WO2019020007A1 - 通过诱导剪接位点碱基突变或多聚嘧啶区碱基置换调控rna剪接的方法 - Google Patents

通过诱导剪接位点碱基突变或多聚嘧啶区碱基置换调控rna剪接的方法 Download PDF

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WO2019020007A1
WO2019020007A1 PCT/CN2018/096810 CN2018096810W WO2019020007A1 WO 2019020007 A1 WO2019020007 A1 WO 2019020007A1 CN 2018096810 W CN2018096810 W CN 2018096810W WO 2019020007 A1 WO2019020007 A1 WO 2019020007A1
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exon
sgrna
interest
enzyme
fusion protein
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PCT/CN2018/096810
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French (fr)
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常兴
袁娟娟
马云青
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中国科学院上海生命科学研究院
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Priority to CA3106738A priority Critical patent/CA3106738A1/en
Priority to EP18837474.8A priority patent/EP3712272A4/en
Priority to US16/982,017 priority patent/US20210355508A1/en
Publication of WO2019020007A1 publication Critical patent/WO2019020007A1/zh

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Definitions

  • the present invention relates to a method of modulating RNA splicing by inducing a splice site base mutation or a polypyrimidine region base substitution.
  • splices are composed of more than 150 small nuclear ribonucleoproteins (snRNPs) such as U1, U2, U4, U5 and U6.
  • snRNPs small nuclear ribonucleoproteins
  • U1snRNP recognizes the GU sequence of the 5' splice site of the intron
  • splicing factor 1 SF1
  • U2AF U2 accessory protein
  • the AG sequence of the 3' splice site, the 65KD subunit binds to the polypyrimidine region sequence, and the exon recognition process is completed; then U5 and U6 proteins catalyze the intron by regulating RNA structural remodeling and RNA-protein interaction Removal process.
  • the RNA splicing process plays an important role in the regulation of gene expression.
  • RNA splicing process can be used as a possible therapeutic target for these diseases, for example, using antisense.
  • Oligonucleotide (ASO) regulation of disease-related gene RNA splicing has a certain alleviation effect on Duchenne muscular atrophy and spinal muscular atrophy.
  • Antisense oligonucleotides can bind to cis-acting elements of RNA (such as exon splicing enhancers) to block exon splicing, but splicing regulation using antisense oligonucleotides requires careful design As well as strict screening, continuous administration is required during the treatment of the disease, and the synthesis process is also very expensive, which is very time consuming and costly. Therefore, there is an urgent need for a treatment strategy for such diseases that can be cured once.
  • RNA such as exon splicing enhancers
  • RNA splicing of a gene of interest in a cell comprising expressing a targeted cytosine deaminase in the cell to induce a sense of a gene of interest in the cell
  • the 3' splice site AG of the interest intron is mutated to AA, or the 5' splice site of the gene of interest is mutated to AT, or the polypyrimidine of the intron of interest of interest Multiple Cs of the region were mutated to T, respectively.
  • the targeted cytosine deaminase for use in the methods described herein can be selected from:
  • cytosine deaminase a fusion protein in which a fragment or a mutant which retains an enzyme activity and a Cas enzyme which is partially or completely deleted but retains a nuclease activity
  • cytosine deaminase a fusion protein of a fragment or mutant that retains an enzyme activity and a TALEN protein that specifically recognizes a targeting sequence
  • cytosine deaminase a fusion protein of a fragment or mutant that retains an enzyme activity and a zinc finger protein that specifically recognizes a targeting sequence
  • cytosine deaminase a fusion protein in which a fragment or a mutant which retains an enzyme activity and a Cpf enzyme which partially or completely deletes the nuclease activity but retains an understanding of the chymase activity;
  • a cytosine deaminase which retains an enzyme-active fragment or a fusion protein of the mutant and the Ago protein.
  • the targeted cytosine deaminase is a cytosine deaminase, a fragment or mutant that retains an enzyme activity and a partial or complete deletion of a nuclease activity but retains an understanding of the chymase activity a fusion protein of the Cas enzyme, or a fusion protein of a cytosine deaminase, a fragment or mutant thereof that retains an enzyme activity and a Cpf enzyme that is partially or completely deleted from the nuclease activity but retains an understanding of the chymase activity; Expressing said targeted cytosine deaminase and sgRNA in said cell, wherein said sgRNA is specifically recognized by said Cas enzyme or Cpf enzyme and binds to an intron splicing of interest containing the gene of interest The sequence of the site or the complementary sequence of the polypyrimidine region of interest.
  • the targeted cytosine deaminase is a cytosine deaminase, a fragment that retains an enzyme activity or a fusion protein of a mutant and an Ago protein; the method comprising The step of expressing the targeted cytosine deaminase and the gDNA recognized by the Ago protein in the cell.
  • RNA splicing of a gene of interest in a cell comprising expressing in the cell (1) a partial or complete deletion of a nuclease activity but retaining an understanding of the chymase a step of a fusion protein of an active Cas protein with a cytosine deaminase AID or a mutant thereof and (2) a sgRNA; wherein a Cas protein recognition region of the sgRNA is specifically recognized by the Cas protein, the sgRNA binding To a sequence containing an intron splice site of interest of interest or a complementary sequence of a polypyrimidine region of interest.
  • the sgRNA binds to a sequence comprising a 5' splice site of an intron of interest of a gene, the fusion protein mutating the GT at the 5' splice site to AT, thereby inducing exon skipping, activating alternative splice sites, inducing a mutually exclusive exon transition or intron inclusion.
  • the sgRNA binds to a sequence comprising a 3' splice site of an intron of interest of a gene, the fusion protein mutating the AG of the 3' splice site to AA , thereby inducing exon skipping, activating alternative splice sites, inducing a mutually exclusive exon transition or intron inclusion.
  • the sgRNA binds to a complementary strand of a polypyrimidine region of interest, inducing a C mutation of the polypyrimidine region to T, thereby enhancing exon inclusion.
  • RNA splicing of a gene of interest in the cell is modulated by transferring the expression vector of the fusion protein and sgRNA into the cell.
  • the method further comprises the step of simultaneously transferring the expression plasmid of Ugi.
  • the method further comprises the step of simultaneously transferring an expression plasmid for a nuclease-deficient or partially defective Cas9 protein, AID or a mutant thereof, and a fusion protein of Ugi.
  • the fusion protein and AID, fragments thereof, or mutants thereof are as described in any portion or any embodiment herein.
  • the cell of interest and the gene of interest are as described in any portion or any embodiment herein.
  • a method of inducing exon skipping comprising: expressing (1) a Cas protein that is partially or completely deleted but retains a known chymase activity in a cell of interest a step of a fusion protein of a cytosine deaminase AID or a mutant thereof and optionally a fusion protein of Ugi and (2) a sgRNA; wherein the Cas protein recognition region of the sgRNA is specifically recognized by the Cas protein, The sgRNA binds to a sequence containing an intron splice site of interest of interest.
  • a method of activating an alternative splice site comprising expressing (1) a Cas protein and a cell that is partially or completely deleted but retains a known chymase activity in a cell of interest. a step of a fusion protein of a pyrimidine deaminase AID or a mutant thereof and optionally a fusion protein of Ugi and (2) a sgRNA; wherein a Cas protein recognition region of the sgRNA is specifically recognized by the Cas protein, The sgRNA binds to a sequence containing an intron splice site of interest of interest, wherein the intron of interest has an alternative splice site.
  • a method of inducing a mutually exclusive exon conversion comprising: expressing in a cell of interest (1) a Cas that is partially or completely deleted but retains a cyclase activity a step of a protein and a cytosine deaminase AID or a mutant thereof and optionally a fusion protein of Ugi and (2) sgRNA; wherein the Cas protein recognition region of the sgRNA is specifically recognized by the Cas protein,
  • the target binding region of the sgRNA comprises a sequence of an intron splice site of interest of interest; wherein the gene of interest is selected from the group consisting of PKM.
  • a method of inducing intron inclusion comprising: expressing in a cell of interest (1) a Cas protein partially or completely deleted but retaining the activity of the chymase a step of a fusion protein of a cytosine deaminase AID or a mutant thereof and optionally a fusion protein of Ugi and (2) a sgRNA; wherein the Cas protein recognition region of the sgRNA is specifically recognized by the Cas protein,
  • the sgRNA contains a cleavage site for an intron of interest; wherein the intron of interest is short in length ( ⁇ 150 bp) and is enriched in G/C bases.
  • a method of enhancing exon inclusion comprising expressing (1) a Cas protein that is partially or completely deleted but retains a known chymase activity in a cell of interest, a step of cytosine deaminase AID or a mutant thereof and a fusion protein of Ugi and (2) sgRNA; wherein the Cas protein recognition region of the sgRNA is specifically recognized by the Cas protein, and the sgRNA is of interest The complement of the polypyrimidine region upstream of the exon.
  • a fusion protein comprising a Cas protein and a cytosine deaminase AID or a mutant thereof that are partially or completely deleted but retain a known chymase activity.
  • the fusion proteins herein further comprise Ugi.
  • RNA splicing for generating a point mutation in a cell, or for regulating a gene of interest in a cell, or for inducing exon skipping, activating alternative splice sites, and inducing a mutual repulsion in a cell of interest.
  • a fusion protein or expression vector thereof and a corresponding sgRNA or expression vector thereof, in the manufacture of a kit for modulating RNA splicing, and the fusion protein described herein or expression thereof and corresponding sgRNA or A kit for expression vectors.
  • TAM induces skipping of exon 5 of CD45 by transforming the invariant guanine at the 3' splice site into adenine.
  • A Schematic representation of the conversion of guanine to adenine at the 3' splice site of the CD45RB exon using TAM and eliciting exon skipping.
  • exon 5 of CD45 was spliced to produce the longest CD45 isoform (CD45RA + RB + RC + , top panel); TAM will be the AG dinucleus at the 3'SS of exon 5 Conversion of the glucosinolate to AA eliminates the splice site and disrupts exon recognition, resulting in a jump in exon 5 and a CD45 subtype lacking CD45RB (CD45RA + RC + , lower panel). (B, C) TAM caused a jump in the exon of CD45RB.
  • Raji cells were transfected with AIDx-nCas9-Ugi and the target sgRNA (CD45-E5-3'SS) or the expression plasmid for control sgRNA (Ctrl) against AAVS1.
  • the target exon CD45RB
  • B exon-specific antibody
  • E upstream exon
  • ERC downstream exon
  • C total CD45
  • Data are representative (B) or summary (C) of two independent experiments. **, p ⁇ 0.01 in the Student's t test.
  • the intron-exon junction was amplified from the genomic DNA of the cells shown in B and the sorted CD45RB hi and CD45RB low cells from TAM-treated cells. Amplicons were analyzed by high-throughput sequencing with over 8000x coverage. The base composition of each nucleotide with a detectable mutation (mutant reading/WT reading > 0.1%) is depicted and the G>A conversion percentage of the mutated Gs is labeled. The positions of the sgRNA and PAM sequences are shown at the top of the intron-exon junction sequence. The dashed line depicts the intron/exon junction. The data is representative of two independent experiments.
  • CD45RB Flow cytometric analysis CD45RB was expressed from TAM-treated cells in control Raji cells or sorted CD45RB hi and CD45RB low cells.
  • F TAM induced CD45RB skipping without altering the coding sequence of CD45.
  • exon-intron junctions were amplified from cDNA and base substitutions were analyzed by high throughput sequencing. Note that both exon mutations are undetectable in the cDNA of TAM treated cells compared to genomic DNA.
  • FIG. 2 TAM induces skipping of the CD45RB exon by converting the invariant guanine at the 5' splice site to adenine.
  • A Schematic diagram of TAM to convert invariant guanine to adenine at the 5'SS of the CD45RB exon and to initiate exon skipping.
  • B, C TAM caused a jump in the exon of CD45RB.
  • Raji cells were transfected with AIDx-nCas9-Ugi and the target sgRNA (E5-5'SS) or the expression plasmid for control sgRNA (Ctrl) against AAVS1.
  • CD45RB target exon
  • B exon-specific antibody
  • E upstream exon
  • C downstream display Expression of exon
  • C total CD45 exon-specific real-time PCR
  • Data are representative (B) or summary (C) of two independent experiments. **, p ⁇ 0.01 in the Student's t test.
  • D G>A mutation enrichment at the 5' position of the CD45RB exon in CD45RB low cells. Intron-exon junctions were amplified from the cells shown in B, as well as sorted CD45RB hi and CD45RB low cells from TAM-treated Raji cells.
  • TAM promotes skipping of RPS24 exon 5 by converting the unchanged guanine at 5'SS to adenine.
  • A TAM converts adenine at the 5' splice site of exon 5 of RPS24 into guanine.
  • 293T cells, E5-5'SS) were transfected with nCas9-AIDx-Ugi and control sgRNA (Ctrl) or expression plasmid for 5'SS sgRNA targeting RPS24 exon 5 (5').
  • sgRNA targeting regions were amplified from genomic DNA (top 2 panels) or cDNA (bottom 2 panels) and analyzed by high throughput sequencing over 8000x coverage.
  • the base composition of a nucleotide with a detectable mutation (>0.1%) is depicted.
  • the top of the exon/intron junction sequence from Refseq shows the location of the sgRNA and PAM sequences. Intron/exon connections are depicted by dashed lines. The data is representative of two independent experiments.
  • TAM promotes the jump of exon 5 of RPS24.
  • splicing linkages were amplified from cDNA and analyzed by high throughput sequencing. The image shows the coverage and percentage of each splice junction treated with control sgRNA (top panel) or E5-5'SS sgRNA (bottom panel).
  • the count and percentage of the node readings are depicted at the top of each connected arc. For the sake of clarity, only arcing representing more than 1% of the total transcript is depicted.
  • C The ratio between the RPS24 isoform and the contained or skipped exon 5 was determined by isomer specific real-time PCR. The data is a summary of three independent experiments. The G to A mutation of (D, E) 5'SS caused a complete jump of RPS24 exon 5. Two single cell clones were obtained from TAM treated cells and analyzed by Sanger sequencing, and the right side (D) represents the genotype of the cells. The expression of the isoform of the contained exon 5 was determined by real-time PCR (E). The data is a summary of three independent experiments.
  • TAM induces skipping of TP53 exon 8 or exon 9 by guanine at the respective splice sites.
  • A-C TAM caused a jump in TP53 exon 8 by mutating its 5'SS.
  • A as shown in Figure 1, using nCas9-AIDx-Ugi and a control sgRNA targeting AAVS1 (Ctrl) or the expression plasmid of 5'SS sgRNA targeting TP53 exon 8 (E8-5'SS) Dye 293T cells.
  • sgRNA targeting regions were amplified from genomic DNA (top 2 panels) or cDNA (bottom 2 panels) and analyzed by high throughput sequencing.
  • the base composition of a nucleotide with a detectable mutation (>0.1%) is depicted.
  • the top of the exon/intron junction sequence from Refseq shows the location of the sgRNA and PAM sequences. Intron/exon connections are depicted by dashed lines. The data is representative of two independent experiments.
  • B Analysis of splicing of TP53 exon 8 by RT-PCR.
  • C As in A, splicing linkages were amplified from cDNA and analyzed by high throughput sequencing. The figure shows the coverage and percentage of each splice junction of cells treated with control sgRNA (top panel) or E8-5'SS sgRNA (bottom panel).
  • FIG. 5 TAM activates alternative splice sites and converts Stat3 ⁇ to Stat3 ⁇ .
  • A Schematic diagram of the use of TAM to eliminate the typical 3'SS of Stat3 exon 23 (Stat3[alpha]) and to facilitate the utilization of downstream substitution 3'SS (Stat3[beta]).
  • B TAM mutation-invariant G at the typical 3'SS of Stat3 exon 23.
  • 293T cells were transfected with AIDx-nCas9-Ugi and an expression plasmid targeting Stat3 exon 23 (E23-3'SS-) or sgRNA (Ctrl) against AAVS1.
  • Intron-exon junctions were amplified from DNA (top 2 panels) or cDNA (bottom 2 panels) and analyzed by high throughput sequencing. The base composition of a nucleotide with a detectable mutation (>0.1%) is depicted. Note that TAM also induced two mutations in exon 23, which were much less than cDNA (54% and 16%) cDNA (26% and 6%). The data is representative of two independent experiments.
  • C TAM enhances the utilization of distal 3'SS in Stat3 exon 23. The splicing junction was amplified from cDNA and analyzed by high throughput sequencing. The figure shows the coverage and percentage of each splice junction treated with control sgRNA (top panel) or E23-3'SS sgRNA (bottom panel).
  • FIG. 6 TAM switches PKM2 to PKM1 by eliminating the 5'SS or 3'SS of exon 10.
  • A Schematic showing the transfer of PKM2 to PKM1 by TAM in C2C12 cells.
  • exon 10, but not exon 9 of the PKM gene was spliced to generate PKM2, whose cDNA was recognized by the restriction enzyme PstI; at the bottom, TAM will be at the 5'SS of exon 10
  • the GT dinucleotide is converted to AT (or the 3'SS AG is converted to AA).
  • exon 9 but exon 10 is spliced to produce PKM1, the cDNA of which is recognized by the restriction enzyme NcoI.
  • TAM increases PKM1 while inhibiting PKM2 expression.
  • C2C12 cells were transfected with TAM and target sgRNA (PKM-E10-5'SS or PKM-E10-3'SS) or control sgRNA (Ctrl). Seven days after transfection, the cells were differentiated into muscle cells, and then PKM was amplified from cDNA, and the amplicons were digested with PstI or NcoI. Fragments corresponding to PKM1 or PKM2 were indicated and included GAPDH and total PKM (amplicons of exon 5 and exon 6) as vector controls.
  • C, D TAM converts invariant G to A at the 3'SS (C) or 5' SS (D) of PKM exon 10.
  • Intron-exon junctions were amplified from genomic DNA (top two panels) or cDNA (bottom two panels) and analyzed by high throughput sequencing. The alkali composition and the percentage of A for each guanine are described. The data is representative of two independent experiments.
  • E Real-time PCR analysis of the ratio of PKM1 to PKM2. The data is representative (B, D, E) or a summary of two independent experiments (C).
  • F TAM moves PKM2 to PKM1.
  • the splicing junction was amplified from cDNA and analyzed by high throughput sequencing. The figure shows the coverage and percentage of each splice junction treated with control sgRNA (top panel) or E10-5'SS sgRNA (bottom panel). The count and percentage of the node readings (in parentheses) are depicted at the top of each connected arc.
  • G, H Similar to the above, TAM can convert PKM2 to PKM1 in undifferentiated C2C12 cells.
  • TAM inhibits PKM1 expression by eliminating the 3'SS or 5'SS of the ninth exon of PKM.
  • A TAM converts invariant G to A at the 3'SS or 5'SS of PKM exon 9.
  • B Genomic DNA from control or TAM-treated cells (E9-3'SS) in muscle cells differentiated from C2C12 cells and analyzed by high-throughput sequencing. The percentage G or A of each guanine with a mutation frequency of more than 0.1% is depicted. The data is representative of two independent experiments. Note that TAM also causes a C>T mutation in exon 9 at this position.
  • C, D, E TAM inhibits PKM1 expression while promoting PKM2 expression.
  • FIG. 8 After the TAM converts the invariant G to A on the 5'SS, the second intron of BAP1 is retained.
  • A Schematic diagram of directing TAM mutation at the 5' splice site of exon 2 of BAP1 without changing G and showing its retention.
  • the second intron of BAP1 may be spliced by intron definition, where the 5'SS is paired with the downstream 3'SS. Converting the invariant G to A, the U1RNP is identified in the 5'SS processing U1 and the intron definition is destroyed, resulting in the inclusion of the intron.
  • B, C TAM induces retention of BAP1 intron 2.
  • 293T cells were transfected with AIDx-nCas9-Ugi and an expression plasmid for AAVS1 (Ctrl) or 5'SS sgRNA for exon 2 of BAP1 (NAP1-E2-5'SS). Seven days after transfection, splicing of BAP1 mRNA was analyzed by RT-PCR (B) or isotype-specific real-time PCR (C). (D) The retained intron contains a 5'SS G>A mutation. Intron-exon junctions were amplified from genomic DNA (top two panels) or cDNA (bottom two panels) of 293T cells treated with control sgRNA (ctrl) or target sgRNA (E2-5'SS).
  • each guanine with detectable mutations is depicted.
  • the positions of the sgRNA and PAM sequences are labeled at the top of the intron-exon junction sequence, and the dashed line depicts the intron/exon junction.
  • the data is representative of two independent experiments. Note that because intron 2 is efficiently spliced in control cells, only cells receiving E2-5'SS sgRNA have reads covering introns, 99% of which contain G>A mutations.
  • E Mutant 5'SS-induced retention of the second intron, rather than skipping the second exon of BAP1. As in D, the splicing junction was amplified from cDNA and analyzed by high throughput sequencing.
  • the figure shows the coverage and percentage of each splice site treated with control sgRNA (top panel) or E2-5'SS sgRNA (bottom panel). The count and percentage of the node readings (in parentheses) are depicted at the top of each connected arc. Note that in sgRNA treated cells, 2.4% of the mRNA was spliced to skip the second exon, while more than 60% retained the second intron. Data are representative (B, D, E) or summary (C) of two independent experiments.
