a nucleotide sequence encoding a guide RNA
  • An expression construct comprising a nucleotide sequence encoding C2c1 protein or a variant thereof, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
  • the guide RNA can form a complex with the C2c1 protein or its variant, and target the C2c1 protein or its variant to the target sequence in the cell genome.
  • the targeting results in the substitution, deletion, and / or addition of one or more nucleotides in the target sequence.
  • Gene as used herein encompasses not only chromosomal DNA present in the nucleus, but also organelle DNA present in the subcellular components (eg, mitochondria, plastids) of the cell.
  • C2c1 nuclease C2c1 protein
  • C2c1 C2c1
  • DSB DNA double-strand breaks
  • NHEJ Non-homologous end joining
  • HR homologous recombination
  • the C2c1 protein is the AaC2c1 protein from Alicyclobacillus acidiphilus, the AkC2c1 protein from Alicyclobacillus kakegawensis, the AmC2c1 protein from Alicyclobacillus macrosporangiidus, the BhC2c1 protein from Bacillus hissashii, the BhC2c1 protein from Bacillus hissashii, Bs3C2c1 protein, DiC2c1 protein from Desulfovibrio inopinatus, LsC2c1 protein from Laceyella sediminis, SbC2c1 protein from Spirochaetes bacteria, TcC2c1 protein from Tuberibacillus calidus.
  • the C2c1 protein is the AaC2c1 protein from Alicyclobacillus acidiphilus NBRC 100859, the AkC2c1 protein from Alicyclobacillus kakegawensis NBRC 103103, the BaCporus protein from Alicyclobacillus SP2, the C1c1 protein from the Bacillus cirrhosis, the MSC2C1 protein from the Bacillus cirrhica, the MSC2C1 protein
  • the C2c1 protein is a C2c1 protein whose natural locus does not have a CRISPR array.
  • the C2c1 protein in which the natural locus does not have a CRISPR array is DiC2c1 or TcC2c1 protein.
  • the C2c1 protein comprises the amino acid sequence shown in any one of SEQ ID NO: 1-10.
  • the AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, TcC2c1 proteins respectively include the amino acid sequences shown in SEQ ID NO: 1-10.
  • the variants of the C2c1 protein comprise at least 80% of wild-type C2c1 protein (e.g., wild-type AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1 or TcC2c1 protein), Amino acid sequences of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, and have wild-type C2c1 proteins (such as wild-type AaC2c1, AkC2c1, AmC2c1 , BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, TcC2c1 protein) genome editing and / targeting activity.
  • wild-type C2c1 protein e.g., wild-type AaC2c1, AkC2c1, AmC2
  • the variants of the C2c1 protein comprise one or more relative to wild-type C2c1 protein (e.g., wild-type AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, TcC2c1 protein)
  • wild-type C2c1 protein e.g., wild-type AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, TcC2c1 protein
  • wild-type C2c1 protein e.g., wild-type AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, TcC2c1 protein
  • target activity e.g
  • the variants of the C2c1 protein include 1, 2, 3 relative to the wild-type C2c1 protein (such as wild-type AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, TcC2c1 protein) , 4, 5, 6, 7, 8, 9 or 10 amino acid residues are substituted, deleted or added.
  • the amino acid substitution is a conservative substitution.
  • Polypeptide “peptide”, and “protein” are used interchangeably in the present invention and refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers.
  • the terms “polypeptide”, “peptide”, “amino acid sequence” and “protein” can also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, gamma carboxylation of glutamic acid residues, hydroxyl And ADP-ribosylation.
  • Sequence "identity” has an art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of the polynucleotide or polypeptide or along the region of the molecule.
  • identity is well known to the skilled person (Carrillo, H. & Lipman, D., SIAM, J Applied, Math 48: 1073 (1988) ).
  • Suitable conservative amino acid substitutions are known to those skilled in the art, and can generally be made without altering the biological activity of the resulting molecule.
  • those skilled in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially change biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub .co., p.224).
  • the variant of the C2c1 protein comprises a nuclease-depleted C2c1 protein (dC2c1).
  • Nuclease-depleted C2c1 protein refers to C2c1 protein that retains guide RNA-mediated DNA binding activity but does not possess double-stranded DNA cleaving activity.
  • the nuclease-depleted C2c1 protein encompasses C2c1 nickase, which cleaves only one strand of double-stranded target DNA.
  • the variant of the C2c1 protein is a fusion protein of a nuclease-dead C2c1 protein and a deaminase.
  • the nuclease-depleted C2c1 protein and the deaminase in the fusion protein can be connected by a linker such as a peptide linker.
  • deaminase refers to an enzyme that catalyzes a deamination reaction.
  • the deaminase refers to cytosine deaminase, which can accept single-stranded DNA as a substrate and can catalyze the deamination of cytidine or deoxycytidine to uracil or deoxyurine Pyrimidine.
  • the deaminase refers to adenine deaminase, which can accept single-stranded DNA as a substrate and can catalyze the formation of inosine (I) from adenosine or deoxyadenosine (A).
  • the C2c1 protein or variants thereof in the genome editing system of the present invention may further include a nuclear localization sequence (NLS).
  • NLS nuclear localization sequence
  • one or more NLSs in the C2c1 protein or its variant should have sufficient strength to drive the accumulation of the C2c1 protein or its variant in an amount that can achieve its gene editing function in the nucleus.
  • the intensity of nuclear localization activity is determined by the number, location of NLS in the C2c1 protein or its variant, the specific NLS (s) used, or a combination of these factors.
  • the NLS of the C2c1 protein or its variant in the genome editing system of the present invention may be located at the N-terminus and / or C-terminus.
  • the C2c1 protein or variant thereof comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS.
  • the C2c1 protein or variant thereof comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the N-terminus.
  • the C2c1 protein or variant thereof comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the C-terminus.
  • the C2c1 protein or variant thereof comprises a combination of these, such as one or more NLSs at the N-terminus and one or more NLSs at the C-terminus. When there is more than one NLS, each can be selected not to depend on other NLS. In some embodiments of the present invention, the C2c1 protein or a variant thereof contains 2 NLSs, for example, the 2 NLSs are located at the N-terminus and the C-terminus, respectively.
  • NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the protein surface, but other types of NLS are also known.
  • Non-limiting examples of NLS include: KKRKV, PKKKRKV, or SGGSPKKKRKV.
  • the C2c1 protein or its variant of the present invention may also include other localization sequences, such as a cytoplasmic localization sequence, a chloroplast localization sequence, a mitochondrial localization sequence, and the like.
  • the target sequence is 18-35 nucleotides in length, preferably 20 nucleotides.
  • the target sequence is flanked at its 5 'end is selected from: 5'TTTN-3', 5'ATTN-3 ', 5'GTTN-3', 5'CTTN-3 ' , 5'TTC-3 ', 5'TTG-3', 5'TTA-3 ', 5'TTT-3', 5'TAN-3 ', 5'TGN-3', 5'TCN-3 'and 5'ATC-3 'PAM (proximal interproximal motif) sequence, where N is selected from A, G, C and T.
  • N proximal interproximal motif
  • the target sequence to be modified may be located at any position in the genome, for example, within a functional gene such as a protein coding gene, or may be located, for example, in a gene expression regulatory region such as a promoter region or an enhancer region, so as to achieve Modification of gene function or modification of gene expression.
  • T7EI, PCR / RE or sequencing methods can be used to detect substitutions, deletions and / or additions in genomic target sequences
  • RNA and gRNA are used interchangeably herein, and are generally composed of partially complementary crRNA and tracrRNA molecules that form a complex, where the crRNA contains sufficient identity to the target sequence to hybridize to the complementary sequence of the target sequence and guide The sequence where the CRISPR complex (C2c1 + crRNA + tracrRNA) binds to this target sequence in a sequence-specific manner.
  • CRISPR complex C2c1 + crRNA + tracrRNA
  • sgRNA unidirectional RNA
  • the guide RNA is sgRNA.
  • the sgRNA is encoded by a nucleic acid sequence selected from one of the following:
  • the sequence Nx (spacer sequence) is capable of specifically hybridizing to the complementary sequence of the target sequence.
  • the sequence other than Nx in the sgRNA is a scaffold sequence of sgRNA.
  • the sgRNA comprises a scaffold sequence encoded by the nucleotide sequence of any one of SEQ ID NO: 31-38.
  • the present invention surprisingly found that the C2c1 protein and guide RNA in different C2c1 systems can be used interchangeably, thereby making it possible to manually design a universal guide RNA.
  • the present invention provides an artificial sgRNA encoded by a nucleotide sequence selected from:
  • the sequence Nx (spacer sequence) is capable of specifically hybridizing to the complementary sequence of the target sequence.
  • the sequence other than Nx in the sgRNA is a scaffold sequence of sgRNA.
  • the artificial sgRNA comprises a scaffold sequence encoded by the nucleotide sequence of any one of SEQ ID NO: 39-75.