  • FIG. 9 Conversion of invariant G to A at the 3'SS of exon 3 of BAP1 results in its retention.
  • A Schematic diagram of directing TAM to mutate the invariant G at the 3'SS of exon 3 of BAP1 and direct its retention.
  • B, C TAM induces retention of BAP1 intron 2.
  • 293T cells were transfected with AIDx-nCas9-Ugi and AAVS1 (Ctrl) or the expression plasmid of 3'SS sgRNA of intron 2 of BAP1. Seven days after transfection, splicing of BAP1 mRNA was analyzed by RT-PCR (B) and isotype-specific real-time PCR (C).
  • the second intron retained contains a G>A mutation at the 3'SS.
  • the base composition of each guanine with detectable mutations is depicted (G > A conversion efficiency over 0.1%).
  • the positions of the sgRNA and PAM sequences are shown at the top of the intron-exon junction sequence.
  • the dotted line depicts the intron/exon junction.
  • the data is representative of two independent experiments.
  • TAM primarily induces retention of the second exon of BAP1.
  • the splicing junction was amplified from cDNA and analyzed by high throughput sequencing. The figure shows the coverage and percentage of each splice junction of cells treated with control sgRNA (top panel) or E3-3'SS sgRNA (bottom panel). The count and percentage of the node readings (in parentheses) are depicted at the top of each connected arc.
  • FIG. 10 Polypyrimidine cluster (PPT) upstream of exon 6 of GANAB converts Cs to Ts to enhance its inclusion.
  • PPT Polypyrimidine cluster
  • A A schematic diagram directing TAM to convert Cs to Ts at the PPT of GANAB exon 6 to enhance the strength of the 3'SS.
  • the polypyrimidine polysaccharide of exon 6 of GANAB contains multiple Cs (left) and converts these C to T (right), increasing the intensity of this 3'SS (from 6.88 to 10.12) and enhancing the exon 6 contain.
  • TAM converts the PPT of GnAB exon 6 to Ts.
  • 293T cells were transfected with AIDx-nCas9--Ugi and control sgRNA (Ctrl) or expression plasmid for sgRNA targeting PPT of GANAB exon 6 (PPT-E6GANAB).
  • sgRNA targeting region was amplified from genomic DNA and analyzed by high throughput sequencing over 8000x coverage.
  • the base composition of a nucleotide with a detectable mutation >0.1%) is depicted.
  • the positions of the sgRNA and PAM sequences are shown at the top of the ligation sequence. Intron/exon connections are depicted by dashed lines. The data is representative of two independent experiments.
  • C, D, E TAM enhances the inclusion of the sixth exon of GANAB.
  • C As in B, the splicing junction was amplified from cDNA and analyzed by high throughput sequencing. The figure shows the coverage and percentage of each splice junction of cells treated with control sgRNA (top panel) or PPT-E6GANAB sgRNA (bottom panel). The count and percentage of the node readings (in parentheses) are depicted at the top of each connected arc.
  • D, E Analysis of splicing of GANAB mRNA by RT-PCR (D) or isoform-specific real-time PCR (E). Data are representative of two independent experiments (C, D) or summary (E).
  • F, G TAM promotes the inclusion of the sixth exon of ThyN1.
  • H, I TAM enhanced the inclusion of the 13th exon of OS9.
  • FIG. 11 Polypyrimidine (PPT) conversion upstream of exon 5 of RPS24 enhances its inclusion by C to T.
  • PPT Polypyrimidine
  • A TAM converts C to T at the PPT of exon 5 of RPS24.
  • 293T cells were transfected with AIDx-nCas9-Ugi and AAVS1 (Ctrl) or an expression plasmid for the sgRNA of the polypyrimidine nucleoside of exon 5 of RPS24 (PPT-E5RPS25). Six days after transfection, the sgRNA targeting region was amplified from genomic DNA and analyzed by high throughput sequencing over 8000x coverage.
  • Figure 12 Using TAM to induce exon skipping in cells of Duchenne muscle-deficient patients, repair the reading frame of the DMD gene, and restore the expression of dystrophin (DMD).
  • A Schematic diagram of directing TAM to convert G to A at the 5'SS of DMD exon 50, restoring expression of dystrophin in patient cells. Compared with normal cells (above), this patient lacks exon 51 due to genetic mutation, destroying the reading frame of dystrophin, resulting in complete deletion of this protein (middle); using TAM mutation exon 50 After the 5'SS GU is AU, the exon 50 skips in the patient's cells, thereby restoring the dystrophin reading frame and protein expression.
  • Figure 13 Schematic representation of the regulation of RNA splicing using TAM technology. Mutation of GT at the 5' splice site of an intron to AT using TAM technology, can induce exon skipping, activate alternative splice sites, induce murine exon transitions or intron inclusions;
  • the AG mutation of the 3' splice site is AA, which can also induce exon skipping, activate alternative splice sites, induce mutual exclusion exon conversion or intron inclusion; induce multimerization at the 3' end of intron
  • the C mutation in the pyrimidine region is T, which enhances the weak splice site, thereby enhancing exon inclusion.
  • FIG 14 Using TAM to induce exon skipping in cells of Duchenne muscle-deficient patients, repair the reading frame of the DMD gene, and restore the expression of dystrophin (DMD).
  • DMD dystrophin
  • mutating a point mutation in a cell in particular by mutating the 3' splice site AG of the gene of interest in the cell to AA, or the 5' splice site of the intron of interest of the gene of interest
  • the GT mutation is AT, or multiple Cs (eg, 2-10) of the polypyrimidine region of the intron of interest of the gene of interest are mutated to T, respectively, thereby regulating RNA splicing of the gene of interest in the cell to induce Exon skipping, activating alternative splice sites, inducing a mutually exclusive exon transition, inducing intron inclusion or enhancing exon inclusion.
  • Modulation as used herein means altering the conventional splicing manner of RNA.
  • the invention can be practiced using a targeted cytosine deaminase.
  • the targeted cytosine deaminase herein is constructed by fusing cytosine deaminase to a protein with targeting.
  • cytosine deaminase refers to various enzymes having cytosine deaminase activity, including but not limited to enzymes of the APOBEC family, such as APOBEC-2, AID, APOBEC-3A, APOBEC-3B, APOBEC-3C, APOBEC -3DE, APOBEC-3G, APOBEC-3F, APOBEC-3H, APOBEC4, APOBEC1 and pmCDA1.
  • Cytosine deaminase suitable for use herein may be from any species, particularly preferably a mammalian, especially human, cytosine deaminase.
  • the cytosine deaminase suitable for use herein is an activated cytosine deaminase, such as a human-activated cytosine deaminase.
  • the APOBEC family of cytosine deaminase is a family of RNA editing enzymes with a nuclear localization signal at the N-terminus and a nuclear export signal at the C-terminus.
  • the catalytic domain is shared by the APOBEC family. It is generally believed that the N-terminal structure is required for somatic hypermutation (SHM).
  • cytosine deaminase The function of cytosine deaminase is to deamination of cytosine, cytosine to uracil, and subsequent DNA repair can turn uracil into other bases. It will be appreciated that cytosine deaminase, or fragments or mutants thereof that retain the biological activity of cytosine deamination, cytosine to uracil, are well known in the art.
  • AID is used herein as a cytosine deaminase in a targeted cytosine deaminase.
  • AID amino acid residues 9-26 are nuclear localization (NLS) domains, in particular amino acid residues 13-26 are involved in DNA binding, amino acid residues 56-94 are catalytic domains, and amino acid residues 109-182 are APOBEC
  • the domain, amino acid residues 193-198 are nuclear export (NES) domains, amino acid residues 39-42 interact with catenin-like protein 1 (CTNNBL1), and amino acid 113-123 residues are hotspot recognition loops.
  • a full length sequence of AID (as indicated by amino acids 1457-1654 of SEQ ID NO: 25) or a fragment of AID can also be used herein.
  • the fragment comprises at least an NLS domain, a catalytic domain and an APOBEC-like domain.
  • the fragment comprises at least amino acid residues 9-182 of the AID (ie, amino acid residues 1465-1638 of SEQ ID NO: 25).
  • the fragment comprises at least amino acid residues 1-182 of the AID (ie, amino acid residues 1457-1638 of SEQ ID NO: 25).
  • an AID fragment as used herein consists of amino acid residues 1-182, consists of amino acid residues 1-164, or consists of amino acid residues 1-190.
  • the AID fragment used herein consists of amino acid residues 1457-1638 of SEQ ID NO: 25, amino acid residues 1457-1642 of SEQ ID NO: 25, or SEQ ID NO: 25 Amino acid residue composition of 1457-1646.
  • variants of AID that retain their cytosine deaminase activity (ie, the biological activity of cytosine deamination, cytosine to uracil) can also be used herein.
  • such variants may have from 1 to 10, such as 1-8, 1-5 or 1-3 amino acid variations relative to the wild-type sequence of the AID, including deletions, substitutions and mutations of amino acids.
  • these amino acid variations do not occur within the above-described NLS domain, catalytic domain and APOBEC-like domain, or even within these domains do not affect the original biological function of these domains.
  • it is preferred that these variations do not occur at positions 24, 27, 38, 56, 58, 87, 90, 112, 140, etc.
  • the variation occurs at positions 10, 82, and 156.
  • substitution mutations occur at positions 10, 82, and 156, and such substitution mutations can be K10E, T82I, and E156G.
  • the amino acid sequence of an exemplary AID mutant comprises the amino acid sequence set forth at positions 1447-1629 of SEQ ID NO: 31, or the amino acid residue set forth at positions 1447-1629 of SEQ ID NO: 31. composition. Examples of other AIDs, fragments or mutants thereof can be found in CN 201710451424.3, the entire contents of which are incorporated herein by reference.
  • the protein having a targeting effect may be a protein well known in the art capable of targeting a gene of interest in a cell genome, including but not limited to a TALEN protein that specifically recognizes a targeting sequence, a zinc finger protein that mutates a recognition targeting sequence, Ago protein, Cpf enzyme and Cas enzyme. This can be carried out using TALEN proteins, zinc finger proteins, Ago proteins, and Cpf enzymes and Cas enzymes well known in the art.
  • a targeted cytosine deaminase suitable for use herein may be selected from: (1) a cytosine deaminase, a fragment or mutant that retains an enzyme activity and a nuclease active moiety or a fusion protein of Casase that is completely deleted but retains the activity of the chymase; (2) a cytosine deaminase, a fusion protein that retains the fragment or mutant of the enzyme and a TALEN protein that specifically recognizes the targeting sequence; (3) a cytosine deaminase, a fusion protein that retains an active fragment or mutant and a zinc finger protein that specifically recognizes a targeting sequence; (4) a cytosine deaminase, a fragment or mutant that retains an enzyme activity and A fusion protein in which the nuclease activity is partially or completely deleted but retains the Cpf enzyme which is known for its chymase activity; and (5) a cytosine deamina
  • Cpf enzyme When a Cpf enzyme is used, it is preferred to use a Cpf enzyme which is partially or completely deleted but retains the activity of the chymase.
  • the Cpf enzyme binds to a specific DNA sequence under the guidance of its recognized sgRNA, allowing the cytosine deaminase to be fused thereto to carry out the mutations described herein.
  • Ago proteins need to bind to specific DNA sequences under the guidance of their recognized gDNA.
  • a cytosine-mediated gene mutation technique is used to mutate an intron splice site to adenine, specifically blocking the exon recognition process.
  • TAM cytosine-mediated gene mutation technique
  • the TAM technique herein uses a fusion protein of a cas protein-deleted Cas protein with a cytosine deaminase AID, an active fragment thereof or a mutant. Under the guidance of sgRNA, the fusion protein is recruited to a specific DNA sequence, and AID, its active fragment or mutant mutates guanine (G) to adenine (A), or cytosine (C) Thymine (T).
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas protein also known as Cas enzyme
  • endonuclease activity and its specifically recognized sgRNA is complementary to the template strand in the target DNA by the pairing region of the sgRNA (ie, the target binding region).
  • the double-stranded DNA is cleaved by Cas at a specific position.
  • a nuclease active moiety (having only DNA single-strand break capability) or a complete deletion (no DNA double-strand break capability), particularly a partial or complete deletion of endonuclease activity, but retaining Cas protein
  • It may be derived from various Cas proteins and variants thereof well known in the art including, but not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10 , Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10 , Csx16, CsaX, Cs
  • a Cas9 enzyme lacking nuclease activity and a single-stranded sgRNA specifically recognized by the same are used.
  • the Cas9 enzyme may be a Cas9 enzyme from a different species including, but not limited to, Cas9 (SpCas9) from S. pyogenes, Cas9 (SaCas9) from S. aureus, and Cas9 (St1 Cas9) from Streptococcus thermophilus, and the like.
  • Various variants of the Cas9 enzyme can be used as long as the Cas9 enzyme specifically recognizes its sgRNA and lacks nuclease activity.
  • the Cas protein with nuclease activity deletion can be prepared by methods well known in the art, including but not limited to deletion of the entire catalytic domain of the endonuclease in the Cas protein or mutation of one or several amino acids in the domain. Thereby producing a Cas protein lacking in nuclease activity.
  • the mutation may be one or several (for example, two or more, three or more, four or more, five or more, more than ten, to the entire catalytic domain) deletion or substitution of amino acid residues, or one or several new amino acids. Insertion of residues (for example, one or more, two or more, three or more, four or more, five or more, ten or more, or 1 to 10, and 1 to 15).
  • Deletion of the above domains or mutation of amino acid residues can be carried out by methods conventional in the art, and whether the Cas protein after the mutation also has nuclease activity.
  • Cas9 its two endonuclease catalytic domains, RuvC1 and HNH, can be mutated, for example, the 10th amino acid of the enzyme (in the RuvC1 domain) is mutated to alanine or other An amino acid that mutates the histidine of amino acid 841 (located in the HNH domain) to alanine or other amino acids.
  • the Cas enzyme is completely nuclease free.
  • the amino acid sequence of the nuclease-free Cas9 enzyme used herein is set forth in SEQ ID NO: 25, pp. 42-1452.
  • the Cas enzyme portion used herein lacks nuclease activity, ie, the Cas enzyme can cause DNA single strand breaks.
  • a representative example of such a Cas enzyme can be shown as amino acid residues 42-14-19 of SEQ ID NO:33.
  • the amino acid sequence of the Cas enzyme used herein is as shown in SEQ ID NO: 23, positions 191-1566, or as shown in SEQ ID NO: 50, amino acid residues 199-1262. Examples of other Cas enzymes can be found in CN 201710451424.3, the entire disclosure of which is incorporated herein by reference.
  • the function of the Cas/sgRNA complex requires a protospacer adjacent motif (PAM) in the non-template strand (3' to 5') of the DNA.
  • PAM protospacer adjacent motif
  • Different Cas enzymes their corresponding PAMs are not identical.
  • the PAM of SpCas9 is typically NGG (SEQ ID NO: 34);
  • the PAM of the SaCas9 enzyme is typically NNGRR (SEQ ID NO: 35);
  • the PAM of the St1Cas9 enzyme is typically NNAGAA (SEQ ID NO: 36); wherein N is A, C, T or G, R is G or A.
  • the PAM of the SaCas9 enzyme is NNGRRT (SEQ ID NO: 37).
  • the PAM of SpCas9 is TGG (SEQ ID NO: 38); in certain preferred embodiments, the PAM of the SaCas9 enzyme KKH mutant is NNNRRT (SEQ ID NO: 39); N is A, C, T or G, and R is G or A.
  • the sgRNA usually comprises two parts: a target binding region and a protein recognition region (such as a Cas enzyme recognition region or a Cpf enzyme recognition region).
  • the target binding region and the protein recognition region are usually joined in the 5' to 3' direction.
  • the target binding region is typically 15 to 25 bases in length, more typically 18 to 22 bases, such as 20 bases.
  • the target binding region specifically binds to the template strand of DNA, thereby recruiting the fusion protein to a predetermined site.
  • the contralateral region of the sgRNA binding region on the DNA template strand is in close proximity to the PAM, or is separated by a few bases (eg, within 10, or within 8 or within 5).
  • the PAM of the enzyme is usually determined according to the splicing enzyme used (such as Cas enzyme), and then the site of PAM can be found on the non-template strand of DNA, and then the non-template strand (3' 5 to 25 bases, more usually 18 to 22, long to the 5') PAM site immediately downstream of the PAM site or within 10 (eg, within 8, within 5, etc.) of the PAM site.
  • a fragment of a base is used as the sequence of the target binding region of the sgRNA.
  • the protein recognition region of the sgRNA is determined based on the splicing enzyme used, as is known to those skilled in the art.
  • the sequence of the target binding region of the sgRNA herein is a DNA strand downstream of the PAM site containing the selected splicing enzyme (such as the Cas enzyme or Cpf enzyme) immediately adjacent to or within 10 of the PAM site.
  • the selected splicing enzyme such as the Cas enzyme or Cpf enzyme
  • the sgRNA designed for use herein.
  • the sgRNA binds to a sequence containing an intron splice site of interest of interest, or to a complementary sequence of a polypyrimidine region of interest.
  • the target binding region of the sgRNA contains the complement of the intron splice site of interest of interest, or the sequence of the polypyrimidine region containing the intron of interest of interest.
  • the sgRNA can be prepared by methods conventional in the art, for example, by conventional chemical synthesis methods.
  • the sgRNA can also be transferred into the cell via an expression vector to express the sgRNA in the cell; or can be introduced into the animal/human by using an adeno-associated virus.
  • Expression vectors for sgRNA can be constructed using methods well known in the art.
  • sgRNA sequences or complementary sequences thereof comprising a target binding region and a protein recognition region, wherein the target binding region binds to a sequence comprising an intron splice site of interest of interest Or a complementary sequence that binds to a polypyrimidine region of interest.
  • the target binding region is 15 to 25 bases in length, such as 18 to 22 bases, preferably 20 bases.
  • X Show.
  • the targeted cytosine deaminase for use herein is preferably a fusion protein of a Cas enzyme as described above with an AID, a fragment thereof or a mutant as described above.
  • the Cas enzyme is usually at the N-terminus of the amino acid sequence of the fusion protein, and the AID, its fragment or mutant is at the C-terminus; of course, the AID, its fragment or mutant can also be at the N-terminus of the amino acid sequence of the fusion protein, while the Cas enzyme is at the C-terminus.
  • fusion proteins formed primarily of Cas enzyme and AID, fragments or mutants thereof are provided herein.
  • a fusion protein or similar expression "formed primarily by" as used herein does not mean that the fusion protein includes only Cas enzyme and AID, fragments or mutants thereof, and the definition is understood to mean that the fusion protein may only include The Cas enzyme and AID, fragments or mutants thereof, or may also contain other components that do not affect the targeting of the Cas enzyme in the fusion protein and the function of the AID, fragment thereof or mutant mutant target sequence, including but not limited to Various linker sequences, nuclear localization sequences, Ugi sequences, and, as described below, by gene cloning procedures, and/or for constructing fusion proteins, promoting expression of recombinant proteins, obtaining recombinant proteins that are automatically secreted outside the host cell, or facilitating recombinant proteins The amino acid sequence introduced in the fusion protein by detection and/or purification or the like.
  • the Cas enzyme can be fused to the AID, a fragment thereof or a mutant by a linker.
  • the linker may be a peptide of 3 to 25 residues, for example, a peptide of 3 to 15, 5 to 15, 10 to 20 residues. Suitable examples of peptide linkers are well known in the art.
  • the linker contains one or more motifs that are repeated before and after, and the motif typically contains Gly and/or Ser.
  • the motif can be SGGS (SEQ ID NO: 40), GSSGS (SEQ ID NO: 41), GGGS (SEQ ID NO: 42), GGGGS (SEQ ID NO: 43), SSSSG (SEQ ID NO: 44) ), GSGSA (SEQ ID NO: 45) and GGSGG (SEQ ID NO: 46).
  • the motif is contiguous in the linker sequence with no amino acid residues inserted between the repeats.
  • the linker sequence may comprise 1, 2, 3, 4 or 5 repeat motifs.
  • the linker sequence is a polyglycine linker sequence.
  • the amount of glycine in the linker sequence is not particularly limited, but is usually 2 to 20, for example, 2 to 15, 2 to 10, and 2 to 8.
  • the linker may also contain other known amino acid residues such as alanine (A), leucine (L), threonine (T), glutamic acid (E), styrene Amino acid (F), arginine (R), glutamine (Q), and the like.
  • the linker sequence is XTEN, the amino acid sequence of which is set forth in amino acid residues 183-198 of SEQ ID NO:29.
  • Other exemplary linker sequences can be found in the linker sequences set forth in CN 201710451424.3, such as SEQ ID NOs: 21-31 and the like of this application.
  • a suitable cleavage site which necessarily introduces one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest.
  • promote expression of a recombinant protein obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus of the recombinant protein or within the protein.
  • Other suitable regions include, for example, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like.
  • the amino terminus or carboxy terminus of the fusion protein herein may also contain one or more polypeptide fragments as a protein tag.
  • Any suitable label can be used in this article.
  • the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE and Ty1. These tags can be used to purify proteins.
  • the fusion proteins herein may also contain a nuclear localization sequence (NLS).
  • NLS nuclear localization sequences of various sources and various amino acids well known in the art can be used.
  • Such nuclear localization sequences include, but are not limited to, NLS of the SV40 viral large T antigen; NLS from nuclear protein, eg, nuclear protein binary NLS; NLS from c-myc; NLS from hRNPA1M9; from input protein - Sequence of the IBB domain of ⁇ ; sequence of fibroid T protein; sequence of mouse c-ablIV; sequence of influenza virus NS1; sequence of hepatitis virus ⁇ antigen; sequence of mouse Mx1 protein; human poly(ADP-ribose) The sequence of the polymerase; and the sequence of the steroid hormone receptor (human) glucocorticoid; and the like.
  • NLS amino acid sequences of these NLS sequences can be found in the sequences set forth in CN 201710451424.3 SEQ ID NO: 33-47.
  • sequence shown by amino acid residues 26-33 of SEQ ID NO: 25 is used herein as the NLS.
  • the NLS may be located at the N-terminus and C-terminus of the fusion protein; or may be located in the fusion protein sequence, such as the N-terminus and/or C-terminus of the Cas9 enzyme in the fusion protein, or the AID, fragment or mutant thereof located in the fusion protein. N-terminal and / or C-terminal.
  • the accumulation of the fusion protein of the invention in the nucleus can be detected by any suitable technique.
  • a detection marker can be fused to the Cas enzyme such that the location of the fusion protein within the cell can be visualized when combined with means for detecting the location of the nucleus (eg, a dye specific for the nucleus, such as DAPI).
  • 3*flag is used herein as a marker, and the peptide sequence can be as shown in amino acid residues 1-23 of SEQ ID NO:25. It will be understood that, in general, if a marker sequence is present, the marker sequence is typically at the N-terminus of the fusion protein.
  • the tag sequence can be directly linked to the NLS or can be joined by a suitable linker sequence.
  • the NLS sequence can be ligated directly to the Cas enzyme or AID, a fragment or mutant thereof, or can be ligated to the Cas enzyme or AID, a fragment thereof or a mutant by a suitable linker sequence.
  • the fusion proteins herein consist of a Cas enzyme and an AID, a fragment thereof or a mutant.
  • the fusion proteins herein are made by Cas enzyme linked to an AID, fragment or mutant thereof via a linker.
  • a fusion protein herein consists of an NLS, a Cas enzyme, an AID or a fragment or mutant thereof, and an optional linker sequence between the Cas enzyme and the AID or a fragment or mutant thereof.
  • the fusion proteins herein may contain phage proteins, such as UGI as an UNG inhibitor, in addition to NLS, Cas enzyme, and AID, fragments or mutations thereof.
  • a fusion protein herein comprises a Cas9 enzyme described herein, an AID or fragment or mutant thereof described herein, UGI and NLS, or an optional linker from these portions and therebetween
  • the sequence and optionally the amino acid sequence composition for detection, isolation or purification can be located at the N-terminus, C-terminus of the fusion protein, or in the fusion protein, for example between the NLS sequence and the Cas enzyme or between the Cas enzyme and the AID, a fragment thereof or a mutant.
  • the fusion proteins herein are AIDs or fragments or mutants thereof, Cas enzymes, Ugi and NLS from N-terminus to C-terminus, or may be Cas enzymes, AIDs or fragments or mutants thereof, Ugi and NLS, which can be connected by a joint between them.
  • the fusion proteins disclosed in CN 201710451424.3 are used herein. More specifically, the amino acid sequences disclosed herein are as set forth in SEQ ID NO: 25, 27, 29, 31, 33, 48 or 50, or as amino acids 26-1654 of SEQ ID NO: 25. Shown, or as shown in SEQ ID NO:27, positions 26-1638, or as shown in SEQ ID NO: 31, amino acids 26-1629, or as shown in SEQ ID NO: 33, amino acids 26-1638. The protein, or as shown in amino acids 26-1636 of SEQ ID NO:48. In certain embodiments, a fusion protein herein is as set forth in SEQ ID NO: 23 of the present application.
  • An expression vector/plasmid expressing the fusion protein described above and a vector/plasmid expressing the desired sgRNA can be constructed and transformed into a cell of interest to regulate its RNA splicing by inducing base mutations at the splice site of the gene of interest.
  • An "expression vector” can be a variety of bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • the expression vector may also include a ribosome binding site for translation initiation and a transcription terminator.
  • the polynucleotide sequences described herein are operably linked to a suitable promoter in an expression vector to direct mRNA synthesis via the promoter.
  • promoters are: lac or trp promoter of E. coli; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti- Promoters for the expression of LTRs of transcriptional viruses and other known controllable genes in prokaryotic or eukaryotic cells or their viruses.
  • the marker gene can be used to provide phenotypic traits for selection of transformed host cells including, but not limited to, dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or for the large intestine Bacillus tetracycline or ampicillin resistance.
  • GFP green fluorescent protein
  • a polynucleotide described herein is expressed in a higher eukaryotic cell, transcription will be enhanced if an enhancer sequence is inserted into the vector.
  • An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.
  • Expression vectors containing the polynucleotide sequences described herein and appropriate transcription/translation control signals can be constructed using methods well known to those of skill in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the fusion protein, its coding sequence or expression vector, and/or sgRNA, its coding sequence or expression vector can be provided as a composition.
  • the composition may contain an expression vector for the fusion protein and sgRNA or sgRNA herein, or an expression vector containing the fusion protein herein and an expression vector for sgRNA or sgRNA.
  • the fusion protein or its expression vector, or sgRNA or its expression vector may be provided as a mixture, or may be packaged separately.
  • the composition may be in the form of a solution or it may be in a lyophilized form.
  • the fusion protein in the composition is a fusion protein of an AID, fragment or mutant thereof described herein and a Cas enzyme as described herein.
  • kits containing the compositions described herein comprising the expression vector of the fusion protein and sgRNA or sgRNA herein, or an expression vector comprising the expression vector of the fusion protein herein and sgRNA or sgRNA.
  • the fusion protein or its expression vector, or sgRNA or its expression vector can be packaged separately or in the form of a mixture.
  • reagents for transferring the fusion protein or its expression vector and/or sgRNA or expression vector thereof into a cell and instructions for directing the skilled person to perform the transfer.
  • the kit can also include instructions for the skilled artisan to practice the various methods and uses described herein using the components contained in the kit.
  • Other reagents such as reagents for PCR and the like are also included in the kit.
  • a fusion protein, coding sequence or expression vector thereof, and/or sgRNA or expression vector thereof can be used to induce a base mutation at a splice site of a gene of interest to modulate its RNA splicing. Accordingly, provided herein is a method of inducing a base mutation in a splice site of a gene of interest in a cell of interest, the method comprising the step of expressing a fusion protein described herein in the cell, according to the expressed A fusion protein, the method further comprising the step of expressing sgRNA or gDNA.
  • the AIDs, fragments or mutants thereof described herein, and fusion proteins of the Cas enzyme and their recognized sgRNAs are expressed in a cell.
  • a cytosine deaminase, a fragment or mutant that retains an enzymatic activity, and a fusion protein that specifically recognizes a TALEN protein of a targeting sequence are expressed in a cell.
  • a cytosine deaminase, a fragment or mutant that retains an enzyme activity, and a fusion protein that specifically recognizes a zinc finger protein of a targeting sequence are expressed in a cell.
  • a cytosine deaminase, a fragment or mutant that retains an enzymatic activity in a cell and a Cpf enzyme that is partially or completely deleted but retains a cyclase activity and a Cpf enzyme are retained in the cell. Identify sgRNA.
  • the cytosine deaminase is expressed in the cell, which retains the enzymatically active fragment or the fusion protein of the mutant with the Ago protein and the gDNA recognized by the Ago protein.
  • cells of interest also include those cells in which a base mutation in the splice site of the gene of interest is required to modulate its RNA splicing.
  • Such cells include prokaryotic cells and eukaryotic cells, such as plant cells, animal cells, microbial cells, and the like.
  • animal cells such as mammalian cells, rodent cells, including humans, horses, cows, sheep, rats, rabbits, and the like.
  • Microbial cells include cells from a variety of microbial species well known in the art, especially those having microbial species of medical research value, production value (e.g., production of fuels such as ethanol, protein production, lipids such as DHA production).
  • the cells may also be cells of various organ origin, such as cells from human liver, kidney, skin, etc., or blood cells.
  • the cells may also be various mature cell lines currently marketed, such as 293 cells, COS cells.
  • the cells are cells from a healthy individual; in other embodiments, the cells are cells from diseased tissue of a diseased individual, such as cells from inflammatory tissue, tumor cells.
  • the cell of interest is an induced pluripotent stem cell.
  • the cells may also be genetically engineered to have a particular function (e. g., to produce a protein of interest) or to produce a phenotype of interest. It should be understood that the cells of interest include somatic and germ cells.
  • the cell is a specific cell in an animal or human.
  • the gene of interest may be any nucleic acid sequence of interest, particularly a variety of diseases associated with, or associated with, the production of various proteins of interest, or various genes or nucleic acid sequences associated with biological functions of interest.
  • Such gene or nucleic acid sequences of interest include, but are not limited to, nucleic acid sequences encoding various functional proteins.
  • a functional protein refers to a protein capable of performing physiological functions of an organism, including a catalytic protein, a transport protein, an immune protein, and a regulatory protein.
  • the functional proteins include, but are not limited to, proteins involved in the development, progression, and metastasis of diseases, proteins involved in cell differentiation, proliferation, and apoptosis, proteins involved in metabolism, development-related proteins. , as well as various drug targets and so on.
  • the functional protein may be an antibody, an enzyme, a lipoprotein, a hormone protein, a transport and storage protein, a motor protein, a receptor protein, a membrane protein, or the like.
  • genes of interest include, but are not limited to, RPS24, CD45, DMD, PKM, BAP1, TP53, STAT3, GANAB, ThyN1, OS9, SMN2, beta hemoglobin gene, LMNA, MDM4, Bcl2, and LRP8, and the like.
  • the methods described herein comprise transferring a fusion protein herein, or an expression vector thereof, and an sgRNA thereof, or an expression vector thereof, or gDNA or an expression vector thereof, into the cell.
  • the cell constitutively expresses a fusion protein described herein, only the corresponding sgRNA or its expression vector or its recognized gDNA or its expression vector can be transferred into the cell.
  • the cells may also be incubated with an inducing agent after administration of the sgRNA or gDNA, or the cells may be subjected to corresponding inducing measures (eg, light).
  • the methods practiced herein are carried out using the AIDs, fragments or mutants thereof described herein, and fusion proteins of the Cas enzymes described herein and their recognized sgRNAs.
  • the fusion protein or its expression vector and/or its recognized sgRNA or its expression vector or gDNA or its expression vector can be transferred into a cell by conventional transfection methods.
  • the cell of interest is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method, the procedures used are well known in the art.
  • Another method is to use MgCl 2 . Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • a plasmid DNA-liposome complex is first prepared, and then the plasmid DNA-liposome complex is co-transfected with the corresponding sgRNA or gDNA.
  • the vectors or plasmids described herein can be transferred into cells of interest using commercially available transfection kits or reagents, including but not limited to such agents. 2000 reagents. After transformation of the cells, the obtained transformants can be cultured in a conventional manner to allow them to express the fusion proteins described herein.
  • the medium used in the culture may be selected from various conventional media depending on the cells used.
  • expression vectors expressing the fusion protein and sgRNA or gDNA herein are designed to be suitable for expression in the cells by different techniques for different cells.
  • a promoter that facilitates expression in the cell and other related regulatory sequences can be provided in an expression vector. These can be selected and implemented by the technician according to the actual situation.
  • a site that can serve as a PAM can be searched for near the splice site of interest of the gene of interest, and the Cas enzyme that recognizes the PAM can be selected according to the PAM and designed, prepared as described herein.
  • the target recognition region of the sgRNA used herein typically contains the complementary sequence of the intron splice site of interest of interest.
  • the splice sites described herein have well-known meanings in the art, including 5' splice sites and 3' splice sites.
  • both the 5' splice site and the 3' splice site are relative to an intron.
  • a site that can serve as a PAM is selected near the splice site of the exon/intron of interest of the gene of interest.
  • the exon or intron of interest of the gene of interest may be exon 5 of RPS, exon 5 of CD45, exon 8 or 9 of the TP53 gene, exon 9 of PKM or 10.
  • Intron 2 of BAP1 and intron 8 of TP53 may be exon 5 of RPS, exon 5 of CD45, exon 8 or 9 of the TP53 gene, exon 9 of PKM or 10.
  • Intron 2 of BAP1 and intron 8 of TP53 may be exon 5 of RPS, exon 5 of CD45, exon 8 or 9 of the TP
  • a site available for PAM is selected adjacent to the polypyrimidine strand present in the intron upstream of the 3' splice site of the gene of interest.
  • the target binding region of such sgRNA contains the sequence of the polypyrimidine region of the intron of interest of interest.
  • the methods herein may be in vitro or in vivo; in addition, the methods herein include methods of therapeutic purposes and methods of non-therapeutic purposes.
  • the fusion protein herein or its expression vector and its recognized sgRNA or expression vector or gDNA or expression vector thereof can be transferred into a subject, such as a corresponding tissue cell, using techniques well known in the art.
  • the subject may be a human or a variety of non-human animals, including various non-human model organisms conventionally employed in the art. In vivo experiments should meet ethical requirements.
  • the method of inducing base mutations at the splice sites of genes of interest in a cell of interest as described herein is a general RNA splice regulation method that can be used for gene therapy.
  • a method of gene therapy comprising administering to a subject in need thereof a therapeutically effective amount of a vector expressing a fusion protein described herein and an expression vector of the corresponding sgRNA or gDNA.
  • the therapeutically effective amount can be determined according to the age, sex, nature and severity of the disease. Generally, administration of a therapeutically effective amount of the carrier will be sufficient to alleviate the symptoms of the disease or to cure the disease.
  • the gene therapy can be used for the treatment of diseases which are caused by mutations in genes, and can also be used for the treatment of diseases which are relieved or cured by regulating different splicing subtypes.
  • diseases caused by genetic variation include, but are not limited to, Duchenne myasthenia caused by DMD gene mutation, thalassemia caused by SMN, ⁇ hemoglobin IVS2 647G>A mutation, premature aging caused by LMNA mutation, and familial high Cholesterol and so on.
  • Diseases that can be relieved or cured by adjusting the proportion of different splice subtypes include tumors, including but not limited to Stat3 ⁇ to Stat3 ⁇ , PKM2 to PKM1, MDM4 exon 6 hopping, Bcl2 Selection of alternative splice sites, LRP8 exon eight jumps.
  • a method of treating a tumor comprising the step of administering to a subject in need thereof a therapeutically effective amount of a vector expressing the fusion protein of any of the embodiments herein and an expression vector for the corresponding sgRNA.
  • the target binding region of the sgRNA comprises the complement of the 3' splice site of Stat3 intron 22.
  • the target binding region of an sgRNA suitable for use in this method is set forth in SEQ ID NO:3.
  • the target binding region of the sgRNA comprises the complement of the 5' or 3' splice site of PKM intron 10.
  • the target binding region of an sgRNA suitable for use in this method is set forth in SEQ ID NO: 15 or 16.
  • a method of treating Duchenne myasthenia caused by a mutation in a DMD gene comprising administering to a subject in need thereof a therapeutically effective amount of a vector expressing a fusion protein described herein and a corresponding A step of an expression vector for sgRNA, wherein the target binding region of the sgRNA comprises the complement of the 5' splice site of DMD exon 50.
  • the target binding region of an sgRNA suitable for use in this method is set forth in SEQ ID NO: 17 or 51.
  • the amino acid sequence of a fusion protein suitable for use in this method can be as set forth in SEQ ID NO: 23 or 50.
  • the route of administration of gene therapy includes the ex vivo pathway and the in vivo pathway.
  • an expression vector expressing a fusion protein described herein and a vector expressing sgRNA or gDNA can be constructed using a suitable backbone vector (e.g., an adeno-associated viral vector), which is administered to a patient, e.g., by injection, in a general manner.
  • a suitable backbone vector e.g., an adeno-associated viral vector
  • blood cells having a genetic variation in the subject may be obtained and treated in vitro by the method described herein, such that the cells are eliminated from the mutation and expanded in vitro, and then returned to the subject.
  • the pluripotent stem cells of the subject can be engineered using the methods described herein and returned to the patient for therapeutic purposes.
  • the fusion protein, coding sequence and/or expression vector thereof, and/or sgRNA and/or expression vector thereof, in the preparation of a reagent or kit for regulating RNA splicing in the preparation of a reagent or kit for regulating RNA splicing.
  • a fusion protein of any of the embodiments herein for use in the regulation of RNA splicing, gene therapy, particularly for treating a disease caused by a genetic mutation or a tumor that benefits from a change in the proportion of different splice isoforms of a functional protein, Its coding sequence and/or expression vector, as well as sgRNA and/or its expression vector.
  • exon skipping can be efficiently induced (eg, RPS24 exon 5, CD45 exon 5, DMD gene exons 50, 23, 51, etc.), regulating the selection of mutually exclusive exons (PKM1/PKM2, etc.), induction of intron retention/inclusion (BAP1 and TP53, etc.) and induction of alternative splice sites (STAT3 ⁇ / ⁇ , etc.) and the like.
  • PAM1/PKM2 regulating the selection of mutually exclusive exons
  • BAP1 and TP53, etc. induction of intron retention/inclusion
  • STAT3 ⁇ / ⁇ , etc. alternative splice sites
  • the content of selective exons can be promoted by mutating C upstream of the 3' splice site to T (RPS24 exon 5, GANAB exon 5, ThyN1 exon 6, OS9 exon 13 and SMN2 exon 7).
  • the method disclosed herein is a universal RNA splicing regulation method, which can be used for disease treatment, especially for gene therapy of the following diseases: Duchenne myasthenia caused by DMD gene mutation, SMN, ⁇ hemoglobin IVS2 647G> A mutation caused by thalassemia, premature aging caused by LMNA mutation, familial hypercholesterolemia.
  • the methods described herein can also regulate the ratio of different splicing subtypes, including but not limited to the induction of Stat3 ⁇ to Stat3 ⁇ , PKM2 to PKM1, MDM4 exon 6 hopping, Bcl2 alternative splicing site selection, LRP8 exon eight jumps, etc., thereby achieving treatment of diseases such as tumors.
  • Plasmids expressing the AIDX-Cas9 or Cas9-AIDX fusion proteins required for expression herein are constructed according to the methods disclosed in the Examples, which are incorporated herein by reference in its entirety.
  • the expression plasmid, ie, MO91-AIDX-XTEN-nCas9-Ugi, which expresses SEQ ID NO, was constructed using the AIDX-nCas9-Ugi fusion protein with reference to the methods of Examples 1-3 and 14 of CN 201710451424.3.
  • the 1654-1657 position is the linker sequence
  • the 1571-1653 is the Ugi sequence
  • the 1658-1664 position is the amino acid sequence of SV40NLS.
  • the coding sequence of the fusion protein is shown in SEQ ID NO:22.
  • sgRNA was cloned into pLX (Addgene) to obtain pLX sgRNA.
  • the following four primers are required, wherein R1 and F2 are sgRNA specific:
  • F1 AAACTCGAGTGTACAAAAAAGCAGGCTTTAAAG (SEQ ID NO: 18)
  • R1 rc(GN 19 )GGTGTTTCGTCCTTTCC (SEQ ID NO: 19)
  • R2 AAAGCTAGCTAATGCCAACTTTGTACAAGAAAGCTG (SEQ ID NO: 21)
  • GN 19 new target binding sequence
  • rc (GN 19 ) reverse complement of the new target binding sequence
  • Transfection was performed when 293T cells reached confluency of 70-90%.
  • first prepare a plasmid DNA-liposome complex including four times the amount 2000 reagent diluted in In the medium, the plasmid expressing the fusion protein described herein and the corresponding sgRNA plasmid are respectively diluted in In the medium, the diluted plasmid is then separately added to the diluted Incubate for 30 minutes in 2000 reagents (1:1). This plasmid DNA-liposome complex was then transfected into 293T cells.
  • the reporter cells constructed according to Example 4 of CN 201710451424.3 were transfected with the plasmid DNA-liposome complex alone, and the puromycin 2ug/ml and blasticidin 20ug/ml were screened for 3d, respectively. Gene expression, splicing and mutation were analyzed by high throughput sequencing on day 7 post transfection.
  • RPS24 is a constituent protein of ribosomes that causes mutations in congenital aplastic anemia. Exon 5 of RPS24 can be alternatively spliced to produce two subtypes with different 3'UTRs, wherein hepatoma cells are more likely to express subtypes containing exon 5, but their physiological functions are not clear.
  • This experiment uses the TAM technique to design the sgRNA (RPS24-E5-5'SS, whose target binding region has the sequence shown in SEQ ID NO: 9) to cleave the 5' splice site or 3' splice site of exon 5 of RPS24.
  • the G mutation is A, which regulates its alternative splicing process.