  • the guide RNA in the genome editing system of the invention is the artificial sgRNA of the invention.
  • the nucleotide sequence encoding the C2c1 protein or its variant is codon-optimized for the organism from which the cell to be genome-edited comes.
  • Codon optimization refers to replacing at least one codon of the natural sequence by a codon that is used more frequently or most frequently in the gene of the host cell (eg, about or more than about 1, 2, 3, 4, 5, 10 , 15, 20, 25, 50 or more codons while maintaining the natural amino acid sequence while modifying the nucleic acid sequence in order to enhance expression in the host cell of interest.
  • a codon that is used more frequently or most frequently in the gene of the host cell eg, about or more than about 1, 2, 3, 4, 5, 10 , 15, 20, 25, 50 or more codons
  • Codon preference difference in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transport RNA
  • codon usage table can be easily obtained, for example, in the codon usage database (“Codon Usage Database”) available on www.kazusa.orjp / codon /, and These tables may be adjusted in different ways applicable See, Nakamura Y. et, "Codon usage tabulated from the international DNA sequence databases:.. Status for the year2000.Nucl.Acids Res, 28: 292 (2000).
  • the organisms from which cells that can be genome edited by the system of the present invention come from are preferably eukaryotes, including but not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; Poultry such as chickens, ducks, geese; plants include monocotyledons and dicotyledons, such as rice, corn, wheat, sorghum, barley, soybeans, peanuts, Arabidopsis, etc.
  • the nucleotide sequence encoding the C2c1 protein or its variants is codon optimized for humans.
  • the nucleotide sequences of the codon optimized encoding AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, TcC2c1 protein are selected from SEQ ID NO: 11-20, respectively.
  • the nucleotide sequence encoding the C2c1 protein or its variant in the expression construct of the system of the present invention and / or the nucleotide sequence encoding the guide RNA and expression control elements such as The promoter is operably linked.
  • expression construct refers to a vector suitable for expression of the nucleotide sequence of interest in an organism, such as a recombinant vector. "Expression” refers to the production of a functional product.
  • expression of a nucleotide sequence may refer to the transcription of the nucleotide sequence (eg, transcription to produce mRNA or functional RNA) and / or translation of the RNA into a precursor or mature protein.
  • the "expression construct” of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, RNA (eg, mRNA) capable of translation.
  • the "expression construct" of the present invention may contain regulatory sequences and nucleotide sequences of interest from different sources, or regulatory sequences and nucleotide sequences of interest from the same source but arranged in a manner different from the normally naturally occurring.
  • regulatory sequence and “regulatory element” are used interchangeably, and refer to the upstream (5 'non-coding sequence), middle or downstream (3' non-coding sequence) of the coding sequence, and affect the transcription, RNA processing or Stability or translated nucleotide sequence. Regulatory sequences can include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • Promoter refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment.
  • the promoter is a promoter capable of controlling gene transcription in a cell, whether or not it is derived from the cell.
  • the promoter may be a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.
  • Constant promoter refers to a promoter that will generally cause expression of a gene in most cell types in most cases.
  • tissue-specific promoter and “tissue-preferred promoter” are used interchangeably and refer to expression in a tissue or organ, but not necessarily exclusively, but also in a specific cell or cell type Promoter.
  • Developmentally regulated promoter refers to a promoter whose activity is determined by developmental events.
  • inducible promoters selectively express operably linked DNA sequences in response to endogenous or exogenous stimuli (environment, hormones, chemical signals, etc.).
  • operably linked refers to a regulatory element (such as, but not limited to, a promoter sequence, transcription termination sequence, etc.) linked to a nucleic acid sequence (eg, coding sequence or open reading frame) such that the nucleotide The transcription of the sequence is controlled and regulated by the transcriptional regulatory elements.
  • a regulatory element such as, but not limited to, a promoter sequence, transcription termination sequence, etc.
  • a nucleic acid sequence eg, coding sequence or open reading frame
  • promoters examples include, but are not limited to, polymerase (pol) I, pol II or pol III promoters.
  • pol I promoter examples include chicken RNA pol I promoter.
  • pol II promoters include, but are not limited to, cytomegalovirus immediate early (CMV) promoter, Rolls sarcoma virus long terminal repeat (RSV-LTR) promoter, and simian virus 40 (SV40) immediate early promoter.
  • pol III promoters include U6 and H1 promoters. Inducible promoters such as metallothionein promoters can be used.
  • promoters include T7 phage promoter, T3 phage promoter, ⁇ -galactosidase promoter and Sp6 phage promoter.
  • the promoter may be cauliflower mosaic virus 35S promoter, corn Ubi-1 promoter, wheat U6 promoter, rice U3 promoter, corn U3 promoter, rice actin promoter.
  • the cells that can be genome edited by the system of the present invention are preferably eukaryotic cells, including but not limited to, mammalian cells such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; poultry such as chicken , Duck, goose cells; plant cells include monocotyledonous plant cells and dicotyledonous plant cells, such as rice, corn, wheat, sorghum, barley, soybean, peanut, Arabidopsis, etc. cells.
  • the cell is a eukaryotic cell, preferably a mammalian cell, and more preferably a human cell.
  • the present invention provides a method for modifying a target sequence in a cell genome, including introducing the genome editing system of the present invention into the cell, whereby the guide RNA targets the C2c1 protein or its variant The target sequence in the genome of the cell.
  • the targeting results in the substitution, deletion, and / or addition of one or more nucleotides in the target sequence.
  • "Introducing" a nucleic acid molecule (eg, plasmid, linear nucleic acid fragment, RNA, etc.) or protein of the genome editing system of the present invention into a cell refers to transforming the cell with the nucleic acid or protein so that the nucleic acid or protein can function in the cell .
  • transformation includes stable transformation and transient transformation.
  • Stable transformation refers to the introduction of foreign nucleotide sequences into the genome, resulting in stable inheritance of foreign genes. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof.
  • Transient transformation refers to the introduction of a nucleic acid molecule or protein into a cell to perform a function without stable inheritance of foreign genes. During transient transformation, the foreign nucleic acid sequence is not integrated into the genome.
  • Methods that can be used to introduce the genome editing system of the present invention into cells include, but are not limited to: calcium phosphate transfection, protoplast fusion, electroporation, liposome transfection, microinjection, viral infection (such as baculovirus, vaccinia virus, adenovirus) Viruses, adeno-associated viruses, lentiviruses and other viruses), gene gun method, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation.
  • the method is performed in vitro.
  • the cell is an isolated cell.
  • the cell is a CAR-T cell.
  • the cells are induced embryonic stem cells.
  • the method can also be performed in vivo.
  • the cell is a cell in an organism, and the system of the present invention can be introduced into the cell in vivo by, for example, a virus-mediated method.
  • the cell may be a tumor cell in a patient.
  • the present invention also provides a method for producing genetically modified cells, including introducing the genome editing system of the present invention into cells, whereby the guide RNA targets the C2c1 protein or its variants The target sequence in the cell genome.
  • the targeting results in substitution, deletion, and / or addition of one or more nucleotides in the target sequence.
  • the invention also provides genetically modified organisms comprising genetically modified cells or their progeny produced by the method of the invention.
  • organism includes any organism suitable for genome editing, preferably eukaryotes.
  • organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cows, cats; poultry such as chickens, ducks, geese; plants include monocotyledons and dicotyledons, For example, rice, corn, wheat, sorghum, barley, soybean, peanut, Arabidopsis, etc.
  • the organism is a eukaryote, preferably a mammal, more preferably a human.
  • genetically modified organism or “genetically modified cell” means an organism or cell that contains an exogenous polynucleotide or modified gene or expression control sequence within its genome.
  • exogenous polynucleotides can be stably integrated into the genome of an organism or cell and inherited for successive generations.
  • the exogenous polynucleotide can be integrated into the genome alone or as part of a recombinant DNA construct.
  • Modified genes or expression control sequences are those in which the sequence of the organism or cell genome contains single or multiple deoxynucleotide substitutions, deletions, and additions.
  • “Exogenous” with respect to a sequence means a sequence from a foreign species, or if from the same species, a sequence that has undergone a significant change in composition and / or locus from its natural form through deliberate human intervention.
  • an expression construct comprising a nucleotide sequence encoding a nuclease-depleted C2c1 protein or a fusion protein with a transcription repressor protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
  • nuclease-dead C2c1 protein or guide RNA The definition of the nuclease-dead C2c1 protein or guide RNA is as described above. The selection of the transcriptional activation protein is within the skill of those skilled in the art.
  • the up-regulation, down-regulation, inactivation, activation or mutation correction of the disease-related genes can be achieved, thereby preventing and / or treating the disease.
  • the target sequence may be located in the protein coding region of a disease-related gene, or for example, may be located in a gene expression regulatory region such as a promoter region or an enhancer region, so that the disease-related gene may be functionally modified or disease-related Modification of gene expression.