  • 293T cells were transfected as described above and analyzed for gene expression, splicing and mutation by high throughput sequencing on day 7 post-transfection.
  • the fusion protein was targeted to the 5' splice site of exon 5 of RPS24 using the UNG inhibitors UGI and sgRNA in the AIDX-nCas9-Ugi fusion protein.
  • CD45 is a receptor tyrosine phosphatase that regulates the development and function of T lymphocytes and B lymphocytes by regulating the signaling of antigen receptors such as TCR or BCR.
  • the CD45 gene consists of approximately 33 exons, wherein exons 4, 5, and 6 encoding the extracellular A, B, and C regions of the CD45 protein, respectively, can be alternatively spliced.
  • the expression pattern of the CD45 subtype depends on the developmental stage of T cells and B cells, and the longest CD45 subtype (B220) containing three selective exons is expressed on the surface of B cells.
  • sgRNA CD45-E5-5'SS and CD45-E5-3'SS for the 5' splice site and 3' splice site of CD45 gene exon 5, the sequence of the target binding region is SEQ ID NO: 1 and 2)
  • editing of exon 5 splice sites was performed in a germinal center B cell line Raji cell expressing an unspliced CD45 subtype.
  • 400 ng of AIDx-nCas9-Ugi expression plasmid, 300 ng of sgRNA expression plasmid and 50 ng of Ugi expression plasmid were electrotransfected to 1 ⁇ 10 5 Raji cells with Neon (Life Technologies) with a voltage of 1,100 V and a pulse of 40 ms. in. 24 h after transfection, 2 ⁇ g/ml puromycin was added to select for transfected cells for 3 days.
  • This experiment uses TAM technology to design sgRNA (TP53-E8-5'SS, whose sequence is shown as SEQ ID NO: 7) to mutate the G of the 3' splice site of exon 8 of TP53 to A, and to regulate its selectivity.
  • the splicing process ( Figure 4). 293T cells were transfected as described above and analyzed for gene expression, splicing and mutation by high throughput sequencing on day 7 post-transfection.
  • the 3'SS mutation caused the exon to jump in 34% of the total transcript and activates the concealed splice site in 23.6% of the mRNA.
  • TAM treated cells also activated the neuroexon (4.3% of the total transcript) in intron 8 (Fig. 4, D-F).
  • RNA may also undergo alternative splice site selection during splicing and will form new protein subtypes with different physiological functions. For example, selection of an alternative splice site on exon 23 of Stat3 results in a truncated STAT3 ⁇ subtype lacking the C-terminal transactivation domain.
  • Full-length STAT3 ⁇ can promote tumorigenesis, while STAT3 ⁇ can exert dominant inactivation, inhibit STAT3 ⁇ function, and promote tumor cell apoptosis.
  • induction of STAT3 ⁇ expression can inhibit cell survival more effectively than knockout STAT3 global expression, indicating that induction of STAT3 ⁇ expression can be used as a tumor treatment strategy.
  • 293T cells used AIDx-nCas9-Ugi and targeted Stat3 exon 23 (STAT3-E23-3'SS, the sequence of its target binding region is shown in SEQ ID NO: 3) or sgRNA (Ctrl) against AAVS1.
  • Intron-exon junctions were amplified from DNA (top 2 panels) or cDNA (bottom 2 panels) and analyzed by high throughput sequencing.
  • TAM and sgRNA were expressed in 293T cells by the above method, and G in which more than 50% of the 3' splice sites were mutated to A (Fig. 5, B). The results show that TAM enhances the utilization of distal 3'SS in Stat3 exon 23 ( Figure 5, C).
  • TAM technology allows us to overcome the shortcomings of conventional double sgRNA splicing methods, accurately disrupt alternative splice sites, and regulate the selection of alternative splice sites.
  • PKM Pyruvate kinase
  • Exons 9 and 10 of PKM can be selectively involved in the production of two subtypes, PKM1 and PKM2, of which PKM1 contains exon 9, Exon 10 is not included, mainly expressed in adult tissues, while PKM2 contains exon 10 and does not contain exon 9, and is mainly expressed in embryonic stem cells as well as tumor cells. Because PKM2 is involved in tumorigenesis, we hope to use TAM technology to switch the PKM splicing pattern of tumor cells from PKM2 to PKM1.
  • FIG. 6 (A) shows a schematic diagram of TAM transferring PKM2 to PKM1 in C2C12 cells.
  • exon 10 of the PKM gene but exon 9 is spliced to generate PKM2, whose cDNA is recognized by the restriction enzyme PstI; in the figure below, TAM will be the GT dinucleus at the 5'SS of exon 10 The glycoside is converted to AT.
  • exon 9 but exon 10 is spliced to produce PKM1, the cDNA of which is recognized by the restriction enzyme NcoI.
  • sgRNA PKM-3'SS-E10 or PKM-5'SS-E10 for the 3' or 5' splice site of intron 10, the sequence of its target binding region is SEQ ID NO: 15 or 16, respectively. Shown), C2C12 cells were infected to mutate G to A (Fig. 6, C, D). We found that PKM2 expression was down-regulated and PKM1 expression was up-regulated in muscle cells differentiated from C2C12 (Fig. 6, B, E, F). Similarly, in undifferentiated C2C12 cells, PKM2 expression was significantly down-regulated and PKM1 expression levels were up-regulated (Fig. 6, G, H).
  • the sgRNA targeting the 5' or 3' splice site of intron 9 (PKM-3'SS-E9, PKM-5'SS-E9, the sequence of its target binding region is SEQ ID NO: 13 or 14, respectively.
  • PKM-3'SS-E9, PKM-5'SS-E9, the sequence of its target binding region is SEQ ID NO: 13 or 14, respectively.
  • G As shown, it is possible to mutate G to A and down-regulate PKM1 expression (Fig. 7), while up-regulation of PKM2 further indicates that mutations at the splice site can alter the selection of splice sites for mutually exclusive exons.
  • Intron inclusion is another form of alternative splicing, and recent studies have demonstrated that inclusion of introns occurs in many human diseases, including tumors. We demonstrate that the use of TAM and sgRNA to disrupt the splice sites of the corresponding introns can specifically induce inclusion of introns.
  • BAP1 is a histone deubiquitinating enzyme whose second intron remains in some tumors, resulting in decreased expression of BAP1.
  • the second intron of BAP1 may be spliced by intron definition, where the 5'SS is paired with the downstream 3'SS. Converting G to A, U1 identifies the U1RNP at 5'SS and destroys the intron definition, resulting in the inclusion of the intron. This experiment directs TAM to mutate G at the 5' splice site of BAP1 intron 2, as shown in Figure 8(A).
  • sgRNA targeting the 5' splice site of intron 2 (BAP1-E2-5'SS, the sequence of its target binding region is shown in SEQ ID NO: 5), using the expression plasmid of AIDx-nCas9-Ugi and 293T cells were transfected with AAVS1 (Ctrl) or an expression plasmid for the sgRNA of BAP1 intron 2. Seven days after transfection, splicing of BAP1 mRNA was analyzed by RT-PCR (Fig. 8, B) or isotype-specific real-time PCR (Fig. 8, C). The results showed that more than 70% of the G mutations were A (Fig. 8, D).
  • intron 2 after induction is induced, and more than 60% of BAP1 mRNA contains intron 2; similarly, the 3' splice site of mutant intron 2 (sgRNA sequence is SEQ ID NO: 6 (BAP1) After -E3-3'SS)), the intron inclusion of BAP1 was also induced (Fig. 9, BE).
  • the expression plasmid shown in 10 was transfected into 293T cells. Six days after transfection, the sgRNA targeting region was amplified from genomic DNA and analyzed by high throughput sequencing over 8000x coverage. The results showed that more than 50% of the C mutations in the polypyrimidine chain were T. We found an increase in the inclusion rate of exon 5 (Fig. 11, B, C). We then sorted out two single-cell clones containing the complete C to T mutation, which increased the inclusion rate of exon 5 by 8-fold and 5-fold, respectively (Fig. 11, E).
  • the expression plasmid shown is transfected into 293T cells. Six days after transfection, the sgRNA targeting region was amplified from genomic DNA and analyzed by high throughput sequencing over 8000x coverage. The results are shown in Figure 10 (BE), in which multiple Cs were induced to mutate to T, the highest was IVS5-6C, and more than 70% of the C mutations were T, while high-throughput sequencing demonstrated increased inclusion of exon 6. 50%.
  • BE Figure 10
  • ThyN1 exon 6 sequence of the sgRNA target binding region as shown in SEQ ID NO: 12, THYN1-E6-PPT
  • Figure 10, FG sequence of the sgRNA target binding region as shown in SEQ ID NO: 12, THYN1-E6-PPT
  • OS9 exon 13 sgRNA
  • TAM technology can restore DMD protein expression (C2C12 and iPS) in human iPS cells and mdx mouse models.
  • Duchenne muscular dystrophy is a muscle wasting disease with one case per 4,000 men in the United States.
  • the skeletal muscle is resistant to dystrophin due to hereditary mutations in the patient's DMD gene resulting in altered open reading frames or the formation of immature codons, leading to disease.
  • the truncated anti-muscle atrophin is capable of exerting some functions, resulting in a milder onset of Becker muscular atrophy. Therefore, studies have been made to use the antisense oligonucleotides or to double-sgRNA-mediated CRISPR technology to skip partial exons to restore the open reading frame of DMD and promote the expression of anti-dystrophin.
  • AIDx-saCas9 (KKH, cleavage enzyme)-Ugi (the coding sequence is shown in SEQ ID NO: 49, the amino acid sequence is shown as SEQ ID NO: 50) and the corresponding sgRNA sequence (the sequence is SEQ) ID NO: 51, the backbone sequence of which is set forth in SEQ ID NO: 52, induces exon skipping of DMD No. 50.
  • control sgRNA (ctrl) or the target sgRNA (E50-5'SS) is combined with AIDx-saCas9 (KKH, cleavage enzyme)-Ugi to treat iPSC cells of patients with Duchenne muscle weakness, and the corresponding DNA is amplified by PCR.
  • AIDx-saCas9 cleavage enzyme

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Abstract