  • the present invention also provides a pharmaceutical composition for treating a disease in a subject in need thereof, which comprises the genome editing system of the present invention and a pharmaceutically acceptable carrier, wherein the genome editing system is used to modify genes related to the disease .
  • Examples of the disease include, but are not limited to, tumor, inflammation, Parkinson's disease, cardiovascular disease, Alzheimer's disease, autism, drug addiction, age-related macular degeneration, schizophrenia, genetic disease, and the like.
  • kits for the method of the present invention includes the genome editing system of the present invention, and instructions for use.
  • the kit generally includes a label indicating the intended use and / or method of use of the contents of the kit.
  • the term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.
  • target sgRNAs for cell transfection assays were constructed by linking annealed oligonucleotides (Table 1) to BsaI digested pUC19-U6-sgRNA (SEQ ID NO: 23-30) vector.
  • a CCR5 site 28 TTTGGCCTGAATAATTGCAG TTC + DNMT1 DNMT1 site 16 CCCTTCAGCTAAAATAAAGG TTT + RNF2 RNF2 site 8 TAGTCATGGTGTTCTTCAAC TTG - - Site 5 GCTCTCAAGACCCACAATCC TTT +
  • the PSI-BLAST program (Altschul, SFet al. Nucleic Acids Res 25, 3389-3402 (1997)) was used to identify new CRISPR-C2c1 orthologs.
  • the coding sequence was humanized (Grote, A. et al., Nucleic Acids Res 33, W526-531 (2005)), and the GeneDesign program (Richardson, SM et al., Genome Res 16, 550-556 (2006) ) Design oligonucleotides for C2c1 gene and sgRNA synthesis.
  • Each C2c1 gene was synthesized according to the literature (Li, G. et al., Methods Mol Biol 1073, 9-17 (2013)).
  • NEB Assembly Master Mix
  • pCAG-2AeGFP SEQ ID NO: 21
  • pUC19-U6 SEQ ID NO: 22
  • the harvested cells were directly lysed with Buffer L (Bimake) supplemented with proteinase K, and incubated at 55 ° C for 3 hours and inactivated at 95 ° C for 10 minutes.
  • PCR amplification was performed on the genomic region around the C2c1 target site of each gene.
  • the 200-400 ng PCR product was mixed with ddH 2 O to a final volume of 10 ⁇ L, and a re-annealing process was performed according to the previous method to form a heteroduplex. After re-annealing, the product was treated with 1/10 volume of NEBuffer TM 2.1 and 0.2 ⁇ L of T7EI (NEB) at 37 ° C. for 30 minutes and analyzed on a 3% agarose gel.
  • Indels are quantified based on relative band intensity (Cong, L. et al., Science 339, 819-823 (2013)).
  • the mutant product identified by the T7EI assay was cloned into TA cloning vectors and transformed into competent E. coli strains (Transgen Biotech). After overnight cultivation, colonies were randomly picked and sequenced.
  • T7 endonuclease (T7EI) assay results show that, in addition to the AaC2c1 and AkC2c1 previously identified by the inventor, four new C2c1 orthologs (AmC2c1, BhC2c1, Bs3C2c1, and LsC2c1) robustly edit the human genome, although their targeting efficiency varies between different orthologs and at different targeting sites ( Figure 1b and Figure 3a).
  • sgRNA derived from the AaC2c1, AkC2c1, AmC2c1, Bs3C2c1, and LsC2c1 loci can replace the original sgRNA for mammalian genome editing, although activity between different C2c1 orthologs and sgRNA may vary Different ( Figure 4c, d and Figure 6). These results demonstrate the interchangeability between different C2c1 and double RNAs from different C2c1 loci.

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Abstract

基于C2c1核酸酶的基因组编辑系统和方法，以及可与不同C2c1核酸酶组合用于基因组编辑的人工向导RNA。

Description

基于C2c1核酸酶的基因组编辑系统和方法 技术领域
本发明涉及基因工程领域。具体而言，本发明涉及基于C2c1核酸酶的基因组编辑系统和方法。本发明还涉及可与不同C2c1核酸酶组合用于基因组编辑的人工向导RNA。
发明背景
随着CRISPR-Cas( Clustered  Regularly  Interspaced  Short  Palindromic  Repeats- CRISPR- associated蛋白)系统的出现，精确的基因组编辑由于其在基因治疗中的光明前景已经成为最令人关注的领域。到目前为止，已成功利用三种类型的CRISPR-Cas系统以促进哺乳动物基因组工程，包括II型Cas9(Cong，L.et al.Science 339，819-823(2013)；Mali，P.et al.Science 339，823-826(2013))、V-A型Cpf1(Zetsche，B.et al.Cell 163，759-771(2015))和V-B型C2c1。对于II型和V型CRISPR-Cas系统，向导RNA和Cas效应蛋白是靶DNA识别和切割的两种核心成分(Wright，A.V.，Nunez，J.K.&Doudna，J.A.Cell 164，29-44(2016)；Shmakov，S.et al.Nat Rev Microbiol 15，169-182(2017))。以前的研究表明在密切相关的Cas9系统(Fonfara，I.et al.Nucleic Acids Res 42，2577-2590(2014))以及Cpf1系统(Zetsche，B.et al.Cell 163，759-771(2015))中，双RNA(crRNA和tracRRNA)和蛋白质组分是可互换的，并能初步优化(Nishimasu，H.et al.Cell 156，935-949(2014)；Zalatan，J.G.et al.Cell 160，339-350(2015))。虽然许多新兴的CRISPR-Cas系统和研究促进CRISPR-Cas系统的广泛应用(Wright，A.V.，Nunez，J.K.&Doudna，J.A.Cell 164，29-44(2016)；Shmakov，S.et al.Nat Rev Microbiol 15，169-182(2017))，但对于如何重新设计甚至从头合成促酶基因组编辑系统仍然知之甚少。
V-B型CRISPR-C2c1系统是一种新兴的具有前景的基因工程技术。然而，可用于哺乳动物基因组编辑的C2c1却很少，大大限制了其应用。本领域仍然需要新的可用于哺乳动物基因组编辑的基于C2c1核酸酶的基因组编辑系统。
发明简述
在一方面，本发明提供了一种用于对细胞基因组中的靶序列进行定点修饰的基因组编辑系统，其包含以下i)至v)中至少一项：
i)C2c1蛋白或其变体，和向导RNA；
ii)包含编码C2c1蛋白或其变体的核苷酸序列的表达构建体，和向导RNA；
iii)C2c1蛋白或其变体，和包含编码向导RNA的核苷酸序列的表达构建体；
iv)包含编码C2c1蛋白或其变体的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；
v)包含编码C2c1蛋白或其变体的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体；
其中所述向导RNA能够与所述C2c1蛋白或其变体形成复合物，将所述C2c1蛋白直系同源物或其变体靶向所述细胞基因组中的靶序列。
在一些实施方案中，所述C2c1蛋白是来自Alicyclobacillus acidiphilus的AaC2c1蛋白、来自Alicyclobacillus kakegawensis的AkC2c1蛋白、来自Alicyclobacillus macrosporangiidus的AmC2c1蛋白、来自Bacillus hisashii的BhC2c1蛋白、来自Bacillus属的BsC2c1蛋白、来自Bacillus属的Bs3C2c1蛋白、来自Desulfovibrio inopinatus的DiC2c1蛋白、来自Laceyella sediminis的LsC2c1蛋白、来自Spirochaetes bacterium的SbC2c1蛋白、来自Tuberibacillus calidus的TcC2c1蛋白。例如，所述C2c1蛋白是来自Alicyclobacillus acidiphilus NBRC 100859的AaC2c1蛋白、来自Alicyclobacillus kakegawensis NBRC 103104的AkC2c1蛋白、来自Alicyclobacillus macrosporangiidus strain DSM 17980的AmC2c1蛋白、来自Bacillus hisashii strain C4的BhC2c1蛋白、来自Bacillus属NSP2.1的BsC2c1蛋白、来自Bacillus属V3-13 contig_40的Bs3C2c1蛋白、来自Desulfovibrio inopinatus DSM 10711的DiC2c1蛋白、来自Laceyella sediminis strain RHA1的LsC2c1蛋白、来自Spirochaetes bacterium GWB1_27_13的SbC2c1蛋白、来自Tuberibacillus calidus DSM 17572的TcC2c1蛋白。
在第二方面，本发明提供了一种对细胞基因组中的靶序列进行定点修饰的方法，包括将本发明的基因组编辑系统导入所述细胞。
在第三方面，本发明提供了一种治疗有需要的对象中的疾病的方法，包括向所述对象递送有效量的本发明的基因组编辑系统以修饰所述对象中与所述疾病相关的基因。
在第四方面，本发明提供了本发明的基因组编辑系统在制备用于治疗有需要的对象中的疾病的药物组合物中的用途，其中所述基因组编辑系统用于修饰所述对象中与所述疾病相关的基因。
在第五方面，本发明提供了用于本发明的方法的试剂盒，该试剂盒包括本发明的基因组编辑系统，以及使用说明。
在第六方面，本发明提供了一种用于治疗有需要的对象中的疾病的药物组合物，其包含本发明的基因组编辑系统和药学可接受的载体，其中所述基因组编辑系统用于修饰所述对象中与所述疾病相关的基因。
附图简述
图1.选择用于基因组编辑测试的非冗余C2c1直系同源物的系统发生树及其基因座。
(a)邻接系统发生树，显示测试的C2c1直系同源物的进化关系。(b)对应于(a)中突出显示的8种C2c1蛋白的细菌基因座图谱crRNADR和推定的tracrRNA的模拟共折叠显示出稳定的二级结构。DR，直接重复。每个细菌基因组间隔区(spacer)的数目在其 CRISPR阵列的上方或下方表示。
图2.C2c1直系同源物的蛋白质比对：测试的10种C2c1直系同源物的氨基酸序列的多序列比对。保守的残基用红色背景突出显示，保守突变用轮廓和红色字体突出显示。
图3.人293T细胞中C2c1直系同源物介导的基因组靶向。
(a)T7EI测定结果表明在人类基因组中与其同源sgRNA结合的八种C2c1蛋白的基因组靶向活性。三角形表示切割的条带。(b)T7EI测定结果表明在人293T细胞中由与其同源sgRNA(Bs3sgRNA)结合的Bs3C2c1介导的同时多重基因组靶向。(c)Sanger测序显示由与Bs3sgRNA结合的Bs3C2c1诱导的代表性插入缺失(indel)。PAM和原间隔区序列分别用红色和蓝色着色。插入缺失和插入分别用紫色破折号和绿色小写字符表示。
图4.用于RNA指导的基因组编辑的C2c1蛋白。
(a)本发明中测试的10种C2c1直系同源物的图形概述。示出其大小(氨基酸数目)。(b)T7EI测定结果表明在人293T细胞中由其同源sgRNA指导的八种C2c1直系同源物的基因组靶向活性。三角形表示切割的条带。(c-d)T7EI测定结果表明在人293T细胞中由AasgRNA(c)和AksgRNA(d)指导的八种C2c1直系同源物的基因组靶向活性。三角形表示切割的条带。
图5.C2c1的sgRNA的DNA比对：测试衍生自10个C2c1基因座的8种sgRNA的DNA序列的多序列比对。
图6.不同C2c1直系同源物与sgRNA之间的可互换性。
T7EI测定结果表明在人293T细胞中由AasgRNA(a)、AksgRNA(b)、AmsgRNA(c)、Bs3sgRNA(d)和LssgRNA(e)指导的八种C2c1直系同源物的基因组靶向活性。红色三角形表示切割的条带。
图7.人工sgRNA介导的多重基因组靶向。
(a)对应于DiC2c1和TcC2c1的细菌基因座的图谱。两个C2c1基因座没有CRISPR阵列。(b-c)T7EI测定结果表明在人293T细胞中由AasgRNA(b)和AksgRNA(c)指导的AaC2c1、DiC2c1和TcC2c1的基因组靶向活性。三角形表示切割的条带。(d)T7EI测定结果表明在人293T细胞中由与AksgRNA结合的TcC2c1介导的同时多重基因组靶向。(e)示意图说明人工sgRNA支架13(artgRNA13)的二级结构。(f)T7EI测定结果表明在人293T细胞中由与artgRNA13结合的TcC2c1介导的同时多重基因组靶向。
图8.不同sgRNA指导C2c1进行基因组编辑。
T7EI测定结果表明在人293T细胞中由AasgRNA(a)、AksgRNA(b)、AmsgRNA(c)、Bs3sgRNA(d)和LssgRNA(e)指导的AaC2c1、DiC2c1和TcC2c1的基因组靶向活性。三角形表示切割的条带。
图9.TcC2c1介导的多重基因组编辑。
(a)T7EI测定结果表明在人293T细胞中由与AmsgRNA结合的TcC2c1介导的同时多重基因组靶向。(b-c)Sanger测序显示由与AksgRNA(b)和AmsgRNA(c)结合的TcC2c1诱导的代表性插入缺失。PAM和原间隔区序列分别用红色和蓝色着色。插入缺失和插 入分别用紫色破折号和绿色小写字符表示。
图10.人工sgRNA指导TcC2c1进行基因组编辑。
(a)示意图说明36种人工sgRNA(artgRNA)支架(支架：1-12和14-37)的二级结构。(b)T7EI测定结果表明在人293T细胞中artsgRNA指导的TcC2c1的基因组靶向活性。三角形表示切割的条带。(c)T7EI测定结果表明在人293T细胞中由与artgRNA13结合的AaC2c1介导的同时多重基因组靶向。
发明详述
一、定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。例如，本发明中使用的标准重组DNA和分子克隆技术为本领域技术人员熟知，并且在如下文献中有更全面的描述：Sambrook，J.，Fritsch，E.F.和Maniatis，T.，Molecular Cloning：A Laboratory Manual；Cold Spring Harbor Laboratory Press：Cold Spring Harbor，1989(下文称为“Sambrook”)。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在一方面，本发明提供了一种用于对细胞基因组中的靶序列进行定点修饰的基因组编辑系统，其包含以下i)至v)中至少一项：
i)C2c1蛋白或其变体，和向导RNA；
ii)包含编码C2c1蛋白或其变体的核苷酸序列的表达构建体，和向导RNA；
iii)C2c1蛋白或其变体，和包含编码向导RNA的核苷酸序列的表达构建体；