提供了一种通过诱导剪接位点碱基突变或多聚嘧啶区碱基置换调控RNA剪接的方法，所述方法包括在细胞中表达靶向性的胞嘧啶脱氨酶，以诱导该细胞中感兴趣基因的感兴趣内含子的3'剪接位点AG突变为AA，或感兴趣基因的感兴趣内含子的5'剪接位点GT突变为AT，或感兴趣基因的感兴趣内含子的多聚嘧啶区的多个C分别突变为T。利用所述方法可特异性阻断外显子识别过程，调控内源性mRNA的选择性剪接过程，可诱导外显子跳读、激活替代剪接位点、诱导互斥外显子转换、诱导内含子包含及增强外显子。

Description

通过诱导剪接位点碱基突变或多聚嘧啶区碱基置换调控RNA剪接的方法 技术领域
本发明涉及通过诱导剪接位点碱基突变或多聚嘧啶区碱基置换调控RNA剪接的方法。
背景技术
真核生物基因的正确表达需要将前体mRNA中的内含子剪接，同时将外显子拼接形成成熟mRNA。而超过98％的内含子是被一种高度动态的蛋白复合体——剪接体切除的。剪接体由超过150种小核核糖核蛋白(snRNPs)组成，如U1，U2，U4，U5和U6。在剪接过程中，U1snRNP识别内含子5’剪接位点的GU序列，剪接因子1(SF1)结合内含子的分叉点，而U2辅助蛋白(U2AF)的35KD亚基结合内含子的3’剪接位点的AG序列，65KD亚基结合在多聚嘧啶区序列，完成外显子识别过程；然后U5和U6蛋白通过调控RNA结构重构以及RNA与蛋白相互作用，催化内含子的去除过程。RNA剪接过程对基因表达调控发挥着重要作用，有研究发现15％的可遗传人类疾病是由于前体mRNA加工异常导致的，因此RNA剪接过程可以作为这些疾病的可能治疗靶点，例如利用反义寡聚核苷酸(ASO)调控疾病相关基因的RNA剪接对杜氏肌肉萎缩症和脊髓性肌萎缩症都有一定的缓解作用。
除了内含子剪接外，75％人类基因在表达过程中会发生选择性RNA剪接，极大地提高了人类蛋白组的丰富性。但是现在仍缺乏便捷有效的方法调控选择性剪接过程，大多数选择性剪接蛋白亚型的功能并不清楚。
反义寡聚核苷酸可以结合在RNA的顺式作用元件(如外显子剪接增强子)上阻断外显子的剪接，但是利用反义寡聚核苷酸进行剪接调控需要谨慎的设计以及严格的筛选，在疾病治疗过程中需要持续给药，其合成过程也非常昂贵，十分耗费时间与金钱。因此对这类疾病急需一种能够一次性治愈的治疗策略。
发明内容
本文提供一种调控细胞中感兴趣基因的RNA剪接的方法，其特征在于，所述方法包括在所述细胞中表达靶向性的胞嘧啶脱氨酶，以诱导该细胞中感兴趣基因的感兴趣内含子的3’剪接位点AG突变为AA，或感兴趣基因的感兴趣内含子的5’剪接位点GT突变为AT，或感兴趣基因的感兴趣内含子的多聚嘧啶区的多个C分别突变为T。
在一个或多个实施方案中，用于本文所述的方法的靶向性胞嘧啶脱氨酶可选自：
(1)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas酶的融合蛋白；
(2)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与特异识别靶向序列的TALEN蛋白的融合蛋白；
(3)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与特异识别靶向序列的锌指蛋白的融合蛋白；
(4)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与核酸酶活性部分或完全缺失但保留了解旋酶活性的Cpf酶的融合蛋白；和
(5)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与Ago蛋白的融合蛋白。
在一个或多个实施方案中，所述靶向性胞嘧啶脱氨酶为胞嘧啶脱氨酶、其保留了酶活的片段或突变体与核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas酶的融合蛋白，或者是胞嘧啶脱氨酶、其保留了酶活的片段或突变体与核酸酶活性部分或完全缺失但保留了解旋酶活性的Cpf酶的融合蛋白；所述方法包括在所述细胞中表达所述靶向性胞嘧啶脱氨酶和sgRNA，其中，所述sgRNA为所述Cas酶或Cpf酶所特异性识别，并结合到含有感兴趣基因感兴趣内含子剪接位点的序列或感兴趣多聚嘧啶区的互补序列。
在一个或多个实施方案中，所述靶向性胞嘧啶脱氨酶是胞嘧啶脱氨酶、其保留了酶活的片段或突变体与Ago蛋白的融合蛋白；所述方法包括在所述细胞中表达所述靶向性胞嘧啶脱氨酶和该Ago蛋白识别的gDNA的步骤。
在一个或多个实施方案中，本文提供一种调控细胞中感兴趣基因的RNA剪接的方法，所述方法包括在所述细胞中表达(1)核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas蛋白与胞嘧啶脱氨酶AID或其突变体的融合蛋白和(2)sgRNA的步骤；其中，所述sgRNA的Cas蛋白识别区为所述Cas蛋白所特异性识别，所述sgRNA结合到含有感兴趣基因感兴趣内含子剪接位点的序列或感兴趣多聚嘧啶区的互补序列。
在一个或多个实施方案中，所述sgRNA结合到含有感兴趣基因的感兴趣内含子的5’剪接位点的序列，所述融合蛋白将所述5’剪接位点处的GT突变为AT，从而诱导外显子跳读、激活替代剪接位点、诱导互斥外显子转换或内含子包含。
在一个或多个实施方案中，所述sgRNA结合到含有感兴趣基因的感兴趣内含子的3’剪接位点的序列，所述融合蛋白将所述3’剪接位点的AG突变为AA，从而诱导外显子跳读、激活替代剪接位点、诱导互斥外显子转换或内含子包含。
在一个或多个实施方案中，所述sgRNA结合到感兴趣多聚嘧啶区的互补链，诱导多聚嘧啶区的C突变为T，从而增强外显子包含。
在一个或多个实施方案中，通过将所述融合蛋白和sgRNA的表达载体转入所述细胞，从而调控该细胞中感兴趣基因的RNA剪接。
在一个或多个实施方案中，所述方法还包括，同时转入Ugi的表达质粒的步骤。
在一个或多个实施方案中，所述方法还包括，同时转入核酸酶缺陷或部分缺陷Cas9蛋白、AID或其突变体以及Ugi的融合蛋白的表达质粒的步骤。
在一个或多个实施方案中，所述融合蛋白和AID、其片段或其突变体如本文任意部分或任一实施方案所述。
在一个或多个实施方案中，所述感兴趣的细胞和感兴趣的基因如本文任意部分或任一实施方案所述。
在某些实施方案中，本文提供一种诱导外显子跳读的方法，所述方法包括在感兴趣细胞中表达(1)核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas蛋白与胞嘧啶脱氨酶AID或其突变体和任选的Ugi的融合蛋白的融合蛋白和(2)sgRNA的步骤；其中，所述sgRNA的Cas蛋白识别区为所述Cas蛋白所特异性识别，所述sgRNA结合到含有感兴趣基因感兴趣内含子剪接位点的序列。
在某些实施方案中，本文提供一种激活替代剪接位点的方法，所述方法包括在感兴趣细胞中表达(1)核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas蛋白与胞嘧啶脱氨酶AID或其突变体和任选的Ugi的融合蛋白的融合蛋白和(2)sgRNA的步骤；其中，所述sgRNA的Cas蛋白识别区为所述Cas蛋白所特异性识别，所述sgRNA结合到含有具有感兴趣基因感兴趣内含子剪接位点的序列，其中，所述感兴趣内含子附近具有替代剪接位点。
在某些实施方案中，本文还提供一种诱导互斥外显子转换的方法，所述方法包括在感兴趣细胞中表达(1)核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas蛋白和胞嘧啶脱氨酶AID或其突变体和任选的Ugi的融合蛋白和(2)sgRNA的步骤；其中，所述sgRNA的Cas蛋白识别区为所述Cas蛋白所特异性识别，所述sgRNA的靶标结合区含有感兴趣基因感兴趣内含子剪接位点的序列；其中，所述感兴趣基因选自PKM。
在某些实施方案中，本文还提供一种诱导内含子包含的方法，所述方法包括在感兴趣细胞中表达(1)核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas蛋白与胞嘧啶脱氨酶AID或其突变体和任选的Ugi的融合蛋白的融合蛋白和(2)sgRNA的步骤；其中，所述sgRNA的Cas蛋白识别区为所述Cas蛋白所特异性识别，所述sgRNA含有感兴趣内含子的剪切位点；其中，所述感兴趣的内含子长度较短(<150bp),并且富含G/C碱基。
在某些实施方案中，本文还提供一种增强外显子包含的方法，所述方法包括在感兴趣细胞中表达(1)核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas蛋白、胞嘧啶脱 氨酶AID或其突变体和Ugi的融合蛋白和(2)sgRNA的步骤；其中，所述sgRNA的Cas蛋白识别区为所述Cas蛋白所特异性识别，所述sgRNA的含有感兴趣外显子的上游多聚嘧啶区的互补序列。
本文还提供一种融合蛋白，所述融合蛋白含有胞核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas蛋白和胞嘧啶脱氨酶AID或其突变体。
在一个或多个实施方案中，本文的融合蛋白还含有Ugi。
本文还提供用于在细胞中产生点突变，或用于调控细胞中感兴趣基因的RNA剪接，或用于在感兴趣的细胞中诱导外显子跳读、激活替代剪接位点、诱导互斥外显子转换、诱导内含子包含、增强外显子包含的融合蛋白，所述融合蛋白含有胞核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas蛋白和胞嘧啶脱氨酶AID或其突变体，任选还含有接头序列、核定位序列以及Ugi。
本文还包括利用本文所述的调控RNA剪接的方法进行疾病治疗的方法。
本文还提供本文所述的融合蛋白或其表达载体及相应的sgRNA或其表达载体在制备调控RNA剪接的试剂盒中的应用，以及含有本文所述的融合蛋白或其表达及相应的sgRNA或其表达载体的试剂盒。
附图说明
图1：TAM通过将3'剪接位点处不变的鸟嘌呤转化为腺嘌呤来诱导CD45的第5外显子的跳读。(A)利用TAM在CD45RB外显子的3'剪接位点的鸟嘌呤转化为腺嘌呤并引发外显子跳跃的示意图。在WT Raji细胞中，组合式剪接CD45的外显子5以产生最长的CD45亚型(CD45RA +RB +RC +，上图)；TAM将外显子5的3'SS处的AG二核苷酸转化为AA，消除该剪接位点并破坏外显子识别，导致外显子5的跳跃并产生缺少CD45RB的CD45亚型(CD45RA +RC +，下图)。(B，C)TAM引起CD45RB外显子的跳跃。用AIDx-nCas9-Ugi和靶标sgRNA(CD45-E5-3'SS)或针对AAVS1的对照sgRNA(Ctrl)的表达质粒转染Raji细胞。转染后7天，使用外显子特异性抗体(B)通过流式细胞术测定靶向外显子(CD45RB)，其上游外显子(外显子4，CD45RA)，下游外显子(外显子6，CD45RC)和总CD45的表达；或通过外显子特异性实时PCR(C)检测相应外显子的表达。数据是两个独立实验的代表性(B)或总结(C)。**，p<0.01在学生t检验。(D)在CD45RB low细胞中，3'SS处的G>A突变富集。从B中显示的细胞的基因组DNA以及来自TAM处理的细胞的分选的CD45RB hi和CD45RB low细胞扩增内含子-外显子连接。扩增子通过高通量测序分析，超过8000x覆盖。描绘了具有可检测突变(突变体读数/WT读数>0.1％)的每个核苷酸的碱基组成，并标记了突变的Gs的G>A转化百分比。在内含子-外显子连接序列的顶部显示了 sgRNA和PAM序列的位置。虚线描绘内含子/外显子连接。数据是两个独立实验的代表。(E)流式细胞分析CD45RB在对照Raji细胞或分选的CD45RB hi和CD45RB low细胞从TAM处理的细胞表达。(F)TAM诱导CD45RB跳跃而不改变CD45的编码序列。如在D中，从cDNA扩增外显子-内含子结，并通过高通量测序分析碱基取代。注意，与基因组DNA相比，两种外显子突变在TAM处理的细胞的cDNA中是不可检测的。
图2：TAM通过将5'剪接位点的不变鸟嘌呤转化为腺嘌呤来诱导CD45RB外显子的跳读。(A)指导TAM在CD45RB外显子的5'SS处将不变鸟嘌呤转化为腺嘌呤并引发外显子跳跃的示意图。(B，C)TAM引起CD45RB外显子的跳跃。用AIDx-nCas9-Ugi和靶标sgRNA(E5-5'SS)或针对AAVS1的对照sgRNA(Ctrl)的表达质粒转染Raji细胞。转染后7天，通过使用外显子特异性抗体(B)或通过流式细胞术测定靶向外显子(CD45RB)，其上游外显子(外显子4，CD45RA)，下游外显子(外显子6，CD45RC)和总CD45的表达外显子特异性实时PCR(C)。数据是两个独立实验的代表性(B)或总结(C)。**，p<0.01在学生t检验。(D)CD45RB low细胞中CD45RB外显子的5'位点的G>A突变富集。从B中显示的细胞扩增内含子-外显子连接，以及来自TAM处理的Raji细胞的分选的CD45RB hi和CD45RB low细胞。扩增子通过高通量测序分析，超过8000x覆盖。描绘了具有可检测突变(突变体读数/WT读数>0.1％)的每个核苷酸的碱基组成，并且在左侧标记了目标G的G>A转化百分比。在内含子-外显子连接序列的顶部标记sgRNA和PAM序列的位置。虚线描绘内含子/外显子连接。数据是两个独立实验的代表。(E)流式细胞分析CD45RB在对照Raji细胞或分选的CD45RB hi和CD45RB low细胞从TAM处理的细胞表达。(F)TAM诱导CD45RB跳跃与CD45蛋白序列的最小变化。从cDNA扩增外显子-内含子连接点，并通过高通量测序分析碱基取代。注意，与基因组DNA相比，TAM处理的细胞的cDNA中的两种突变突变显着降低。
图3：TAM通过在将5'SS处不变的鸟嘌呤转化为腺嘌呤来促进RPS24外显子5的跳读。(A)TAM将RPS24外显子5的5'剪接位点的腺嘌呤转化为鸟嘌呤。用nCas9-AIDx-Ugi和对照sgRNA(Ctrl)或靶向RPS24外显子5(5')的5'SS的sgRNA的表达质粒转染293T细胞，E5-5’SS)。转染后6天，从基因组DNA(顶部2个小组)或cDNA(底部2个小组)扩增sgRNA靶向区域，并通过超过8000x覆盖的高通量测序进行分析。描绘了具有可检测突变(>0.1％)的核苷酸的碱基组成。来自Refseq的外显子/内含子连接序列的顶部显示了sgRNA和PAM序列的位置。用虚线描绘内含子/外显子连接。数据是两个独立实验的代表。(B)TAM促进RPS24外显子5的跳跃。如在A中，从cDNA扩增剪接连接并通过高通量测序进行分析。图片显示对照sgRNA(上图)或E5-5'SS sgRNA(下图)处理细胞的每个剪接结的覆盖率和百分比。结点读数的计数和百分比(括号中)描绘在每个连接弧的 顶部。为了清楚起见，仅描绘了代表总转录物超过1％的结弧。(C)通过异构体特异性实时PCR测定RPS24同种型与包含或跳过的外显子5之间的比例。数据是三个独立实验的总结。(D，E)5’SS的G至A突变引起RPS24外显子5的完全跳跃。从TAM处理的细胞中获得两个单细胞克隆，并通过Sanger测序进行分析，右侧(D)表示细胞的基因型。通过实时PCR(E)测定包含的外显子5的同种型的表达。数据是三个独立实验的总结。
图4：TAM通过在突变各自的剪接位点的鸟嘌呤，诱导TP53外显子8或外显子9的跳读。(A-C)TAM通过突变其5'SS引起TP53外显子8的跳跃。(A)如图1所示，用nCas9-AIDx-Ugi和靶向AAVS1(Ctrl)的对照sgRNA或靶向TP53外显子8(E8-5'SS)的5'SS的sgRNA的表达质粒转染293T细胞。转染后6天，从基因组DNA(顶部2个小组)或cDNA(底部2个小组)扩增sgRNA靶向区域，并通过高通量测序进行分析。描绘了具有可检测突变(>0.1％)的核苷酸的碱基组成。来自Refseq的外显子/内含子连接序列的顶部显示了sgRNA和PAM序列的位置。用虚线描绘内含子/外显子连接。数据是两个独立实验的代表。(B)通过RT-PCR分析TP53外显子8的剪接。(C)如在A中，从cDNA扩增剪接连接并通过高通量测序进行分析。图片显示了对照sgRNA(上图)或E8-5'SS sgRNA(下图)处理的细胞的每个剪接结的覆盖率和百分比。为了清楚起见，仅描绘了代表总转录物超过1％的结弧。结点读数的计数和百分比(括号中)描绘在每个连接弧的顶部。注意，在TAM处理的细胞中，总转录物的42.1％跳过了外显子8，而1.1％激活了外显子8内的隐蔽剪接位点。(D-F)TAM通过突变其3'SS引起TP53外显子9的跳跃。(D)如(A)所示，用TAM和靶向TP53外显子9的3'SS的sgRNA转染293T细胞。转染7天后，从基因组DNA扩增内含子-外显子连接并通过高通量测序进行分析。(E)通过RT-PCR分析TP53的剪接。(F)如在D中，从cDNA扩增剪接连接并通过高通量测序进行分析。描绘了占总誊本超过1％的交点。注意，3’SS突变导致外显子在总转录物的34％中跳跃，并在23.6％的mRNA中激活隐蔽剪接位点。TAM处理的细胞也激活了内含子8内的神经外显子(总转录物的4.3％)。(A-F)数据代表两个独立实验。
图5：TAM激活替代剪接位点并将Stat3α转化为Stat3β。(A)利用TAM消除Stat3外显子23(Stat3α)的典型3'SS并促进下游替代3'SS(Stat3β)的利用的示意图。(B)TAM在Stat3外显子23的典型3'SS处突变不变G。如图1所示，293T细胞用AIDx-nCas9-Ugi和靶向Stat3外显子23(E23-3'SS-)或针对AAVS1的sgRNA(Ctrl)的表达质粒转染。从DNA(顶部2个小组)或cDNA(底部2个小组)扩增内含子-外显子连接，并通过高通量测序进行分析。描绘了具有可检测突变(>0.1％)的核苷酸的碱基组成。注意，TAM也在外显子23中诱导了两个突变，其比cDNA(54％和16％)的cDNA(26％和6％)少得多。数据是两个独立实验的代表。(C)TAM在Stat3外显子23增强了远端3'SS的利用。 从cDNA扩增剪接连接并通过高通量测序进行分析。图片显示了对照sgRNA(上图)或E23-3'SS sgRNA(下图)处理细胞的每个剪接结的覆盖率和百分比。描绘了占总誊本超过1％的交点。连接读数的计数和百分比(括号中)描绘在每个连接弧的顶部。注意仅在用Stat3-E23-3'SS处理的细胞中，sgRNA是在～10％转录物中活化的隐蔽剪接位点。数据是两个独立实验的代表。(E-F)TAM将Stat3α转化为Stat3β。通过RT-PCR(D)，同种型特异性实时荧光定量PCR(E)检测Stat3α和Stat3β在TAM处理细胞中的表达，并测定Stat3α与Stat3β之间的比例(F)。
图6：TAM通过消除外显子10的5'SS或3’SS将PKM2切换到PKM1。(A)显示TAM在C2C12细胞中将PKM2转移到PKM1的示意图。顶板，在WT C2C12细胞中，外显子10，但不是PKM基因的外显子9被剪接以产生PKM2，其cDNA被限制酶PstI识别；底部，TAM将外显子10的5'SS处的GT二核苷酸转化为AT(或3’SS的AG转化为AA)。因此，外显子9而不是外显子10被剪接以产生PKM1，其cDNA被限制酶NcoI识别。(B)TAM增加PKM1，同时抑制PKM2表达。用TAM和目标sgRNA(PKM-E10-5'SS或PKM-E10-3’SS)或对照sgRNA(Ctrl)转染C2C12细胞。转染后7天，将细胞向肌肉细胞分化，而后从cDNA扩增PKM，扩增子用PstI或NcoI消化。指出了对应于PKM1或PKM2的片段，并且包括GAPDH和总PKM(外显子5和外显子6的扩增子)作为载体对照。(C，D)TAM在PKM外显子10的3'SS(C)或5’SS(D)处将不变G转换为A。从基因组DNA(顶部两个图)或cDNA(底部两个图)扩增内含子-外显子连接，并通过高通量测序进行分析。描述了每种鸟嘌呤的碱组成和A的百分比。数据是两个独立实验的代表。(E)PKM1与PKM2比值的实时PCR分析。数据是代表性的(B，D，E)或两个独立实验(C)的总结。(F)TAM将PKM2移至PKM1。如在C中，从cDNA扩增剪接连接并通过高通量测序进行分析。图片显示了对照sgRNA(上图)或E10-5'SS sgRNA(下图)处理细胞的每个剪接结的覆盖率和百分比。结点读数的计数和百分比(括号中)描绘在每个连接弧的顶部。(G，H)和上面类似，TAM可以在未分化的C2C12细胞中，将PKM2转化为PKM1。
图7：TAM通过消除PKM的第9个外显子的3'SS或5’SS来抑制PKM1的表达。(A)TAM在PKM外显子9的3'SS或5’SS处将不变G转化为A。(B)C2C12细胞分化来的肌肉细胞中，从对照或TAM处理的细胞(E9-3'SS)的基因组DNA，并通过高通量测序进行分析。描绘了突变频率超过0.1％的每个鸟嘌呤的百分比G或A。数据是两个独立实验的代表。注意，TAM在这个位点也引起外显子9内的C>T突变。(C、D、E)TAM抑制PKM1表达同时促进PKM2的表达。(C)从cDNA扩增PKM，扩增子用NcoI消化。指出了对应于PKM1或PKM2的片段，并且包括GAPDH和总PKM(外显子5和外显子6的扩增子)作为载体对照。(D)通过实时PCR测定PKM1和PKM2的表达，计算PKM1和PKM2的比 例。(E)从cDNA扩增剪接连接并通过高通量测序进行分析。图片显示了对照sgRNA(上图)或E9-3'SS sgRNA(下图)处理的细胞的每个剪接结的覆盖率和百分比。结点读数的计数和百分比(括号中)描绘在每个连接弧的顶部。数据是两个独立实验的总结。***，p<0.0001在学生的t检验。(F)如上，C2C12细胞分化来的肌肉细胞中，从对照或TAM处理的细胞(E9-5'SS)的基因组DNA，并通过高通量测序进行分析。描绘了突变频率超过0.1％的每个鸟嘌呤的百分比G或A。数据是两个独立实验的代表。(G)实时定量PCR分析PKM1和PKM2的表达。