iv)包含编码C2c1蛋白或其变体的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；
v)包含编码C2c1蛋白或其变体的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体；
其中所述向导RNA能够与所述C2c1蛋白或其变体形成复合物，将所述C2c1蛋白或其变体靶向所述细胞基因组中的靶序列。在一些实施方案中，所述靶向导致所述靶序列中的一或多个核苷酸的取代、缺失和/或添加。
“基因组”如本文所用不仅涵盖存在于细胞核中的染色体DNA，而且还包括存在于细胞的亚细胞组分(如线粒体、质体)中的细胞器DNA。
“C2c1核酸酶”、“C2c1蛋白”和“C2c1”在本文中可互换使用，指的是包括C2c1蛋白或其片段的RNA指导的核酸酶。C2c1具有向导RNA介导的DNA结合活性以及DNA切割活性，能在向导RNA的指导下靶向并切割DNA靶序列形成DNA双链断裂(DSB)。DSB能够激活细胞内固有的修复机制非同源末端连接(Non-homologous end joining，NHEJ)和同源重组(Homologous recombination，HR)对细胞中的DNA损伤进行修 复，在修复过程中，对该特定的DNA序列进行定点编辑。
在一些实施方案中，所述C2c1蛋白是来自Alicyclobacillus acidiphilus的AaC2c1蛋白、来自Alicyclobacillus kakegawensis的AkC2c1蛋白、来自Alicyclobacillus macrosporangiidus的AmC2c1蛋白、来自Bacillus hisashii的BhC2c1蛋白、来自Bacillus属的BsC2c1蛋白、来自Bacillus属的Bs3C2c1蛋白、来自Desulfovibrio inopinatus的DiC2c1蛋白、来自Laceyella sediminis的LsC2c1蛋白、来自Spirochaetes bacterium的SbC2c1蛋白、来自Tuberibacillus calidus的TcC2c1蛋白。
例如，所述C2c1蛋白是来自Alicyclobacillus acidiphilus NBRC 100859的AaC2c1蛋白、来自Alicyclobacillus kakegawensis NBRC 103104的AkC2c1蛋白、来自Alicyclobacillus macrosporangiidus strain DSM 17980的AmC2c1蛋白、来自Bacillus hisashii strain C4的BhC2c1蛋白、来自Bacillus属NSP2.1的BsC2c1蛋白、来自Bacillus属V3-13 contig_40的Bs3C2c1蛋白、来自Desulfovibrio inopinatus DSM 10711的DiC2c1蛋白、来自Laceyella sediminis strain RHA1的LsC2c1蛋白、来自Spirochaetes bacterium GWB1_27_13的SbC2c1蛋白、来自Tuberibacillus calidus DSM 17572的TcC2c1蛋白。
在本发明一些实施方式中，所述C2c1蛋白是其天然基因座不具有CRISPR阵列的C2c1蛋白。在一些实施方式中，所述天然基因座不具有CRISPR阵列的C2c1蛋白是DiC2c1或TcC2c1蛋白。
在一些实施方案中，所述C2c1蛋白包含SEQ ID NO：1-10中任一所示的氨基酸序列。例如，所述AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1、TcC2c1蛋白分别包含SEQ ID NO：1-10所示氨基酸序列。
在一些实施方案中，所述C2c1蛋白的变体分别包含与野生型C2c1蛋白(如野生型AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1或TcC2c1蛋白)具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列，并且分别具有野生型C2c1蛋白(如野生型AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1、TcC2c1蛋白)的基因组编辑和/靶向活性。
在一些实施方案中，所述C2c1蛋白的变体分别包含相对于野生型C2c1蛋白(如野生型AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1、TcC2c1蛋白)具有一或多个氨基酸残基取代、缺失或添加的氨基酸序列，并且分别具有野生型C2c1蛋白(如野生型AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1、TcC2c1蛋白)的基因组编辑和/或靶向活性。例如，所述C2c1蛋白的变体分别包含相对于野生型C2c1蛋白(如野生型AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1、TcC2c1蛋白)具有1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸残基取代、缺失或添加的氨基酸序列。在一些实施方案中，所述氨基酸取代是保守型取代。
“多肽”、“肽”、和“蛋白质”在本发明中可互换使用，指氨基酸残基的聚合物。 该术语适用于其中一个或多个氨基酸残基是相应的天然存在的氨基酸的人工化学类似物的氨基酸聚合物，以及适用于天然存在的氨基酸聚合物。术语“多肽”、“肽”、“氨基酸序列”和“蛋白质”还可包括修饰形式，包括但不限于糖基化、脂质连接、硫酸盐化、谷氨酸残基的γ羧化、羟化和ADP-核糖基化。
序列“相同性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性。(参见，例如：Computational Molecular Biology，Lesk，A.M.，ed.，Oxford University Press，New York，1988；Biocomputing：Informatics and Genome Projects，Smith，D.W.，ed.，Academic Press，New York，1993；Computer Analysis of Sequence Data，Part I，Griffin，A.M.，and Griffin，H.G.，eds.，Humana Press，New Jersey，1994；Sequence Analysis in Molecular Biology，von Heinje，G.，Academic Press，1987；and Sequence Analysis Primer，Gribskov，M.and Devereux，J.，eds.，M Stockton Press，New York，1991)。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo，H.&Lipman，D.，SIAM J Applied Math 48：1073(1988))。
在肽或蛋白中，合适的保守型氨基酸取代是本领域技术人员已知的，并且一般可以进行而不改变所得分子的生物活性。通常，本领域技术人员认识到多肽的非必需区中的单个氨基酸取代基本上不改变生物活性(参见，例如，Watson et al.，Molecular Biology of the Gene，4th Edition，1987，The Benjamin/Cummings Pub.co.，p.224)。
在一些实施方案中，所述C2c1蛋白的变体包含核酸酶死亡的C2c1蛋白(dC2c1)。核酸酶死亡的C2c1蛋白指的是保留向导RNA介导的DNA结合活性但是不具备双链DNA切割活性的C2c1蛋白。在一些实施方案中，所述核酸酶死亡的C2c1蛋白涵盖C2c1切口酶，其只切割双链靶DNA的一条链。
在一些实施方案中，所述C2c1蛋白的变体是核酸酶死亡的C2c1蛋白与脱氨酶的融合蛋白。例如，所述融合蛋白中的核酸酶死亡的C2c1蛋白与脱氨酶可以通过接头例如肽接头连接。
如本发明所用，“脱氨酶”是指催化脱氨基反应的酶。在本发明一些实施方式中，所述脱氨酶指的是胞嘧啶脱氨酶，其能够接受单链DNA作为底物并能够催化胞苷或脱氧胞苷分别脱氨化为尿嘧啶或脱氧尿嘧啶。在本发明一些实施方式中，所述脱氨酶指的是腺嘌呤脱氨酶，其能够接受单链DNA作为底物并能够催化腺苷或脱氧腺苷(A)形成肌苷(I)。通过使用核酸酶死亡的C2c1蛋白与脱氨酶的融合蛋白，可以实现靶DNA序列中的碱基编辑，例如C至T的转换或A至G的转换。
在本发明的一些实施方案中，本发明的基因组编辑系统中的C2c1蛋白或其变体还可以包含核定位序列(NLS)。一般而言，所述C2c1蛋白或其变体中的一个或多个NLS应具有足够的强度，以便在细胞核中驱动所述C2c1蛋白或其变体以可实现其基因编辑功能的量积聚。一般而言，核定位活性的强度由所述C2c1蛋白或其变体中NLS的数目、位置、所使用的一个或多个特定的NLS、或这些因素的组合决定。
在本发明的一些实施方案中，本发明的基因组编辑系统中的C2c1蛋白或其变体的NLS可以位于N端和/或C端。在一些实施方案中，所述C2c1蛋白或其变体包含约1、2、3、4、5、6、7、8、9、10个或更多个NLS。在一些实施方案中，所述C2c1蛋白或其变体包含在或接近于N端的约1、2、3、4、5、6、7、8、9、10个或更多个NLS。在一些实施方案中，所述C2c1蛋白或其变体包含在或接近于C端约1、2、3、4、5、6、7、8、9、10个或更多个NLS。在一些实施方案中，所述C2c1蛋白或其变体包含这些的组合，如包含在N端的一个或多个NLS以及在C端的一个或多个NLS。当存在多于一个NLS时，每一个可以被选择为不依赖于其他NLS。在本发明的一些实施方式中，所述C2c1蛋白或其变体包含2个NLS，例如所述2个NLS分别位于N端和C端。