图8：TAM在5'SS上将不变G转换为A后，保留了BAP1的第二个内含子。(A)引导TAM在BAP1外显子2的5'剪接位点处突变不变G并示出其保留的示意图。BAP1的第二个内含子可能通过内含子定义方式进行剪接，其中5'SS与下游3'SS配对。将不变量G转换为A，在5'SS处理U1识别U1RNP并破坏内含子定义，导致包含该内含子。(B，C)TAM诱导BAP1内含子2的保留。用AIDx-nCas9-Ugi和针对AAVS1(Ctrl)或针对BAP1(NAP1-E2-5'SS)的外显子2的5'SS的sgRNA的表达质粒转染293T细胞。转染7天后，通过RT-PCR(B)或同种型特异性实时PCR(C)分析BAP1mRNA的剪接。(D)保留内含子含有5'SS G>A突变。从用对照sgRNA(ctrl)或目标sgRNA(E2-5'SS)处理的293T细胞的基因组DNA(顶部两个图)或cDNA(底部两个图)扩增内含子-外显子连接。描绘了具有可检测突变的每个鸟嘌呤的碱基组成。在内含子-外显子连接序列的顶部标记sgRNA和PAM序列的位置，虚线描绘内含子/外显子连接。数据是两个独立实验的代表。注意，因为内含子2在对照细胞中有效剪接，只有接受E2-5'SS sgRNA的细胞具有覆盖内含子的读数，其中99％含有G>A突变。(E)突变5’SS诱导的第二内含子的保留，而不是跳过BAP1的第二外显子。如在D中，从cDNA扩增剪接连接并通过高通量测序进行分析。图片显示了对照sgRNA(上图)或E2-5'SS sgRNA(下图)处理细胞的每个剪接位的覆盖率和百分比。结点读数的计数和百分比(括号中)描绘在每个连接弧的顶部。注意在sgRNA处理的细胞中，2.4％的mRNA被剪接以跳过第二个外显子，而超过60％保留了第二个内含子。数据是两个独立实验的代表性(B，D，E)或总结(C)。
图9：在BAP1的外显子3的3'SS处将不变G转化为A导致其保留。(A)引导TAM突变BAP1外显子3的3'SS处的不变G并引导其保留的示意图。(B，C)TAM诱导BAP1内含子2的保留。用AIDx-nCas9-Ugi和AAVS1(Ctrl)或BAP1的内含子2的3'SS的sgRNA的表达质粒转染293T细胞。转染7天后，通过RT-PCR(B)和同种型特异性实时PCR(C)分析BAP1mRNA的剪接。(D)保留的第二个内含子在3'SS处含有G>A突变。从使用对照sgRNA(Ctrl)处理的293T细胞的基因组DNA(顶部2个小组)或cDNA(底部2个小区)中扩增出5'SS，或者以3'ss(E3-3'SS)为靶标的sgRNA扩增。描绘了具有可检测突变的每 个鸟嘌呤的碱基组成(G>A转化效率超过0.1％)。在内含子-外显子连接序列的顶部显示了sgRNA和PAM序列的位置。虚线描绘了内含子/外显子结。数据是两个独立实验的代表。注意，因为内含子2在Ctrl细胞中有效拼接，只有接受E3-3'SSsgRNA的细胞具有覆盖内含子的读数。(E)TAM主要诱导BAP1的第二外显子的保留。如在D中，从cDNA扩增剪接连接并通过高通量测序进行分析。图片显示了对照sgRNA(上图)或E3-3'SS sgRNA(下图)处理的细胞的每个剪接结的覆盖率和百分比。结点读数的计数和百分比(括号中)描绘在每个连接弧的顶部。注意在sgRNA处理的细胞中，4.7％的mRNA跳过第3外显子，8.7％使用下游隐窝剪接位点，而超过20％保留了第2个内含子。数据是两个独立实验的代表性(B，D，E)或总结(C)。
图10：在GANAB外显子6上游的聚嘧啶簇(PPT)将Cs转化为Ts增强其包含。(A)指导TAM在GANAB外显子6的PPT处将Cs转化为Ts的示意图，以增强3'SS的强度。GANAB外显子6的聚嘧啶多糖包含多个C(左)，并将这些C转化为T(右)，增加这种3'SS的强度(从6.88到10.12)，并增强外显子6的包含。(B)TAM将GnAB外显子6的PPT转化为Ts。用AIDx-nCas9--Ugi和对照sgRNA(Ctrl)或靶向GANAB外显子6(PPT-E6GANAB)的PPT的sgRNA的表达质粒转染293T细胞。转染后6天，从基因组DNA扩增sgRNA靶向区域，并通过超过8000x覆盖的高通量测序进行分析。描绘了具有可检测突变(>0.1％)的核苷酸的碱基组成。在连接序列的顶部显示了sgRNA和PAM序列的位置。用虚线描绘内含子/外显子连接。数据是两个独立实验的代表。(C，D，E)TAM加强了GANAB第六外显子的包含。(C)如在B中，从cDNA扩增剪接连接并通过高通量测序进行分析。图片显示了对照sgRNA(上图)或PPT-E6GANAB sgRNA(下图)处理的细胞的每个剪接结的覆盖率和百分比。结点读数的计数和百分比(括号中)描绘在每个连接弧的顶部。(D，E)通过RT-PCR(D)或同工型特异性实时PCR(E)分析GANAB mRNA的剪接。数据是两个独立实验的代表(C，D)或总结(E)。(F，G)TAM促进ThyN1的第六外显子的包含。(H，I)TAM增强了OS9的第13个外显子的包含。
图11：在RPS24外显子5上游的聚嘧啶(PPT)转化C至T增强其包含。(A)TAM在RPS24外显子5的PPT处将C转化为T。293T细胞用AIDx-nCas9-Ugi和AAVS1(Ctrl)或针对RPS24(PPT-E5RPS25)第5外显子的聚嘧啶核苷的sgRNA的表达质粒转染。转染后6天，从基因组DNA扩增sgRNA靶向区域，并通过超过8000x覆盖的高通量测序进行分析。描绘了具有可检测突变(>0.1％)的每个胞嘧啶的百分比，数据是两个独立实验的代表。(B，C)如A中，TAM增强了RPS24的第5外显子的包含。通过从cDNA(B)或同种型特异性实时PCR(C)扩增的连接的高通量测序来分析RPS24mRNA的剪接。(D，E)PPT的C至T转化增加了RPS24的外显子6的含量。从TAM处理的细胞衍生出两个单细胞克 隆，并通过Sanger测序(D)进行分析，右侧表示克隆细胞的基因型。(E)通过同种型特异性实时PCR测定RPS24的外显子6的含量。数据是两个独立实验的代表性(A，B，D)或总结(C，E)。
图12：利用TAM在杜氏肌无力病人细胞中诱导外显子跳读，修复DMD基因的读码框，恢复抗肌萎缩蛋白(DMD)的表达。(A)指导TAM在DMD外显子50的5’SS处将G转化为A,恢复病人细胞中抗肌萎缩蛋白的表达的示意图。和正常细胞相比(上图)，此病人因为遗传突变缺失外显子51，破坏了抗肌萎缩蛋白的读码框，造成此蛋白的完全缺失(中图)；利用TAM突变外显子50的5’SS的GU为AU后，在病人细胞中外显子50跳读，从而恢复抗肌萎缩蛋白的读码框和蛋白表达。(B)用对照sgRNA(ctrl)或目标sgRNA(E50-5'SS)处理杜氏肌无力病人的iPSC细胞后，利用PCR扩增相应的DNA，进行高通量测序分析诱导的突变。数据是两个独立实验的代表。(C，D)分别将正常人来源的iPSCs,病人来源的iPSCs和修复后病人来源的iPSCs向心肌细胞分化，而后分别利用RT-PCR(C)或western blot(D)检测DMD基因的表达。(E)修复后的细胞精确发生49和52号外显子的剪接。
图13：利用TAM技术调控RNA剪接的示意图。利用TAM技术将内含子5’剪接位点处的GT突变为AT，可诱导外显子跳读、激活替代剪接位点、诱导互斥外显子转换或内含子包含；将所述内含子3’剪接位点的AG突变为AA，也可诱导外显子跳读、激活替代剪接位点、诱导互斥外显子转换或内含子包含；诱导内含子3’端的多聚嘧啶区中的C突变为T，可增强弱剪接位点，从而增强外显子包含。
图14：利用TAM在杜氏肌无力病人细胞中诱导外显子跳读，修复DMD基因的读码框，恢复抗肌萎缩蛋白(DMD)的表达。
具体实施方式
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
本文通过在细胞中产生点突变，尤其是将细胞中感兴趣基因的感兴趣内含子的3’剪接位点AG突变为AA，或感兴趣基因的感兴趣内含子的5’剪接位点GT突变为AT，或感兴趣基因的感兴趣内含子的多聚嘧啶区的多个C(例如2－10个)分别突变为T，从而调控该细胞中感兴趣基因的RNA剪接，以诱导外显子跳读、激活替代剪接位点、诱导互斥外显子转换、诱导内含子包含或增强外显子包含。“调控”在本文中意指改变RNA的常规的剪接方式。
可利用靶向性的胞嘧啶脱氨酶来实施本发明。本文中，通过将胞嘧啶脱氨酶与具有靶向作用的蛋白融合，构建本文的靶向性胞嘧啶脱氨酶。
本文中，胞嘧啶脱氨酶指具有胞嘧啶脱氨酶活性的各种酶，包括但不限于APOBEC家族的酶，如APOBEC-2、AID、APOBEC-3A、APOBEC-3B、APOBEC-3C、APOBEC-3DE、APOBEC-3G、APOBEC-3F、APOBEC-3H、APOBEC4、APOBEC1和pmCDA1。适用于本文的胞嘧啶脱氨酶可以来自任何物种，尤其优选是哺乳动物尤其是人的胞嘧啶脱氨酶。优选的是，适用于本文的胞嘧啶脱氨酶是激活型胞嘧啶脱氨酶，如人源激活型胞嘧啶脱氨酶。APOBEC家族的胞嘧啶脱氨酶是一种RNA编辑酶家族，N端有核定位信号，C端有核输出信号，其催化结构域为APOBEC家族所共有。一般认为N端结构为体细胞超变(SHM)所必须。胞嘧啶脱氨酶的功能是对胞嘧啶脱氨基，将胞嘧啶变成尿嘧啶，随后的DNA修复可以将尿嘧啶变成其它碱基。应理解的是，本领域周知的胞嘧啶脱氨酶或其保留了对胞嘧啶脱氨基、将胞嘧啶变成尿嘧啶的生物学活性的片段或突变体均可用于本文。
在某些实施方案中，本文使用AID作为靶向性胞嘧啶脱氨酶中的胞嘧啶脱氨酶。AID的氨基酸残基9－26为核定位(NLS)结构域，尤其是氨基酸残基13－26参与了DNA的结合，氨基酸残基56－94为催化结构域，氨基酸残基109－182为APOBEC样结构域，氨基酸残基193－198为核输出(NES)结构域，氨基酸残基39－42与连环蛋白样蛋白1(CTNNBL1)相互作用，氨基酸113－123残基是hotspot识别环。
本文可使用AID的全长序列(如SEQ ID NO:25第1457－1654位氨基酸所示)，也可使用AID的片段。优选的是，所述片段至少包括NLS结构域、催化结构域和APOBEC样结构域。因此，在某些实施方案中，所述片段至少包含AID第9－182位氨基酸残基(即SEQ ID NO:25第1465－1638位氨基酸残基)。在其他实施方案中，所述片段至少包含AID第1－182位氨基酸残基(即SEQ ID NO:25第1457－1638位氨基酸残基)。例如，在某些实施方案中，本文使用的AID片段由第1－182位氨基酸残基组成，由第1－186位氨基酸残基组成，或由第1－190位氨基酸残基组成。因此，在某些实施方案中，本文使用的AID片段由SEQ ID NO:25第1457－1638位氨基酸残基、SEQ ID NO:25第1457－1642位氨基酸残基，或由SEQ ID NO:25第1457－1646位氨基酸残组成。
本文还可使用AID的保留了其胞嘧啶脱氨酶活(即对胞嘧啶脱氨基、将胞嘧啶变成尿嘧啶的生物学活性)的变体。例如，这样的变体相对于AID的野生型序列可具有1－10个，如1－8个，1－5个或1－3个氨基酸变异，包括氨基酸的缺失、取代和突变。优选的是，这些氨基酸变异不发生在上述NLS结构域、催化结构域和APOBEC样结构域内，或即便发生在这些结构域内也不影响到这些结构域原本的生物学功能。例如，优选的是，这些变异不发生在AID氨基酸序列的第24、27、38、56、58、87、90、112、140等位置上。在某些实施方案中，这些变异也不发生在氨基酸39－42、氨基酸113－123之内。因此，例如，变异可发生在氨基酸1－8、氨基酸28－37、氨基酸43－55和/或氨基酸183－198 之中。在某些实施方案中，变异发生在第10、82和156位。例如，在第10、82和156位发生取代突变，这类取代突变可以是K10E、T82I和E156G。在这些实施方案中，示例性的AID突变体的氨基酸序列含有SEQ ID NO:31第1447-1629位所示的氨基酸序列，或由SEQ ID NO:31第1447-1629位所示的氨基酸残基组成。其它AID、其片段或突变体的例子可参见CN 201710451424.3，本文将其全部内容以引入的方式纳入本文。
本文中，具有靶向作用的蛋白可以是本领域周知的能靶向细胞基因组中感兴趣基因的蛋白，包括但不限于特异识别靶向序列的TALEN蛋白、突变识别靶向序列的锌指蛋白、Ago蛋白、Cpf酶以及Cas酶。可利用本领域周知的TALEN蛋白、锌指蛋白、Ago蛋白和Cpf酶和Cas酶来实施本文。
因此，在某些实施方案中，适用于本文的靶向性胞嘧啶脱氨酶可选自：(1)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas酶的融合蛋白；(2)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与特异识别靶向序列的TALEN蛋白的融合蛋白；(3)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与特异识别靶向序列的锌指蛋白的融合蛋白；(4)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与核酸酶活性部分或完全缺失但保留了解旋酶活性的Cpf酶的融合蛋白；和(5)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与Ago蛋白的融合蛋白。
当使用Cpf酶的时候，优选使用核酸酶活性部分或完全缺失但保留了解旋酶活性的Cpf酶。Cpf酶在其识别的sgRNA的指引下，结合到特定的DNA序列，允许与其融合的胞嘧啶脱氨酶实施本文所述的突变。Ago蛋白则需要在其识别的gDNA的指引下结合到特定的DNA序列。
在某些实施方案中，本文利用靶向胞嘧啶脱氨酶AID介导基因突变技术(TAM)将内含子剪接位点的鸟嘌呤突变成腺嘌呤，特异性阻断外显子识别过程，调控内源性mRNA的选择性剪接过程。本文的TAM技术使用一种核酸酶活性缺失的Cas蛋白与胞嘧啶脱氨酶AID、其活性片段或突变体的融合蛋白。在sgRNA的指引下，所述融合蛋白被招募到特定的DNA序列，AID、其活性片段或突变体将鸟嘌呤(G)突变成腺嘌呤(A)，或将胞嘧啶突变(C)成胸腺嘧啶(T)。
CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)是细菌抵御病毒侵袭或躲避哺乳动物免疫反应的基因编辑系统。该系统经过改造和优化，目前已被广泛应用在体外生化反应、细胞与个体的基因编辑中。通常，具有核酸内切酶活性的Cas蛋白(也称为Cas酶)与其特异性识别的sgRNA形成的复合物通过sgRNA的配对区(即靶标结合区)与靶标DNA中的模板链进行互补配对，由Cas在特定位置将双链DNA切断。本文利用Cas/sgRNA的上述特性，即利用sgRNA与靶标的特异性结合而将Cas定位到期望的位置， 在该位置由融合蛋白中的AID、其活性片段或突变体将鸟嘌呤(G)突变成腺嘌呤(A)，或将胞嘧啶突变(C)成胸腺嘧啶(T)。
适用于本文的核酸酶活性部分(仅具有DNA单链断裂能力)或完全缺失(无DNA双链断裂能力)，尤其是核酸内切酶活性部分或完全缺失、但保留了解旋酶活性的Cas蛋白可以是衍生自本领域周知的各种Cas蛋白及其变异体，包括但不限于Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csn1和Csx12)、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4、Cpf1其同源物或其修饰形式。
在一些实施方案中，使用核酸酶活性缺失的Cas9酶和其特异性识别的单链sgRNA。Cas9酶可以是来自不同物种的Cas9酶，包括但不限于来自化脓链球菌的Cas9(SpCas9)、来自金黄色葡萄球菌的Cas9(SaCas9)，以及来自嗜热链球菌的Cas9(St1Cas9)等。可以使用Cas9酶的各种变体，只要该Cas9酶能特异性识别它的sgRNA，并缺失核酸酶活性即可。
可采用本领域周知的方法制备核酸酶活性缺失的Cas蛋白，这些方法包括但不限于使Cas蛋白中核酸内切酶的整个催化结构域缺失或使该结构域中的一个或数个氨基酸发生突变，从而产生核酸酶活性缺失的Cas蛋白。突变可以是一个或数个(例如2个以上、3个以上、4个以上、5个以上、10个以上，至整个催化结构域)氨基酸残基的缺失或取代，或一个或数个新氨基酸残基(例如1个以上、2个以上、3个以上、4个以上、5个以上、10个以上，或者1～10个、1～15个不等)的插入。可采用本领域常规的方法进行上述结构域的缺失或氨基酸残基的突变，以及检测突变后的Cas蛋白是否还具有核酸酶活性。例如，对于Cas9，可将它的两个核酸内切酶催化结构域RuvC1和HNH分别突变，例如将该酶的第10个氨基酸(位于RuvC1结构域中)天冬酰胺突变为丙氨酸或其它氨基酸，将第841位氨基酸(位于HNH结构域中)组氨酸突变为丙氨酸或其它氨基酸。这两处突变使Cas9失去核酸内切酶活性。优选的是，Cas酶完全无核酸酶活性。在一个或多个实施方案中，本文使用的无核酸酶活性的Cas9酶的氨基酸序列如SEQ ID NO:25第42－1452所示。在其他实施方案中，本文使用的Cas酶部分缺失核酸酶活性，即该Cas酶可引起DNA单链断裂。这类Cas酶的代表性例子可如SEQ ID NO:33第42-1419位氨基酸残基所示。在其它实施方案中，本文使用的Cas酶的氨基酸序列如SEQ ID NO:23第199-1566位所示，或如SEQ ID NO:50第199-1262位氨基酸残基所示。其它Cas酶的例子可参见CN201710451424.3，本文将其全部内容以引入的方式纳入本文。
Cas/sgRNA复合物行使功能需要在DNA的非模板链(3’到5’)有前间区序列邻近基 序(protospacer adjacent motif，PAM)。不同Cas酶，其对应的PAM并不完全相同。例如，SpCas9的PAM通常是NGG(SEQ ID NO:34)；SaCas9酶的PAM通常是NNGRR(SEQ ID NO:35)；St1Cas9酶的PAM通常是NNAGAA(SEQ ID NO:36)；其中，N为A、C、T或G，R为G或A。
在某些优选的实施方式中，SaCas9酶的PAM是NNGRRT(SEQ ID NO:37)。在某些优选的实施方式中，SpCas9的PAM是TGG(SEQ ID NO:38)；在某些优选的实施方式中，SaCas9酶KKH突变体的PAM是NNNRRT(SEQ ID NO:39)；其中，N为A、C、T或G，R为G或A。
sgRNA通常包括两部分：靶标结合区和蛋白识别区(如Cas酶识别区或Cpf酶识别区)。靶标结合区与蛋白识别区通常以5’到3’的方向连接。靶标结合区的长度通常为15～25个碱基，更通常为18～22个碱基，如20个碱基。靶标结合区与DNA的模板链特异性结合，从而将融合蛋白招募到预定位点。通常，DNA模板链上sgRNA结合区域的对侧区紧邻PAM，或者隔开数个碱基(例如10个以内，或8个以内，或5个以内)。因此，在设计sgRNA时，通常先根据所用的剪接酶(如Cas酶)确定该酶的PAM，然后在DNA的非模板链上寻找可作为PAM的位点，之后将该非模板链(3’到5’)PAM位点下游紧邻该PAM位点或与该PAM位点隔开10个以内(例如8个以内、5个以内等)的长15～25个碱基、更通常长18～22个碱基的片段作为sgRNA的靶标结合区的序列。sgRNA的蛋白识别区则根据所使用的剪接酶而确定，这为本领域所技术人员所掌握。
因此，本文的sgRNA的靶标结合区的序列为含所选剪接酶(如Cas酶或Cpf酶)识别的PAM位点的DNA链下游紧邻该PAM位点或与该PAM位点隔开10个以内(例如8个以内、5个以内等)的长15～25个碱基、更通常长18～22个碱基的片段；其蛋白识别区为所选剪接酶所特异性识别。
鉴于本文的目的是将内含子剪接位点的鸟嘌呤突变成腺嘌呤，或将3’剪接位点上游的多聚嘧啶链中的C突变为T，因此，在设计用于本文的sgRNA时，需考虑剪接位点附近是否存在PAM序列，以及PAM序列与剪接位点的距离。因此，通常，sgRNA结合到含有感兴趣基因感兴趣内含子剪接位点的序列，或结合到感兴趣多聚嘧啶区的互补序列。或者，sgRNA的靶标结合区含有感兴趣基因感兴趣内含子剪接位点的互补序列，或含有感兴趣基因感兴趣内含子的多聚嘧啶区的序列。
可采用本领域常规的方法制备sgRNA，例如，采用常规的化学合成方法合成。sgRNA也可经由表达载体转入细胞，在细胞内表达出该sgRNA；或利用腺相关病毒导入动物/人类体内。可采用本领域周知的方法构建sgRNA的表达载体。
在某些实施方案中，本文也提供sgRNA序列或其互补序列，其包括靶标结合区和蛋 白识别区，其中，所述靶标结合区结合到含有感兴趣基因感兴趣内含子剪接位点的序列，或结合到感兴趣多聚嘧啶区的互补序列。通常，所述靶标结合区长度为15～25个碱基，如18～22个碱基，优选20个碱基。在某些实施方案中，所述sgRNA的靶标结合区结合到DMD外显子50的含3’剪接位点的序列；优选地，所述sgRNA的靶标结合区如SEQ ID NO:17或51所示。
用于本文的靶向性的胞嘧啶脱氨酶优选是前文所述的Cas酶与前文所述的AID、其片段或突变体的融合蛋白。Cas酶通常在融合蛋白氨基酸序列的N端，AID、其片段或突变体在C端；当然，AID、其片段或突变体也可在融合蛋白氨基酸序列的N端，而Cas酶在C端。在某些实施方案中，本文提供主要由Cas酶和AID、其片段或突变体形成的融合蛋白。应理解的是，本文所述的“主要由……形成”的融合蛋白或类似表述并不意指融合蛋白仅包括Cas酶和AID、其片段或突变体，该限定应理解为融合蛋白可仅包括Cas酶和AID、其片段或突变体，或还可含有其他不影响到该融合蛋白中的Cas酶的靶向作用及AID、其片段或突变体突变靶序列的功能的部分，包括但不限于各种接头序列、核定位序列、Ugi序列以及如下文所述因基因克隆操作、和/或为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的检测和/或纯化等而在融合蛋白中引入的氨基酸序列。
Cas酶可通过接头与AID、其片段或突变体融合。接头可以是3～25个残基的肽，例如3～15、5～15、10～20个残基的肽。肽接头的适合的实例是本领域中公知的。通常，接头含有一个或多个前后重复的基序，该基序通常含有Gly和/或Ser。例如，该基序可以是SGGS(SEQ ID NO:40)、GSSGS(SEQ ID NO:41)、GGGS(SEQ ID NO:42)、GGGGS(SEQ ID NO:43)、SSSSG(SEQ ID NO:44)、GSGSA(SEQ ID NO:45)和GGSGG(SEQ ID NO:46)。优选地，该基序在接头序列中是相邻的，在重复之间没有插入氨基酸残基。接头序列可以包含1、2、3、4或5个重复基序组成。在某些实施方案中，接头序列是多甘氨酸接头序列。接头序列中甘氨酸的数量无特别限制，通常为2～20个，例如2～15、2～10、2～8个。除甘氨酸和丝氨酸来，接头中还可含有其它已知的氨基酸残基，例如丙氨酸(A)、亮氨酸(L)、苏氨酸(T)、谷氨酸(E)、苯丙氨酸(F)、精氨酸(R)、谷氨酰胺(Q)等。在某些实施方案中，接头序列为XTEN，其氨基酸序列如SEQ ID NO:29第183-198位氨基酸残基所示。其它示例性的接头序列可参见CN 201710451424.3中所列出的接头序列，如该申请的SEQ ID NO:21－31等。
应理解，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的氨基酸序列末端引入了一个或多个不相干的残基，而这并不影响目的序列的活性。为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白 的纯化，常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内，例如，包括但不限于，适合的接头肽、信号肽、前导肽、末端延伸等。因此，本文融合蛋白的氨基端或羧基端还可含有一个或多个多肽片段，作为蛋白标签。任何合适的标签都可以用于本文。例如，所述的标签可以是FLAG，HA，HA1，c-Myc，Poly-His，Poly-Arg，Strep-TagII，AU1，EE，T7，4A6，ε，B，gE以及Ty1。这些标签可用于对蛋白进行纯化。
本文的融合蛋白还可含有核定位序列(NLS)。可使用本领域周知的各种来源和各种氨基酸组成的核定位序列。这类核定位序列包括但不限于：SV40病毒大T抗原的NLS；来自核质蛋白的NLS，例如，的核质蛋白二分NLS；来自c-myc的NLS；来自hRNPA1M9的NLS；来自输入蛋白-α的IBB结构域的序列；肌瘤T蛋白的序列；小鼠c-ablIV的序列；流感病毒NS1的序列；肝炎病毒δ抗原的序列；小鼠Mx1蛋白的序列；人聚(ADP-核糖)聚合酶的序列；以及类固醇激素受体(人)糖皮质激素的序列；等等。这些NLS序列的氨基酸序列可参见CN 201710451424.3SEQ ID NO:33-47所列出的序列。在某些具体实施方案中，本文使用SEQ ID NO:25第26－33位氨基酸残基所示的序列作为NLS。NLS可位于融合蛋白的N端、C端；也可位于融合蛋白序列中，例如位于融合蛋白中Cas9酶的N端和/或C端，或位于融合蛋白中的AID、其片段或突变体的N端和/或C端。
可以通过任何适合的技术检测本发明融合蛋白在细胞核中的积聚。例如，可将检测标记融合到Cas酶上，使得在与检测细胞核的位置的手段(例如，对于细胞核特异的染料，如DAPI)相结合时融合蛋白在细胞内的位置可以被可视化。在某些实施方案中，本文使用3*flag作为标记，该肽段序列可如SEQ ID NO:25第1－23位氨基酸残基所示。应理解，通常，若存在标记序列时，标记序列通常在融合蛋白的N端。标记序列与NLS之间可直接连接，也可通过适当的接头序列连接。NLS序列可直接与Cas酶或AID、其片段或突变体连接，也可通过适当的接头序列与Cas酶或AID、其片段或突变体连接。
因此，在某些实施方案中，本文的融合蛋白由Cas酶和AID、其片段或突变体组成。在其它实施方案中，本文的融合蛋白由Cas酶通过接头与AID、其片段或突变体连接而成。在某些实施方案中，本文的融合蛋白由NLS、Cas酶、AID或其片段或突变体以及Cas酶和AID或其片段或突变体之间的任选的接头序列组成。在某些实施方案中，本文的融合蛋白除含有NLS、Cas酶和AID、其片段或突变体外，还可含有噬菌体蛋白，如作为UNG抑制剂的UGI。示例性的UGI的氨基酸序列可如本申请SEQ ID NO:23第1576-1659位氨基酸残基所示。因此，在某些实施方案中，本文的融合蛋白含有本文所述的Cas9酶、本文所述的AID或其片段或突变体、UGI以及NLS，或由这些部分及它们之间的任选的接头序列以及任选的用于检测、分离或纯化的氨基酸序列组成。Ugi序列可位于融合蛋白的N端、 C端，或者位于融合蛋白之中，例如位于NLS序列与Cas酶之间或位于Cas酶与AID、其片段或突变体之间。在某些实施方案中，本文的融合蛋白从N端到C端依次为AID或其片段或突变体、Cas酶、Ugi和NLS，或者可为Cas酶、AID或其片段或突变体、Ugi和NLS，它们之间可由接头连接。
在某些实施方案中，本文使用CN 201710451424.3所公开的融合蛋白。更具体而言，本文使用本申请所公开的其氨基酸序列如SEQ ID NO:25、27、29、31、33、48或50所示，或如SEQ ID NO:25第26－1654位氨基酸所示，或如SEQ ID NO:27第26－1638位所示，或如SEQ ID NO:31第26－1629位氨基酸所示，或如SEQ ID NO:33第26－1638位氨基酸所示的融合蛋白，或如SEQ ID NO:48第26－1629位氨基酸所示。在某些实施方案中，本文的融合蛋白如本申请SEQ ID NO:23所示。
可构建表达上述融合蛋白的表达载体/质粒和表达所需sgRNA的载体/质粒，将其转入感兴趣的细胞中，以通过诱导感兴趣基因剪接位点的碱基突变来调控其RNA剪接。
“表达载体”可以是本领域熟知的各种细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其它载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。表达载体还可包括翻译起始用的核糖体结合位点和转录终止子。本文所述的多核苷酸序列可操作性地连接到表达载体中的适当启动子上，以经由该启动子指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其它一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。标记基因可用于提供用于选择转化的宿主细胞的表型性状，包括但不限于真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。当本文所述的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列，则将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。
本领域一般技术人员清楚如何选择适当的载体、启动子、增强子和宿主细胞。可采用本领域技术人员熟知的方法构建含本文所述的多核苷酸序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。
本文的融合蛋白、其编码序列或表达载体，和/或sgRNA、其编码序列或表达载体可以组合物的形式提供。例如，组合物可含有本文的融合蛋白和sgRNA或sgRNA的表达载体，或可含有本文融合蛋白的表达载体和sgRNA或sgRNA的表达载体。在组合物中，融合蛋白或其表达载体、或sgRNA或其表达载体可以混合物的形式提供，或者可单独包装。 组合物可以是溶液的形式，也可以是冻干形式。优选的是，组合物中的融合蛋白是本文所述的AID、其片段或突变体与本文所述的Cas酶的融合蛋白。
组合物可提供在试剂盒中。因此，本文提供含有本文所述组合物的试剂盒。或者，本文也提供一种试剂盒，该试剂盒含有本文的融合蛋白和sgRNA或sgRNA的表达载体，或含有本文融合蛋白的表达载体和sgRNA或sgRNA的表达载体。试剂盒中，融合蛋白或其表达载体、或sgRNA或其表达载体可独立包装，或以混合物的形式提供。试剂盒中还可包括例如用于将所述融合蛋白或其表达载体和/或sgRNA或其表达载体转入细胞的试剂，以及指导技术人员进行所述转入的说明书。或者，试剂盒还可包括指导技术人员采用试剂盒所含成分实施本文所述的各种方法和用途的说明书。试剂盒中还包括其它的试剂，例如用于PCR的试剂等。
本文的融合蛋白、其编码序列或表达载体，和/或sgRNA或其表达载体可用于诱导感兴趣基因剪接位点的碱基突变来调控其RNA剪接。因此，本文提供一种诱导感兴趣的细胞内感兴趣的基因的剪接位点发生碱基突变的方法，所述方法包括在所述细胞内表达本文所述的融合蛋白的步骤，根据所表达的融合蛋白，该方法还包括表达sgRNA或gDNA的步骤。例如，在某些实施方案中，在细胞中表达本文所述的AID、其片段或突变体与Cas酶的融合蛋白及其识别的sgRNA。在某些实施方案中，在细胞中表达胞嘧啶脱氨酶、其保留了酶活的片段或突变体与特异识别靶向序列的TALEN蛋白的融合蛋白。在某些实施方案中，在细胞中表达胞嘧啶脱氨酶、其保留了酶活的片段或突变体与特异识别靶向序列的锌指蛋白的融合蛋白。在某些实施方案中，在细胞中表达胞嘧啶脱氨酶、其保留了酶活的片段或突变体与核酸酶活性部分或完全缺失但保留了解旋酶活性的Cpf酶的融合蛋白以及Cpf酶识别的sgRNA。在其它实施方案中，在细胞中表达胞嘧啶脱氨酶、其保留了酶活的片段或突变体与Ago蛋白的融合蛋白以及Ago蛋白识别的gDNA。
本文中，感兴趣的细胞尤其还包括那些需要在其中使感兴趣基因的剪接位点发生碱基突变以调控其RNA剪接的细胞。这类细胞包括原核细胞和真核细胞，例如植物细胞、动物细胞、微生物细胞等。尤其优选的是动物细胞，例如哺乳动物细胞、啮齿类动物细胞，包括人、马、牛、羊、鼠、兔等等。微生物细胞包括本领域周知的来自各种微生物种类的细胞，尤其是那些具有医疗研究价值、生产价值(例如燃料如乙醇的生产、蛋白质生产、油脂如DHA生产)的微生物种类的细胞。细胞还可以是各种器官来源的细胞，例如来自人肝脏、肾脏、皮肤等处的细胞，或者是血液细胞。细胞还可以是目前在售的各种成熟的细胞系，例如293细胞、COS细胞。在某些实施方案中，细胞是来自健康个体的细胞；在其他实施方案中，细胞是来自患病个体的患病组织的细胞，例如来自炎症组织的细胞、肿瘤细胞。在某些实施方案中，感兴趣的细胞是诱导型多能干细胞。细胞还可以是经基因工 程改造过，以使其具有某种特定功能(例如生产感兴趣的蛋白)或产生感兴趣的表型的细胞。应理解的是，感兴趣的细胞包括体细胞和生殖细胞。在某些实施方案中，细胞是动物或人体内特定的细胞。
感兴趣的基因可以是任何感兴趣的核酸序列，尤其是各种与疾病相关，或与各种感兴趣的蛋白质的生产相关，或各种与感兴趣的生物学功能相关的基因或核酸序列。这类感兴趣的基因或核酸序列包括但不限于编码各种功能蛋白的核酸序列。本文中，功能蛋白指能够完成生物体的生理功能的蛋白质，包括催化蛋白、运输蛋白、免疫蛋白和调节蛋白等。在某些具体实施方式中，所述功能蛋白包括但不限于：疾病的发生、发展和转移中涉及的蛋白，细胞分化、增殖与凋亡中涉及的蛋白，参与新陈代谢的蛋白，发育相关的蛋白，以及各种药物靶点等等。例如，功能蛋白可以是抗体、酶、脂蛋白、激素类蛋白、运输和贮存蛋白、运动蛋白、受体蛋白、膜蛋白等。
作为示范性的例子，感兴趣的基因包括但不限于RPS24、CD45、DMD、PKM、BAP1、TP53、STAT3、GANAB、ThyN1、OS9、SMN2、β血红球蛋白基因、LMNA、MDM4、Bcl2和LRP8等。
在某些实施方案中，本文所述的方法包括将本文的融合蛋白或其表达载体和其识别的sgRNA或其表达载体或gDNA或其表达载体转入所述细胞内。在细胞组成型表达本文所述融合蛋白的情况下，可仅将相应的sgRNA或其表达载体或其识别的gDNA或其表达载体转入细胞中。在细胞诱导型表达本文所述融合蛋白的情况下，在转入sgRNA或gDNA之后，还可用诱导剂孵育细胞，或对细胞施与相应的诱导措施(例如光照)。优选地是，利用本文所述的AID、其片段或突变体与本文所述的Cas酶的融合蛋白及其识别的sgRNA来实施本文实施的方法。
可采用常规的转染方法将所述融合蛋白或其表达载体和/或其识别的sgRNA或其表达载体或gDNA或其表达载体转入细胞中。例如，当感兴趣的细胞为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。例如，转染时，首先制备质粒DNA-脂质体复合物，然后将该质粒DNA-脂质体复合物和相应的sgRNA或gDNA共同转染细胞。可采用市售的转染试剂盒或试剂将本文所述的载体或质粒转入感兴趣的细胞中，这类试剂包括但不限于
Figure PCTCN2018096810-appb-000001
2000试剂。转化细胞后，获得的转化子可以用常规方法培养，以允许其表达本文所述的融合蛋白。根据所用的细胞，培养中所用的培养基可选自各种常规培养基。
通常，针对不同的细胞，可采用已知技术设计表达本文融合蛋白和sgRNA或gDNA 的表达载体，以使这些表达载体适于在该细胞中表达。例如，可在表达载体中提供利于在该细胞中启动表达的启动子以及其他相关的调控序列。这些都可由技术人员根据实际情况加以选择和实施。
对于用于本文的sgRNA，可在感兴趣基因的感兴趣的剪接位点附近寻找可作为PAM的位点，根据该PAM选择能识别该PAM的Cas酶，并依本文所述设计、制备含该Cas酶的本发明融合蛋白以及相应的sgRNA。因此，用于本文的sgRNA的靶标识别区通常含有感兴趣基因感兴趣内含子剪接位点的互补序列
本文所述的剪接位点具有本领域周知含义，包括5’剪接位点和3’剪接位点。本文中，所述5’剪接位点和3’剪接位点均是相对于内含子而言。通常，在感兴趣基因的感兴趣外显子/内含子的剪接位点附近选择可作为PAM的位点。例如，感兴趣的基因的感兴趣的外显子或内含子可以是RPS的外显子5、CD45的外显子5、TP53基因的外显子8或9、PKM的外显子9或10、BAP1的内含子2和TP53的内含子8等。或者，在某些实施方案中，在感兴趣基因3’剪接位点的上游存在于内含子内的多聚嘧啶链附近选择可作为PAM的位点。因此，这类sgRNA的靶标结合区含有感兴趣基因感兴趣内含子的多聚嘧啶区的序列。
本文的方法可以是体外方法，也可以是体内方法；此外，本文的方法包括治疗目的的方法和非治疗目的的方法。当体内实施时，可采用本领域周知的手段将本文的融合蛋白或其表达载体和其识别的sgRNA或其表达载体或gDNA或其表达载体转入实验对象体内，如相应的组织细胞内。应理解，体内实施时，实施对象可以是人或各种非人动物，包括本领域惯常采用的各种非人模式生物。体内实验应满足伦理要求。
本文所述的诱导感兴趣的细胞内感兴趣的基因的剪接位点发生碱基突变的方法是一种通用的RNA剪接调控方法，可用于基因治疗。因此，本文提供一种基因治疗方法，所述方法包括给予有需要的对象治疗有效量的表达本文所述融合蛋白的载体和相应的sgRNA或gDNA的表达载体。治疗有效量可根据对象的年龄、性别、所患疾病的性质和严重程度等方面予以确定。通常，给予治疗有效量的所述载体应足以缓和所述疾病的症状或治愈所述疾病。该基因治疗可用于因基因发生变异而致病的疾病的治疗，也可以用于通过调节不同剪接亚型而得以缓解症状或治愈的疾病的治疗。例如，因基因变异导致的疾病包括但不限于：DMD基因变异造成的杜氏肌无力症，SMN，β血红球蛋白IVS2 647G>A突变造成的地中海贫血症，LMNA突变造成的早衰症和家族性高胆固醇血症等。可通过调节不同剪接亚型的比例而实现症状缓解或治愈的疾病包括肿瘤，所述剪接亚型包括但不限于Stat3α向Stat3β的转换，PKM2向PKM1的转换，MDM4外显子6的跳跃，Bcl2可变剪接位点的选择，LRP8外显子八跳跃。
在某些实施方案中，本文提供一种肿瘤治疗方法，所述方法包括给予有需要的对象治 疗有效量的表达本文任一实施方案所述的融合蛋白的载体和相应的sgRNA的表达载体的步骤。在某些实施方案中，所述sgRNA的靶标结合区包含Stat3内含子22的3’剪接位点的互补序列。在某些实施方案中，适用于此方法的sgRNA的靶标结合区如SEQ ID NO:3所示。或者，所述sgRNA的靶标结合区包含PKM内含子10的5’或3’剪接位点的互补序列。在某些实施方案中，适用于此方法的sgRNA的靶标结合区如SEQ ID NO:15或16所示。
在某些实施方案中，本文还提供一种治疗由于DMD基因变异造成的杜氏肌无力症的方法，所述方法包括给予有需要的对象治疗有效量的表达本文所述融合蛋白的载体和相应的sgRNA的表达载体的步骤，其中，所述sgRNA的靶标结合区包含DMD外显子50的5’剪接位点的互补序列。在某些实施方案中，适用于此方法的sgRNA的靶标结合区如SEQ ID NO:17或51所示。在某些实施方案中，适用于此方法的融合蛋白的氨基酸序列可如SEQ ID NO:23或50所示。
可采用本领域周知的手段实施本文所述的基因治疗方法。通常，基因治疗的给药途径包括离体途径和在体途径。例如，可采用合适的骨架载体(例如腺相关病毒载体)构建表达本文所述融合蛋白的表达载体和表达sgRNA或gDNA的载体，将其以通用的方式例如给予患者，例如注射。或者，在涉及血液疾病时，可获取对象存在基因变异的血液细胞，体外采用本文所述方法处理，使所述细胞消除所述变异后体外扩增，再回输给该对象。此外，还可利用本文所述的方法改造对象的多能干细胞，回输至患者，以达到治疗目的。
本文再一方面还提供本文任一实施方案所述的融合蛋白、其编码序列和/或表达载体，和/或sgRNA和/或其表达载体在制备用于调控RNA剪接的试剂或试剂盒、在制备用于基因治疗的试剂、或在制备用于治疗因基因变异导致的疾病或受益于功能蛋白不同剪接亚型的比例改变的肿瘤的药物中的应用。本文也涉及一种用于调控RNA剪接、基因治疗(尤其是治疗因基因变异导致的疾病或受益于功能蛋白不同剪接亚型的比例改变的肿瘤)的本文任一实施方案所述的融合蛋白、其编码序列和/或表达载体，以及sgRNA和/或其表达载体。
利用本文所述方法，可有效的诱导外显子跳读(例如RPS24外显子5，CD45外显子5，DMD基因外显子50，23，51等)，调控互斥外显子的选择(PKM1/PKM2等)，诱导内含子保留/包含(BAP1和TP53等)以及诱导可变剪接位点的利用(STAT3α/β等)等。同时可通过将3’剪接位点上游的C突变为T，促进选择性外显子的包含比例(RPS24外显子5，GANAB外显子5，ThyN1外显子6，OS9外显子13和SMN2外显子7)。此外，本文还证明，通过这一方法可以有效的纠正人类遗传突变造成的基因剪接缺陷。因此，本文所公开的方法是一种通用的RNA剪接调控方法，可用于疾病治疗，尤其可用于以下疾病的基因治疗：DMD基因变异造成的杜氏肌无力症，SMN，β血红球蛋白IVS2 647G>A突变造 成的地中海贫血症，LMNA突变造成的早衰症，家族性高胆固醇血症。同时本文所述方法还可通过调节不同剪接亚型的比例，包括但不限于诱导Stat3α向Stat3β的转换，PKM2向PKM1的转换，MDM4外显子6的跳跃，Bcl2可变剪接位点的选择，LRP8外显子八跳跃等，从而实现对肿瘤等疾病的治疗。
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是示例性的，而非限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook&Russell所著的Molecular Cloning:A Laboratory Manual(分子克隆实验指南第三版)中所述的条件，或按照制造厂商所建议的条件。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。
一、材料和方法
(1)表达AIDX-Cas9或Cas9-AIDX的融合蛋白的质粒的构建
参照CN 201710451424.3(本文将其全部内容以引用的方式纳入本文)实施例中所披露的方法构建表达本文所需的表达AIDX-Cas9或Cas9-AIDX融合蛋白的质粒。
在以下的实验中，使用到AIDX-nCas9-Ugi融合蛋白，参照CN 201710451424.3实施例1-3和14的方法构建其表达质粒，即MO91-AIDX-XTEN-nCas9-Ugi，该质粒表达SEQ ID NO:23所示的融合蛋白，其中，第1-182位为AIDX的氨基酸序列，第183-198位是接头XTEN的氨基酸序列，第199-1566位为nCas9的氨基酸序列，第1567-1570位和第1654-1657位为接头序列，第1571-1653为Ugi序列，第1658-1664位为SV40NLS的氨基酸序列。该融合蛋白的编码序列如SEQ ID NO:22所示。
(2)gRNA的制备
1、寻找20bp的靶标序列。如果该20bp的靶标序列的起始碱基不是G，需将一个G加到其5’端以使其能被RNA聚合酶III U6启动子有效转录。需注意的是该靶标序列不能含有XhoI或NheI的识别位点。
2、将sgRNA克隆到pLX(Addgene)中，获得pLX sgRNA。需如下4个引物，其中R1和F2是sgRNA特异性的：
F1：AAACTCGAGTGTACAAAAAAGCAGGCTTTAAAG(SEQ ID NO:18)
R1：rc(GN 19)GGTGTTTCGTCCTTTCC(SEQ ID NO:19)
F2：GN 19GTTTTAGAGCTAGAAATAGCAA(SEQ ID NO:20)
R2：AAAGCTAGCTAATGCCAACTTTGTACAAGAAAGCTG(SEQ ID NO:21)
其中，GN 19＝新的靶标结合序列，rc(GN 19)＝新靶标结合序列的反向互补序列。
3、分别使用F1+R1和F2+R2扩增pLX sgRNA；
4、凝胶纯化两次扩增获得的产物，合并，用于F1+R2进行第三次PCR；
5、使用NheI和XhoI消化步骤4进行的PCR获得的产物；和
6、连接和转化，从而制备得到sgRNA的表达载体。
(3)细胞的转染
293T细胞至70－90％的汇合度时进行转染。转染时，首先制备质粒DNA-脂质体复合物，包括将四倍量的
Figure PCTCN2018096810-appb-000002
2000试剂稀释在
Figure PCTCN2018096810-appb-000003
培养基中，分别将表达本文所述融合蛋白的质粒及相应的sgRNA质粒稀释在
Figure PCTCN2018096810-appb-000004
培养基中，然后将稀释的质粒分别加到稀释的
Figure PCTCN2018096810-appb-000005
2000试剂中(1：1)孵育30分钟。之后将该质粒DNA-脂质体复合物转染293T细胞。作为对照，仅用所述质粒DNA-脂质体复合物转染依照CN 201710451424.3实施例4所构建的报告细胞，加嘌呤霉素2ug/ml和杀稻瘟菌素20ug/ml，筛选3d，分别在转染后第7天通过高通量测序分析基因表达，剪接和突变。
(4)定量PCR和高通量测序等
本文中的定量PCR和高通量测序等生物学方法，除非另有说明，否则均采用本领域惯常采用的方法和试剂实施。
二、结果
1、剪接位点G突变为A会导致外显子的跳读。
RPS24是核糖体的组成蛋白，其突变会导致先天性再生障碍性贫血。RPS24的外显子5可以被选择性剪接，产生具有不同3’UTR的两种亚型，其中肝癌细胞更倾向于表达包含外显子5的亚型，但是其生理功能并不清楚。
本实验利用TAM技术设计sgRNA(RPS24-E5-5’SS，其靶标结合区的序列如SEQ ID NO:9所示)将RPS24的外显子5的5’剪接位点或者3’剪接位点的G突变为A，调控其选择性剪接过程。如前文所述转染293T细胞，并分别在转染后第7天通过高通量测序分析基因表达，剪接和突变。
在293T细胞中，利用AIDX-nCas9-Ugi融合蛋白中的UNG抑制因子UGI和sgRNA使该融合蛋白靶向RPS24外显子5的5’剪接位点。通过测序发现内含子5的第一位(IVS5+1)有40％以上的G到A的突变，同时外显子5的最后一位碱基有30％的G到A的突变，在 外显子5上还有两个位点有不到10％的G到A的突变(图3，A)。通过外显子拼接位点的测序发现，相对于对照组，RPS24sgRNA转染的细胞中外显子5的包含比例下调(图3，B)，定量PCR结果也得到一致的结论(图3，C)，并且在成熟RNA中并没有发现外显子的突变(图3，A)。