一般而言，NLS由暴露于蛋白表面上的带正电的赖氨酸或精氨酸的一个或多个短序列组成，但其他类型的NLS也是已知的。NLS的非限制性实例包括：KKRKV、PKKKRKV，或SGGSPKKKRKV。
此外，根据所需要编辑的DNA位置，本发明的C2c1蛋白或其变体还可以包括其他的定位序列，例如细胞质定位序列、叶绿体定位序列、线粒体定位序列等。
在本发明的一些实施方案中，所述靶序列长度为18-35个核苷酸，优选20个核苷酸。在本发明的一些实施方案中，所述靶序列在其5’端侧翼为选自：5’TTTN-3’、5’ATTN-3’、5’GTTN-3’、5’CTTN-3’、5’TTC-3’、5’TTG-3’、5’TTA-3’、5’TTT-3’、5’TAN-3’、5’TGN-3’、5’TCN-3’和5’ATC-3’的PAM(前间区邻近基序)序列，其中N选自A、G、C和T。
在本发明中，待进行修饰的靶序列可以位于基因组的任何位置，例如位于功能基因如蛋白编码基因内，或者例如可以位于基因表达调控区如启动子区或增强子区，从而实现对所述基因功能修饰或对基因表达的修饰。可以通过T7EI、PCR/RE或测序方法检测基因组靶序列中的取代、缺失和/或添加
“向导RNA”和“gRNA”在本文中可互换使用，通常由部分互补形成复合物的crRNA和tracrRNA分子构成，其中crRNA包含与靶序列具有足够相同性以便与靶序列的互补序列杂交并且指导CRISPR复合物(C2c1+crRNA+tracrRNA)与该靶序列以序列特异性方式结合的序列。然而，可以设计并使用单向导RNA(sgRNA)，其同时包含crRNA和tracrRNA的特征。
在本发明的一些实施方案中，所述向导RNA是sgRNA。在一些具体实施方案中，所述sgRNA由选自以下之一的核酸序列编码：
Figure PCTCN2018118458-appb-000001
Figure PCTCN2018118458-appb-000002
其中Nx表示X个连续的核苷酸组成的核苷酸序列(spacer序列)，N各自独立地选自A、G、C和T；X为18≤X≤35的整数。优选地，X＝20。在一些实施方案中，序列Nx(spacer序列)能够与靶序列的互补序列特异性杂交。所述sgRNA中除Nx之外的序列为sgRNA的支架(scaffold)序列。在一些实施方案中，所述sgRNA包含由SEQ ID NO：31-38中任一项的核苷酸序列编码的支架序列。
本发明令人惊奇地发现，不同的C2c1系统中的C2c1蛋白以及向导RNA可以互换使用，从而使得可以人工设计通用的向导RNA。
因此在另一方面，本发明提供一种人工sgRNA，其由选自以下的核苷酸序列编码：
Figure PCTCN2018118458-appb-000003
Figure PCTCN2018118458-appb-000004
Figure PCTCN2018118458-appb-000005
其中Nx表示X个连续的核苷酸组成的核苷酸序列(spacer序列)，N各自独立地选自A、G、C和T；X为18≤X≤35的整数。优选地，X＝20。在一些实施方案中，序列 Nx(spacer序列)能够与靶序列的互补序列特异性杂交。所述sgRNA中除Nx之外的序列为sgRNA的支架(scaffold)序列。
在一些实施方案中，所述人工sgRNA包含由SEQ ID NO：39-75中任一项的核苷酸序列编码的支架序列。
在一些实施方案中，本发明的基因组编辑系统中的向导RNA是本发明的人工sgRNA。
为了在靶细胞中获得有效表达，在本发明的一些实施方式中，所述编码C2c1蛋白或其变体的核苷酸序列针对待进行基因组编辑的细胞所来自的生物体进行密码子优化。
密码子优化是指通过用在宿主细胞的基因中更频繁地或者最频繁地使用的密码子代替天然序列的至少一个密码子(例如约或多于约1、2、3、4、5、10、15、20、25、50个或更多个密码子同时维持该天然氨基酸序列而修饰核酸序列以便增强在感兴趣宿主细胞中的表达的方法。不同的物种对于特定氨基酸的某些密码子展示出特定的偏好。密码子偏好性(在生物之间的密码子使用的差异)经常与信使RNA(mRNA)的翻译效率相关，而该翻译效率则被认为依赖于被翻译的密码子的性质和特定的转运RNA(tRNA)分子的可用性。细胞内选定的tRNA的优势一般反映了最频繁用于肽合成的密码子。因此，可以将基因定制为基于密码子优化在给定生物中的最佳基因表达。密码子利用率表可以容易地获得，例如在www.kazusa.orjp/codon/上可获得的密码子使用数据库(“Codon Usage Database”)中，并且这些表可以通过不同的方式调整适用。参见，Nakamura Y.等，“Codon usage tabulated from the international DNA sequence databases：status for the year2000.Nucl.Acids Res.，28：292(2000)。
可通过本发明的系统进行基因组编辑的细胞所来自的生物体优选是真核生物，包括但不限于，哺乳动物如人、小鼠、大鼠、猴、犬、猪、羊、牛、猫；家禽如鸡、鸭、鹅；植物包括单子叶植物和双子叶植物，例如水稻、玉米、小麦、高粱、大麦、大豆、花生、拟南芥等。
在本发明的一些具体实施方式中，所述编码C2c1蛋白或其变体的核苷酸序列针对人进行密码子优化。在一些具体实施方式中，所述密码子优化的编码AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1、TcC2c1蛋白的核苷酸序列分别选自SEQ ID NO：11-20。
根据本发明的一些实施方式，本发明所述系统的表达构建体中所述编码C2c1蛋白或其变体的核苷酸序列和/或所述编码向导RNA的核苷酸序列与表达调控元件如启动子可操作地连接。
如本发明所用，“表达构建体”是指适于感兴趣的核苷酸序列在生物体中表达的载体如重组载体。“表达”指功能产物的产生。例如，核苷酸序列的表达可指核苷酸序列的转录(如转录生成mRNA或功能RNA)和/或RNA翻译成前体或成熟蛋白质。本发明的“表达构建体”可以是线性的核酸片段、环状质粒、病毒载体，或者，在一些实施方式中，可以是能够翻译的RNA(如mRNA)。
本发明的“表达构建体”可包含不同来源的调控序列和感兴趣的核苷酸序列，或相同来源但以不同于通常天然存在的方式排列的调控序列和感兴趣的核苷酸序列。
“调控序列”和“调控元件”可互换使用，指位于编码序列的上游(5′非编码序列)、中间或下游(3′非编码序列)，并且影响相关编码序列的转录、RNA加工或稳定性或者翻译的核苷酸序列。调控序列可包括但不限于启动子、翻译前导序列、内含子和多腺苷酸化识别序列。
“启动子”指能够控制另一核酸片段转录的核酸片段。在本发明的一些实施方案中，启动子是能够控制细胞中基因转录的启动子，无论其是否来源于所述细胞。启动子可以是组成型启动子或组织特异性启动子或发育调控启动子或诱导型启动子。
“组成型启动子”指一般将引起基因在多数细胞类型中在多数情况下表达的启动子。“组织特异性启动子”和“组织优选启动子”可互换使用，并且指主要但非必须专一地在一种组织或器官中表达，而且也可在一种特定细胞或细胞型中表达的启动子。“发育调控启动子”指其活性由发育事件决定的启动子。“诱导型启动子”响应内源性或外源性刺激(环境、激素、化学信号等)而选择性表达可操纵连接的DNA序列。
如本文中所用，术语“可操作地连接”指调控元件(例如但不限于，启动子序列、转录终止序列等)与核酸序列(例如，编码序列或开放读码框)连接，使得核苷酸序列的转录被所述转录调控元件控制和调节。用于将调控元件区域可操作地连接于核酸分子的技术为本领域已知的。
本发明可使用的启动子的实例包括但不限于聚合酶(pol)I、pol II或pol III启动子。pol I启动子的实例包括鸡RNA pol I启动子。pol II启动子的实例包括但不限于巨细胞病毒立即早期(CMV)启动子、劳斯肉瘤病毒长末端重复(RSV-LTR)启动子和猿猴病毒40(SV40)立即早期启动子。pol III启动子的实例包括U6和H1启动子。可以使用诱导型启动子如金属硫蛋白启动子。启动子的其他实例包括T7噬菌体启动子、T3噬菌体启动子、β-半乳糖苷酶启动子和Sp6噬菌体启动子。当用于植物时，启动子可以是花椰菜花叶病毒35S启动子、玉米Ubi-1启动子、小麦U6启动子、水稻U3启动子、玉米U3启动子、水稻肌动蛋白启动子。
可通过本发明的系统进行基因组编辑的细胞优选是真核生物细胞，包括但不限于，哺乳动物细胞如人、小鼠、大鼠、猴、犬、猪、羊、牛、猫；家禽如鸡、鸭、鹅的细胞；植物细胞包括单子叶植物细胞和双子叶植物细胞，例如水稻、玉米、小麦、高粱、大麦、大豆、花生、拟南芥等的细胞。在本发明的一些实施方案中，所述细胞是真核生物细胞，优选哺乳动物细胞，更优选是人细胞。
在另一方面，本发明提供了一种修饰细胞基因组中靶序列的方法，包括将本发明的基因组编辑系统导入所述细胞，由此所述向导RNA将所述C2c1蛋白或其变体靶向所述细胞基因组中的靶序列。在一些实施方案中，所述靶向导致所述靶序列中的一或多个核苷酸的取代、缺失和/或添加。
将本发明的基因组编辑系统的核酸分子(例如质粒、线性核酸片段、RNA等)或蛋白 质“导入”细胞是指用所述核酸或蛋白质转化细胞，使得所述核酸或蛋白质在细胞中能够发挥功能。本发明所用的“转化”包括稳定转化和瞬时转化。“稳定转化”指将外源核苷酸序列导入基因组中，导致外源基因稳定遗传。一旦稳定转化，外源核酸序列稳定地整合进所述生物体和其任何连续世代的基因组中。“瞬时转化”指将核酸分子或蛋白质导入细胞中，执行功能而没有外源基因稳定遗传。瞬时转化中，外源核酸序列不整合进基因组中。
可用于将本发明的基因组编辑系统导入细胞的方法包括但不限于：磷酸钙转染、原生质融合、电穿孔、脂质体转染、微注射、病毒感染(如杆状病毒、痘苗病毒、腺病毒、腺相关病毒、慢病毒和其他病毒)、基因枪法、PEG介导的原生质体转化、土壤农杆菌介导的转化。
在一些实施方式中，所述方法在体外进行。例如，所述细胞是分离的细胞。在一些实施方式中，所述细胞是CAR-T细胞。在一些实施方式中，所述细胞是诱导的胚胎干细胞。
在另一些实施方式中，所述方法还可以在体内进行。例如，所述细胞是生物体内的细胞，可以通过例如病毒介导的方法将本发明的系统体内导入所述细胞。例如，所述细胞可以是患者体内的肿瘤细胞。
在另一方面，本发明还提一种产生经遗传修饰的细胞的方法，包括将本发明的基因组编辑系统导入细胞中，由此所述向导RNA将所述C2c1蛋白或其变体靶向所述细胞基因组中的靶序列。在一些实施方式中，所述靶向导致所述靶序列中的一或多个核苷酸取代、缺失和/或添加。
在另一方面，本发明还提供经遗传修饰的生物体，其包含通过本发明的方法产生的经遗传修饰的细胞或其后代。
如本文所用，“生物体”包括适于基因组编辑的任何生物体，优选真核生物。生物体的实例包括但不限于，哺乳动物如人、小鼠、大鼠、猴、犬、猪、羊、牛、猫；家禽如鸡、鸭、鹅；植物包括单子叶植物和双子叶植物，例如水稻、玉米、小麦、高粱、大麦、大豆、花生、拟南芥等。在本发明的一些实施方案中，所述生物体是真核生物，优选哺乳动物，更优选人。
如本文所用，“经遗传修饰的生物体”或“经遗传修饰的细胞”意指在其基因组内包含外源多核苷酸或修饰的基因或表达调控序列的生物体或细胞。例如外源多核苷酸能够稳定地整合进生物体或细胞的基因组中，并遗传连续的世代。外源多核苷酸可单独地或作为重组DNA构建体的部分整合进基因组中。修饰的基因或表达调控序列为在生物体或细胞基因组中所述序列包含单个或多个脱氧核苷酸取代、缺失和添加。针对序列而言的“外源”意指来自外来物种的序列，或者如果来自相同物种，则指通过蓄意的人为干预而从其天然形式发生了组成和/或基因座的显著改变的序列。
在另一方面，本发明提供了一种基因表达调控系统，其基于本发明的核酸酶死亡的C2c1蛋白。此系统尽管并没有改变靶基因的序列，在本文范围内也定义为基因组编辑 系统。
在一些实施方案中，本发明的基因表达调控系统是基因抑制或沉默系统，其可以包含以下之一：
i)核酸酶死亡的C2c1蛋白或其与转录阻遏蛋白的融合蛋白，和向导RNA；
ii)包含编码核酸酶死亡的C2c1蛋白或其与转录阻遏蛋白的融合蛋白的核苷酸序列的表达构建体，和向导RNA；
iii)核酸酶死亡的C2c1蛋白或其与转录阻遏蛋白的融合蛋白，和包含编码向导RNA的核苷酸序列的表达构建体；
iv)包含编码核酸酶死亡的C2c1蛋白或其与转录阻遏蛋白的融合蛋白的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；或
v)包含编码核酸酶死亡的C2c1蛋白或其与转录阻遏蛋白的融合蛋白的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体。
所述核酸酶死亡的C2c1蛋白或向导RNA的定义如上所述。所述转录阻遏蛋白的选择属于本领域技术人员的技能范围。
如本文所用，基因抑制或沉默是指基因表达水平的下调或消除，优选在转录水平。
然而，本发明的基因表达调控系统还可以使用核酸酶死亡的C2c1蛋白和转录激活蛋白的融合蛋白。在此种情况下，所述基因表达调控系统是基因表达激活系统。例如，本发明的基因表达激活系统可以包含以下之一：
i)核酸酶死亡的C2c1蛋白和转录激活蛋白的融合蛋白，和向导RNA；
ii)包含编码核酸酶死亡的C2c1蛋白和转录激活蛋白的融合蛋白的核苷酸序列的表达构建体，和向导RNA；
iii)核酸酶死亡的C2c1蛋白和转录激活蛋白的融合蛋白，和包含编码向导RNA的核苷酸序列的表达构建体；
iv)包含编码核酸酶死亡的C2c1蛋白和转录激活蛋白的融合蛋白的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；或
v)包含编码核酸酶死亡的C2c1蛋白和转录激活蛋白的融合蛋白的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体。
所述核酸酶死亡的C2c1蛋白或向导RNA的定义如上所述。所述转录激活蛋白的选择属于本领域技术人员的技能范围。
如本文所用，基因激活是指基因表达水平的上调，优选在转录水平。
在另一方面，本发明还涵盖本发明的基因组编辑系统在疾病治疗中的应用。
通过本发明的基因组编辑系统对疾病相关基因进行修饰，可以实现疾病相关基因的上调、下调、失活、激活或者突变纠正等，从而实现疾病的预防和/或治疗。例如，本发明中靶序列可以位于疾病相关基因的蛋白编码区内，或者例如可以位于基因表达调控区如启动子区或增强子区，从而可以实现对所述疾病相关基因功能修饰或对疾病相关基因表达的修饰。
“疾病相关”基因是指与非疾病对照的组织或细胞相比，在来源于疾病影响的组织的细胞中以异常水平或以异常形式产生转录或翻译产物的任何基因。在改变的表达与疾病的出现和/或进展相关的情况下，它可以是以异常高的水平被表达的基因；它可以是以异常低的水平被表达的基因。疾病相关基因还指具有一个或多个突变或直接负责或与一个或多个负责疾病的病因学的基因连锁不平衡的遗传变异的基因。转录的或翻译的产物可以是已知的或未知的，并且可以处于正常或异常水平。
因此，本发明还提供治疗有需要的对象中的疾病的方法，包括向所述对象递送有效量的本发明的基因组编辑系统以修饰与所述疾病相关的基因。
本发明还提供本发明的基因组编辑系统在制备用于治疗有需要的对象中的疾病的药物组合物中的用途，其中所述基因组编辑系统用于修饰与所述疾病相关的基因。
本发明还提供用于治疗有需要的对象中的疾病的药物组合物，其包含本发明的基因组编辑系统和药学可接受的载体，其中所述基因组编辑系统用于修饰与所述疾病相关的基因。
在一些实施方式中，所述对象是哺乳动物，例如人。
所述疾病的实例包括但不限于肿瘤、炎症、帕金森病、心血管疾病、阿尔茨海默病、自闭症、药物成瘾、年龄相关性黄斑变性、精神分裂症、遗传性疾病等。
在仍另一方面，本发明的范围内还包括用于本发明的方法的试剂盒，该试剂盒包括本发明的基因组编辑系统，以及使用说明。试剂盒一般包括表明试剂盒内容物的预期用途和/或使用方法的标签。术语标签包括在试剂盒上或与试剂盒一起提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。
实施例
为了便于理解本发明，下面将参照相关具体实施例及附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
材料与方法
1.DNA操作
根据Molecular Cloning：A Laboratory Manual并进行一些修改进行DNA操作，包括DNA制备、消化、连接、扩增、合成、纯化、琼脂糖凝胶电泳等。简而言之，通过连接退火的寡核苷酸(表1)到BsaI消化的pUC19-U6-sgRNA(SEQ ID NO：23-30)载体中构建用于细胞转染测定的靶向sgRNA。
表1.人类基因组靶靶序列。
基因 原间隔区ID 原间隔区序列(5’-3’) 5’PAM 链
CCR5 CCR5 site 10 TCCTTCTCCTGAACACCTTC TTG +
  CCR5 site 28 TTTGGCCTGAATAATTGCAG TTC +
DNMT1 DNMT1 site 16 CCCTTCAGCTAAAATAAAGG TTT +
RNF2 RNF2 site 8 TAGTCATGGTGTTCTTCAAC TTG -
- Site 5 GCTCTCAAGACCCACAATCC TTT +
2.从头基因合成和质粒构建
采用PSI-BLAST程序(Altschul，S.F.et al.Nucleic Acids Res 25，3389-3402(1997))鉴定新的CRISPR-C2c1直系同源物。其编码序列进行人源化(Grote，A.et al.，Nucleic Acids Res 33，W526-531(2005))，并且使用GeneDesign程序(Richardson，S.M.et al.，Genome Res 16，550-556(2006))设计用于C2c1基因和sgRNA合成的寡核苷酸。根据文献(Li，G.et al.，Methods Mol Biol 1073，9-17(2013))合成各C2c1基因。使用
Figure PCTCN2018118458-appb-000006
Assembly Master Mix(NEB)通过体外同源重组体外组装纯化的产物成表达载体。将pCAG-2AeGFP(SEQ ID NO：21)和pUC19-U6(SEQ ID NO：22)载体分别用于C2c1蛋白和sgRNA的哺乳动物表达。
3.细胞培养和转染
将人胚胎肾293T细胞在补充有10％胎牛血清(FBS，Gibco)和1％抗生素-抗真菌剂(Gibco)的Dulbecco′s Modified Eagle培养基(DMEM，Gibco)中于37℃，5％CO 2孵育下培养。按照制造商推荐的方案，使用Lipofectamine LTX(Invitrogen)转染293T细胞。对于48孔板的每个孔，使用总共400ng质粒(C2c1∶sgRNA＝2∶1)。然后在转染后48小时，直接收获细胞用于基因组DNA提取。
4.T7核酸内切酶I(T7EI)测定和Sanger测序
将收获的细胞直接用补充有蛋白酶K的Buffer L(Bimake)裂解，并在55℃下孵育3小时并在95℃下灭活10分钟。对每个基因的C2c1靶位点周围的基因组区域进行PCR扩增。将200-400ng PCR产物与ddH 2O混合至终体积10μL，并根据先前方法进行再退火过程以形成异源双链体。重新退火后，用1/10体积的NEBuffer TM 2.1和0.2μL T7EI(NEB)在37℃处理产物30分钟，并在3％琼脂糖凝胶上进行分析。基于相对条带强度对插入缺失进行定量(Cong，L.et al.，Science 339，819-823(2013))。将T7EI测定鉴定的突变产物克隆到TA克隆载体中，并转化到感受态大肠杆菌菌株(Transgen Biotech)中。过夜培养后，随机挑出菌落并测序。