同时我们获得了两株同基因型的单克隆细胞系，它们的5’剪接位点完全突变为A，同时在外显子上也包含有一个G到A的突变(图3，D)。在这两株克隆中，RPS24外显子5包含的亚型完全检测不到，说明5’剪接位点的G到A的突变可以导致RPS24外显子5的跳读(图3，E)。
通过以上结果说明TAM技术可以有效地将剪接位点的G突变为A，导致外显子的跳读(RPS外显子5的5’剪接位点突变)。
2、CD45五号外显子剪接位点G突变为A会导致外显子的跳读。
为进一步验证是否能够有效地破坏剪接位点并且调控外显子跳读，我们选择了CD45基因的三个选择性外显子作为靶基因。CD45是一种受体酪氨酸磷酸酶，可以通过调控抗原受体(如TCR或者BCR)的信号传导调控T淋巴细胞和B淋巴细胞的发育与功能。CD45基因约由33个外显子组成，其中分别编码CD45蛋白胞外A，B，C区域的外显子4，5，6可以被选择性剪接。CD45亚型的表达模式取决于T细胞和B细胞的发育阶段，在B细胞表面表达包含有三个选择性外显子的最长CD45亚型(B220)。
我们针对CD45基因外显子5的5’剪接位点和3’剪接位点的G设计sgRNA(CD45-E5-5’SS和CD45-E5-3’SS，靶标结合区的序列分别如SEQ ID NO:1和2所示)，在一种表达未剪接CD45亚型的生发中心B细胞系Raji细胞中进行外显子5剪接位点的编辑。将400ng的AIDx-nCas9-Ugi的表达质粒，300ng的sgRNA的表达质粒和50ng的Ugi表达质粒用具有1,100V电压和一个脉冲40ms的Neon(Life Technologies)电转染到1×10 5个Raji细胞中。转染后24h，加入2μg/ml嘌呤霉素以选择转染细胞3天。
我们发现这两个sgRNA分别可以诱导53.6％和73.4％的DNA发生剪接位点的G>A突变(图1和2)，发现当破坏外显子5的剪接位点后，CD45RB表达明显下调，并且CD45RA与CD45RC的表达未发生明显改变，说明在诱导外显子跳读时剪接位点是独立的，并且5’SS或者3’SS的突变都可以造成外显子跳读。
4、TP53基因8号外显子剪接位点G突变为A会导致外显子的跳读。
本实验利用TAM技术设计sgRNA(TP53-E8-5’SS，其序列如SEQ ID NO:7所示)将TP53的外显子8的3’剪接位点的G突变为A，调控其选择性剪接过程(图4)。如前文所述转 染293T细胞，并分别在转染后第7天通过高通量测序分析基因表达，剪接和突变。
通过测序发现内含子8的第一位(IVS8+1)有80％以上的G到A的突变(图4，A)。通过外显子拼接位点的测序发现，sgRNA转染的细胞中TP53有超过40％发生外显子8的跳读，定量PCR结果(图4，B、C)也得到一致的结论，并且在成熟RNA中并没有发现外显子的突变。对照组没有可检测到的外显子8的跳读。
5、TP53基因9号外显子剪接位点G突变为A会导致外显子的跳读。
我们还证明利用同样的方法，可以造成TP53基因外显子外显子9的跳读。具体而言，用TAM和靶向TP53外显子9的3'SS的sgRNA(TP53-E9-3’SS,其靶标结合区序列如SEQ ID NO:8所示)转染293T细胞。转染7天后，从基因组DNA扩增内含子-外显子连接并通过高通量测序进行分析。通过RT-PCR分析TP53的剪接。从cDNA扩增剪接连接并通过高通量测序进行分析。3’SS突变导致外显子在总转录物的34％中跳跃，并在23.6％的mRNA中激活隐蔽剪接位点。TAM处理的细胞也激活了内含子8内的神经外显子(总转录物的4.3％)(图4，D-F)。
6、剪接位点的准确编辑可以改变可变剪接位点的选择
除了外显子跳读外，RNA在剪接过程中还可能发生可变剪接位点的选择，并且会形成具有不同生理功能的新蛋白亚型。例如Stat3外显子23上一个可变剪接位点的选择会形成一种截短的缺少C端反式激活结构域的STAT3β亚型。全长的STAT3α可以促进肿瘤发生，而STAT3β可以发挥显性失活作用，抑制STAT3α功能，促进肿瘤细胞凋亡。尤其在乳腺癌细胞中，相对于敲除STAT3整体表达而言，诱导STAT3β表达能够更有效地抑制细胞存活，预示着诱导STAT3β表达可以作为肿瘤治疗策略。因为STAT3常规剪接位点与选择性剪接位点之间只有50bp，较难使用常规的双sgRNA剪接方法诱导STAT3β表达，而TAM技术可以提供更为准确的基因编辑方法。本实验设计sgRNA破坏常规剪接位点，利用TAM消除Stat3外显子23(Stat3α)的典型3'SS，并促进下游替代3'SS(Stat3β)的利用，其示意图如图5(A)所示。293T细胞用AIDx-nCas9-Ugi和靶向Stat3外显子23(STAT3-E23-3'SS，其靶标结合区的序列如SEQ ID NO:3所示)或针对AAVS1的sgRNA(Ctrl)。从DNA(顶部2个小组)或cDNA(底部2个小组)扩增内含子-外显子连接，并通过高通量测序进行分析。利用上述方法在293T细胞中表达TAM和sgRNA,使其中超过50％的3’剪接位点的G突变为A(图5，B)。结果显示，TAM在Stat3外显子23增强了远端3'SS的利用(图5，C)。通过定量PCR以及免疫印迹分析发现STAT3β表达水平上调而STAT3α表达水平下调(图5，E-F)。与预期一致，相对于敲除STAT3整体蛋白表达，TAM编辑的细胞增殖速率更为 明显地被抑制。
以上结果显示针对可变剪接位点极为靠近的情况，TAM技术可以使我们克服常规双sgRNA剪接方法的缺陷，准确地破坏选择性剪接位点，调控可变剪接位点的选择。
7、互斥外显子
互斥外显子是另一种主要的选择性剪接形式，互斥外显子可以被选择性包含在不同转录本中产生功能不同的蛋白。丙酮酸激酶(PKM)是糖酵解过程的限速酶，在剪接过程中PKM的外显子9和10可以被选择性包含产生两种亚型PKM1和PKM2，其中PKM1包含外显子9，不包含外显子10，主要表达在成人组织中，而PKM2包含外显子10，不包含外显子9，主要表达在胚胎干细胞以及肿瘤细胞中。因为PKM2与肿瘤发生有关，我们希望能够利用TAM技术将肿瘤细胞的PKM剪接方式从PKM2切换为PKM1。
图6(A)显示了TAM在C2C12细胞中将PKM2转移到PKM1的示意图。上图中，PKM基因的外显子10而不是外显子9被剪接以产生PKM2，其cDNA被限制酶PstI识别；下图中，TAM将外显子10的5'SS处的GT二核苷酸转化为AT。因此，外显子9而不是外显子10被剪接以产生PKM1，其cDNA被限制酶NcoI识别。
我们设计针对内含子10的3’或5’剪接位点的sgRNA(PKM-3’SS-E10或PKM-5'SS-E10，其靶标结合区的序列分别如SEQ ID NO:15或16所示)，感染C2C12细胞使其中的G突变为A(图6，C、D)。我们发现在C2C12分化得到的肌肉细胞中，PKM2表达明显下调而PKM1表达水平上调(图6,B、E、F)。同样，在未分化的C2C12细胞中，PKM2表达明显下调而PKM1表达水平上调(图6，G,H)。
而利用针对内含子9的5’或3’剪接位点的sgRNA(PKM-3'SS-E9，PKM-5’SS-E9,其靶标结合区的序列分别如SEQ ID NO:13或14所示)，可以使其G突变为A，同时PKM1表达水平下调(图7)，同时PKM2的表达上调进一步说明剪接位点的突变能够改变互斥外显子的剪接位点的选择。
8、诱导内含子包含
內含子包含是另一种可变剪接的形式，最近的研究证明內含子的包含在很多人类疾病包括肿瘤中发生。我们证明，利用TAM和sgRNA破坏相应的内含子的剪接位点，可以特异诱导内含子的包含。
BAP1是组蛋白去泛素化酶，其第二内含子在一些肿瘤中保留，引起BAP1的表达降低。BAP1的第二个内含子可能通过内含子定义方式进行剪接，其中5'SS与下游3'SS配对。将G转换为A，在5'SS处理U1识别U1RNP并破坏内含子定义，导致包含该内含子。本 实验引导TAM在BAP1内含子2的5'剪接位点处突变G，其示意图如图8(A)所示。
我们设计针对內含子2的5’剪接位点的sgRNA(BAP1-E2-5'SS，其靶标结合区的序列如SEQ ID NO:5所示),用AIDx-nCas9-Ugi的表达质粒和针对AAVS1(Ctrl)或针对BAP1内含子2的该sgRNA的表达质粒转染293T细胞。转染7天后，通过RT-PCR(图8，B)或同种型特异性实时PCR(图8，C)分析BAP1mRNA的剪接。结果显示，超过70％的G突变为A(图8，D)。突变后内含子2的包含被诱导，有超过60％的BAP1mRNA含有内含子2；与此类似，突变内含子2的3’剪接位点(sgRNA的序列如SEQ ID NO:6(BAP1-E3-3'SS)所示)后，也诱导BAP1的内含子包含(图9，B-E)。
9、3’剪接位点-3位C到T的突变可以促进外显子包含
除了剪接位点，mRNA上其他顺式作用元件也可以改变前体mRNA剪接过程，因此我们还可以利用TAM技术编辑其他剪接调控元件。因为内含子元件的改变不会影响基因表达序列，我们聚焦在内含子的剪接调控元件的编辑上。在3’剪接位点上游，存在着一个多聚嘧啶链，由胞嘧啶(C)和胸腺嘧啶(T)组成，我们的实验证明可以通过TAM和相应的sgRNA把多聚嘧啶链中的C突变为T，增强3’剪接位点的强度，促进下游外显子的包含。
用AIDx-nCas9-Ugi的表达质粒和针对AAVS1(Ctrl)的sgRNA或针对RPS24第5外显子的聚嘧啶核苷的sgRNA(RPS24-E5-PPT，其靶标结合区的序列如SEQ ID NO:10所示)的表达质粒转染293T细胞。转染后6天，从基因组DNA扩增sgRNA靶向区域，并通过超过8000x覆盖的高通量测序进行分析。结果显示，多聚嘧啶链中超过50％的C突变为T。我们发现外显子5的包含率提高(图11，B,C)。之后我们分选得到了两个包含完全的C到T突变的单细胞克隆，其外显子5的包含率分别提高了8倍与5倍(图11，E)。
此外，我们利用AIDx-nCas9-Ugi的表达质粒和对照sgRNA(Ctrl)或靶向GANAB外显子6的PPT的sgRNA(GANAB-E6-PPT，其靶标结合区的序列如SEQ ID NO:4所示)的表达质粒转染293T细胞。转染后6天，从基因组DNA扩增sgRNA靶向区域，并通过超过8000x覆盖的高通量测序进行分析。结果如图10(B-E)所示，其中多个C被诱导突变为T，最高的是IVS5-6C，其超过70％的C突变为T，同时高通量测序证明外显子6的包含增加了50％。类似的方法也可以造成ThyN1外显子6(sgRNA靶标结合区的序列如SEQ ID NO:12所示，THYN1-E6-PPT)的包含增加(图10，F-G)和OS9外显子13(sgRNA靶标结合区的序列如SEQ ID NO:11所示，OS9-E13-PPT)的包含增加(图10，H-I)。
10、TAM技术可以在人类iPS细胞以及mdx小鼠模型中恢复DMD蛋白表达(C2C12和iPS)
杜氏肌肉萎缩症(DMD)是一种肌肉萎缩疾病，在美国每4000个男性就有1个病例。由于病人的DMD基因的可遗传性突变导致其开放阅读框改变或者未成熟密码子的形成而导致骨骼肌抗肌肉萎缩蛋白缺陷，导致疾病的发生。相对于突变的DMD基因，截短的抗肌肉萎缩蛋白能够发挥部分功能，导致发病程度较轻的贝克肌萎缩症。因此有研究利用反义寡聚核苷酸或者利用双sgRNA介导的CRISPR技术跳读部分外显子从而使DMD的开放阅读框恢复，促进抗抗肌萎缩蛋白的表达。这种通过跳读DMD基因的非必需区域以部分恢复抗抗肌萎缩蛋白表达的方法，预计可以使80％的DMD病人从中获利。但是利用反义寡聚核苷酸进行治疗需要进行持续性给药，极度耗费时间与金钱，因此开发新的DMD基因治疗策略十分必要。
为了验证TAM技术是否可以调控DMD基因的外显子跳读，我们利用一个缺少外显子51的DMD病人的iPS细胞进行实验，根据序列分析发现当利用sgRNA(其靶标结合区的序列如SEQ ID NO:17所示，DMD EXON50 5'SS)跳读外显子50后抗肌萎缩蛋白的开放阅读框可以恢复(图12)。我们在病人来源的iPSC中转染sgRNA(其靶标结合区的序列如SEQ ID NO:17所示)的表达质粒和AIDx-nCas9-Ugi的表达质粒,高通量测序发现可以诱导12％以上的G>A突变(图12，B)，而后我们分离出一株单克隆细胞具有完全的G>A突变(图12B)。而后我们将iPSCs细胞向心肌细胞分化，发现TAM编辑过的细胞发生了外显子50的跳读(图12C，D)，进一步western实验发现抗肌肉萎缩蛋白蛋白的表达在TAM修复的细胞中获得了恢复(图12，E)。
采用相同的实验，利用AIDx-saCas9(KKH，切割酶)-Ugi(编码序列如SEQ ID NO:49所示，氨基酸序列如SEQ ID NO:50所示)和相应的sgRNA序列(其序列如SEQ ID NO:51所示，其骨架序列如SEQ ID NO:52所示)诱导DMD第50号外显子跳读。具体而言，用对照sgRNA(ctrl)或目标sgRNA(E50-5'SS)结合AIDx-saCas9(KKH，切割酶)-Ugi处理杜氏肌无力病人的iPSC细胞后，利用PCR扩增相应的DNA，进行高通量测序分析诱导的突变。数据是两个独立实验的代表。结果如图14(A)所示。分别将正常人来源的iPSCs、病人来源的iPSCs和修复后病人来源的iPSCs向心肌细胞分化，而后分别利用RT-PCR或western blot或免疫荧光染色检测DMD基因和抗肌萎缩蛋白的表达，结果分别如图14的B、C和D所示。图14的E、F和G显示，修复后的心肌细胞逆转了肌无力的表型，心肌细胞分别通过低渗透压诱导的肌酸激酶释放(E)、miR31表达(F)、以及β-肌萎缩蛋白聚糖蛋白的表达(G)，证明修复后的心肌细胞逆转了肌无力的表型。此外，全基因组测序证明基因编辑的高度特异性，两次全基因组测序只发现一个脱靶位点(图14，H和I)。
本文涉及的序列信息如下：
Figure PCTCN2018096810-appb-000006

Claims (15)

  1. 一种调控细胞中感兴趣基因的RNA剪接的方法，其特征在于，所述方法包括在所述细胞中表达靶向性胞嘧啶脱氨酶，以诱导该细胞中感兴趣基因的感兴趣内含子的3’剪接位点AG突变为AA，或感兴趣基因的感兴趣内含子的5’剪接位点GT突变为AT，或感兴趣基因的感兴趣内含子的多聚嘧啶区的多个C分别突变为T。
  2. 如权利要求1所述的方法，其特征在于，所述靶向性胞嘧啶脱氨酶选自：
    (1)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas酶的融合蛋白；
    (2)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与特异识别靶向序列的TALEN蛋白的融合蛋白；
    (3)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与特异识别靶向序列的锌指蛋白的融合蛋白；
    (4)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与核酸酶活性部分或完全缺失但保留了解旋酶活性的Cpf酶的融合蛋白；和
    (5)胞嘧啶脱氨酶、其保留了酶活的片段或突变体与Ago蛋白的融合蛋白。
  3. 如权利要求2所述的方法，其特征在于，所述靶向性胞嘧啶脱氨酶为胞嘧啶脱氨酶、其保留了酶活的片段或突变体与核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas酶的融合蛋白，或者是胞嘧啶脱氨酶、其保留了酶活的片段或突变体与核酸酶活性部分或完全缺失但保留了解旋酶活性的Cpf酶的融合蛋白；所述方法包括在所述细胞中表达所述靶向性胞嘧啶脱氨酶和sgRNA，其中，所述sgRNA为所述Cas酶或Cpf酶所特异性识别，并结合到含有感兴趣基因感兴趣内含子剪接位点的序列或结合到感兴趣多聚嘧啶区的互补序列。
  4. 如权利要求3所述的方法，其特征在于，
    所述sgRNA结合到含有所述感兴趣基因的感兴趣内含子的5’剪接位点的序列，所述融合蛋白将所述5’剪接位点处的GT突变为AT，从而诱导外显子跳读、激活替代剪接位点、诱导互斥外显子转换或内含子包含；或
    所述sgRNA结合到含有所述感兴趣基因的感兴趣内含子的3’剪接位点的序列，所述融合蛋白将所述3’剪接位点的AG突变为AA，从而诱导外显子跳读、激活替代剪接位点、诱导互斥外显子转换或内含子包含；或
    所述sgRNA结合到感兴趣多聚嘧啶区的互补序列，诱导该多聚嘧啶区的C突变为T，从而增强外显子的包含。
  5. 如权利要求2所述的方法，其特征在于，所述靶向性胞嘧啶脱氨酶是胞嘧啶脱氨酶、其保留了酶活的片段或突变体与Ago蛋白的融合蛋白；所述方法包括在所述细胞中表达所述靶向性胞嘧啶脱氨酶和该Ago蛋白识别的gDNA的步骤。
  6. 如权利要求3所述的方法，其特征在于，所述融合蛋白还含有Ugi，或所述方法还包括同时转入Ugi的表达质粒的步骤；
    或者，所述方法包括直接导入所述融合蛋白和所述sgRNA的步骤。
  7. 如权利要求2－3中任一项所述的方法，其特征在于，
    所述Cas酶的核酸酶活性全部缺失，无DNA双链断裂能力，或部分缺失，仅具有DNA单链断裂能力；和/或
    所述Cas酶选自：Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csn1和Csx12)、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4、其同源物或其修饰形式；优选地，所述Cas酶为Cas9酶，优选选自：来自化脓链球菌的Cas9、来自金黄色葡萄球菌的Cas9，以及来自嗜热链球菌的Cas9；和/或
    所述胞嘧啶脱氨酶为全长人源激活型胞嘧啶脱氨酶(hAID)、或其保留了酶活的片段或突变体，其中所述片段至少包括胞嘧啶脱氨酶的NLS结构域、催化结构域和APOBEC样结构域；和/或所述融合蛋白还包含以下序列中的一种或多种：接头，核定位序列，Ugi，以及为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化而引入的氨基酸残基或氨基酸序列。
  8. 如权利要求7所述的方法，其特征在于，
    所述Cas酶为Cas9酶，该酶的两个核酸内切酶催化结构域RuvC1和/或HNH发生突变，导致该酶核酸酶活性缺失、保留了解旋酶活性；优选地，所述Cas9酶的RuvC1和HNH都发生突变，导致该酶核酸酶活性缺失、保留了解旋酶活；更优选地，所述Cas9酶的第10个氨基酸天冬酰胺突变为丙氨酸或其它氨基酸，第841位氨基酸组氨酸突变为丙氨酸或其它氨基酸；更优选地，所述Cas9酶的氨基酸序列如SEQ ID NO:23第199-1566位所示，或如SEQ ID NO:25第42－1452位氨基酸残基所示，或如SEQ ID NO:33第42-1419位氨基酸残基所示，或如SEQ ID NO:50第199-1262位氨基酸残基所示；和/或
    所述胞嘧啶脱氨酶的片段至少包含胞嘧啶脱氨酶的第9－182位氨基酸残基，例如至少包含第1－182位氨基酸；优选地，所述片段由第1－182位氨基酸残基组成，由第1－186位氨基酸残基组成，或由第1－190位氨基酸残基组成；或者，所述胞嘧啶脱氨酶的氨基酸序列如SEQ ID NO:25第1457－1654位氨基酸残基所示，所述片段至少包含SEQ ID  NO:25的第1465－1638位氨基酸残基，例如至少包含SEQ ID NO:25第1457－1638位氨基酸残基，优选地，所述片段由SEQ ID NO:25第1457－1638位氨基酸残基、SEQ ID NO:25第1457－1642位氨基酸残基，或SEQ ID NO:25第1457－1646位氨基酸残组成；所述突变体在第10、82和156位具有取代突变，优选地，所述取代突变是K10E、T82I和E156G，更优选地，所述突变体含有如SEQ ID NO:31第1447-1629位所示的氨基酸序列，或由如SEQ ID NO:31第1447-1629位所示的氨基酸残基组成。
  9. 如权利要求8所述的方法，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID NO:23、25、27、29、31、33、48或50所示，或如SEQ ID NO:25第26－1654位氨基酸所示，或如SEQ ID NO:27第26－1638位所示，或如SEQ ID NO:31第26－1629位氨基酸所示，或如SEQ ID NO:33第26－1638位氨基酸所示，或如SEQ ID NO:48第26－1629位氨基酸所示。
  10. 一种融合蛋白，其特征在于，所述融合蛋白含有胞核酸酶活性部分或完全缺失但保留了解旋酶活性的Cas酶，胞嘧啶脱氨酶或其保留了酶活的片段或突变体，以及Ugi，和任选的核定位序列和接头序列。
  11. 如权利要求10所述的融合蛋白，其特征在于，
    所述Cas酶如权利要求7或8所述；
    所述胞嘧啶脱氨酶或其保留了酶活的片段或突变体如权利要求7或8所述；
    所述Ugi的氨基酸序列如SEQ ID NO:23第1576-1659位氨基酸残基所示。
  12. 一种组合物或含有该组合物的试剂盒，其中，
    所述组合物含有权利要求10或11所述的融合蛋白或其表达载体；
    所述试剂盒还任选地含有能被该组合物中的融合蛋白识别的sgRNA或其表达载体；
    优选地，所述试剂盒含有能使该组合物中的融合蛋白和sgRNA表达的病毒颗粒。
  13. 一种sgRNA，其包含蛋白识别区和靶标识别区，其特征在于，所述靶标结合区结合到含有感兴趣基因感兴趣内含子剪接位点的序列，或结合到感兴趣基因的感兴趣多聚嘧啶区的互补序列。
  14. 如权利要求13所述的sgRNA，其特征在于，所述sgRNA的靶标结合区结合到DMD外显子50的含5’剪接位点的序列；优选地，所述sgRNA的靶标结合区如SEQ ID NO:17或51所示。
  15. 权利要求10或11所述的融合蛋白或其表达载体及能被该融合蛋白识别的sgRNA或其表达载体在制备用于调控RNA剪接的试剂或试剂盒、用于基因治疗的试剂、或用于治疗因基因变异导致的疾病或受益于功能蛋白不同剪接亚型的比例改变的肿瘤的药物中 的应用；
    优选地，所述因基因变异导致的疾病选自：DMD基因变异造成的杜氏肌无力症，SMN，β血红球蛋白IVS2 647G>A突变造成的地中海贫血症，LMNA突变造成的早衰症和家族性高胆固醇血症；所述剪接亚型选自：Stat3α向Stat3β的转换，PKM2向PKM1的转换，MDM4外显子6的跳跃，Bcl2可变剪接位点的选择和LRP8外显子八跳跃。
PCT/CN2018/096810 2017-07-25 2018-07-24 通过诱导剪接位点碱基突变或多聚嘧啶区碱基置换调控rna剪接的方法 WO2019020007A1 (zh)

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