实施例1、新C2c1蛋白鉴定
选择并从头合成来自不同细菌的六种代表性C2c1蛋白，以及之前报道的四种C2c1直系同源物，在人胚胎肾293T细胞中进行基因组编辑(图1、2和SEQ ID NO：1-10)。在这10种C2c1直系同源物中，来自D.inopinatus(DiC2c1)和T.calidus(TcC2c1)的C2c1 既没有可预测的前体CRISPR RNA(pre-crRNA)也没有反式激活crRNA(tracrRNA)(图1b)，提示这两种C2c1蛋白可能不适合基因组编辑应用。
为了进行哺乳动物基因组编辑，用单独的C2c1酶和其靶向含有适当PAM的人内源基因座的同源单向导RNA(sgRNA)共转染293T细胞(图1)。T7核酸内切酶(T7EI)测定的结果显示，除了发明人先前已经鉴定的AaC2c1和AkC2c1之外，四种新的C2c1直系同源物(AmC2c1、BhC2c1、Bs3C2c1和LsC2c1)稳健地编辑人类基因组，尽管它们的靶向效率在不同的直系同源物之间和在不同的靶向位点不同(图1b和图3a)。还通过简单地使用多个sgRNA，使用Bs3C2c1实现多重基因组编辑，同时编辑人类基因组中的四个位点(图3b，c)。这些新发现的C2c1直系同源物扩展对基于C2c1的基因组编辑的选择。
实施例2、不同C2c1及双RNA的可互换性
为了研究C2c1系统中双RNA(crRNA和tracRRNA)和蛋白质组分之间的可互换性，首先分析C2c1蛋白和双RNA两者的保守性。除了C2c1直系同源物的保守氨基酸序列外(图4a和图2)，前体crRNA：tracrRNA双链体的DNA序列及其二级结构也表现出高保守性(图1b和图5)。接下来，用分别与来自8个C2c1系统的各sgRNA复合的8种C2c1直系同源物，在293T细胞中进行基因组编辑。如T7EI测定的结果所示，衍生自AaC2c1、AkC2c1、AmC2c1、Bs3C2c1和LsC2c1基因座的sgRNA可以替代原始sgRNA用于哺乳动物基因组编辑，尽管在不同C2c1直系同源物和sgRNA之间的活性有所不同(图4c，d和图6)。这些结果证明不同C2c1和来自不同C2c1基因座的双RNA之间的可互换性。
实施例3、利用天然基因座无CRISPR阵列的C2c1进行基因组编辑
本发明进一步选择两个基因座没有携带CRISPR阵列的C2c1直系同源物DiC2c1和TcC2c1进行后续实验(图7a)。基因座没有携带CRISPR阵列使得它们的crRNA：tracrRNA双链体的序列不可预测。在293T细胞中共转染与靶向不同基因组位点的衍生自其他8种C2c1直系同源物的基因座的sgRNA组合的DiC2c1和TcC2c1以及AaC2c1。T7EI测定结果表明衍生自AaC2c1、AkC2c1、AmC2c1、Bs3C2c1和LsC2c1的sgRNA使TcC2c1能够稳健地编辑人类基因组(图7b、c和图8)。此外，AasgRNA或AksgRNA能够使TcC2c1实现多重基因组编辑(图7d和图9)。上述结果表明在来自不同系统的C2c1和双链RNA之间可互换性使得可能利用天然基因座不具有CRISPR阵列的C2c1直系同源物来编辑哺乳动物基因组。
实施例4、设计用于C2c1介导的基因组编辑的人工sgRNA
不同C2c1系统中C2c1蛋白和双RNA之间的可互换性有利于设计新的人工sgRNA(artsgRNA)支架以促进C2c1介导的基因组编辑。考虑到C2c1直系同源物中DNA序列和二级结构的保守性(图1b和3)，设计并从头合成37种sgRNA支架(SEQ ID NO：39-75)，用于靶向人CCR5基因座(图7e，图10a)。T7EI测定的结果表明22种artsgRNA 支架有效地工作(图10b)。为了验证artgRNA的普遍适用性，使用artsgRNA13指导TcC2c1或AaC2c1进行多重基因组编辑(图10a)。T7EI测定结果表明，artsgRNA13同时促进TcC2c1和AaC2c1两者的多重基因组编辑(图7f和图10c)。结果表明通过设计和合成artsgRNA能促进C2c1介导的基因组编辑特别是多重基因组编辑。
表2本发明涉及的序列及信息
Figure PCTCN2018118458-appb-000007
Figure PCTCN2018118458-appb-000008

Claims (17)

  1. 一种用于对细胞基因组中的靶序列进行定点修饰的基因组编辑系统，其包含以下i)至v)中至少一项：
    i)C2c1蛋白或其变体，和向导RNA；
    ii)包含编码C2c1蛋白或其变体的核苷酸序列的表达构建体，和向导RNA；
    iii)C2c1蛋白或其变体，和包含编码向导RNA的核苷酸序列的表达构建体；
    iv)包含编码C2c1蛋白或其变体的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；
    v)包含编码C2c1蛋白或其变体的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体；
    其中所述向导RNA能够与所述C2c1蛋白或其变体形成复合物，将所述C2c1蛋白直系同源物或其变体靶向所述细胞基因组中的靶序列。
  2. 权利要求1的系统，其中所述C2c1蛋白是来自Alicyclobacillus acidiphilus的AaC2c1蛋白、来自Alicyclobacillus kakegawensis的AkC2c1蛋白、来自Alicyclobacillus macrosporangiidus的AmC2c1蛋白、来自Bacillus hisashii的BhC2c1蛋白、来自Bacillus属的BsC2c1蛋白、来自Bacillus属的Bs3C2c1蛋白、来自Desulfovibrio inopinatus的DiC2c1蛋白、来自Laceyella sediminis的LsC2c1蛋白、来自Spirochaetes bacterium的SbC2c1蛋白或来自Tuberibacillus calidus的TcC2c1蛋白。
  3. 权利要求1的系统，其中所述C2c1蛋白是来自Alicyclobacillus acidiphilusNBRC100859的AaC2c1蛋白、来自Alicyclobacillus kakegawensis NBRC 103104的AkC2c1蛋白、来自Alicyclobacillus macrosporangiidus strain DSM 17980的AmC2c1蛋白、来自Bacillus hisashii strain C4的BhC2c1蛋白、来自Bacillus属NSP2.1的BsC2c1蛋白、来自Bacillus属V3-13contig_40的Bs3C2c1蛋白、来自Desulfovibrio inopinatus DSM 10711的DiC2c1蛋白、来自Laceyella sediminis strain RHA1的LsC2c1蛋白、来自Spirochaetes bacterium GWB1_27_13的SbC2c1蛋白或来自Tuberibacillus calidus DSM 17572的TcC2c1蛋白。
  4. 权利要求1-3中任一项的系统，其中所述C2c1蛋白包含SEQ ID NO：1-10中任一所示的氨基酸序列，或所述C2c1蛋白的变体包含与SEQ ID NO：1-10中任一所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列。
  5. 权利要求1-4任一项的系统，其中所述向导RNA是sgRNA。
  6. 权利要求5的系统，所述sgRNA包含由选自SEQ ID NO：31-38或39-75的核苷酸序列编码的sgRNA支架。
  7. 权利要求1-6中任一项的系统，其中所述C2c1蛋白是其天然基因座不具有CRISPR阵列的C2c1蛋白。
  8. 权利要求1-7中任一项的系统，其中编码C2c1蛋白或其变体的核苷酸序列经密码子优化。
  9. 权利要求8的系统，其中所述编码C2c1蛋白或其变体的核苷酸序列选自SEQ ID NO：11-20。
  10. 一种对细胞基因组中的靶序列进行定点修饰的方法，包括将权利要求1-9中任一项的系统导入细胞中。
  11. 权利要求10的方法，其中所述细胞来自哺乳动物如人、小鼠、大鼠、猴、犬、猪、羊、牛、猫；家禽如鸡、鸭、鹅；植物，包括单子叶植物和双子叶植物，例如水稻、玉米、小麦、高粱、大麦、大豆、花生、拟南芥等。
  12. 权利要求10或11的方法，其中所述系统通过选自以下的方法导入所述细胞：磷酸钙转染、原生质融合、电穿孔、脂质体转染、微注射、病毒感染(如杆状病毒、痘苗病毒、腺病毒、腺相关病毒、慢病毒和其他病毒)、基因枪法、PEG介导的原生质体转化、土壤农杆菌介导的转化。
  13. 一种治疗有需要的对象中的疾病的方法，包括向所述对象递送有效量的权利要求1-9中任一项的基因组编辑系统以修饰所述对象中与所述疾病相关的基因。
  14. 权利要求1-9中任一项的基因组编辑系统在制备用于治疗有需要的对象中的疾病的药物组合物中的用途，其中所述基因组编辑系统用于修饰所述对象中与所述疾病相关的基因。
  15. 一种用于治疗有需要的对象中的疾病的药物组合物，其包含权利要求1-9中任一项的基因组编辑系统和药学可接受的载体，其中所述基因组编辑系统用于修饰所述对象中与所述疾病相关的基因。
  16. 权利要求13-15中任一项的方法、用途或药物组合物，其中所述对象是哺乳动物，例如人。
  17. 权利要求16的方法、用途或药物组合物，其中所述疾病选自肿瘤、炎症、帕金森病、心血管疾病、阿尔茨海默病、自闭症、药物成瘾、年龄相关性黄斑变性、精神分裂症和遗传性疾病。
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