WO2023050158A1 - 一种实现多碱基编辑的方法 - Google Patents
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
Definitions
- the invention belongs to the field of biotechnology, and in particular relates to a method for realizing polybase editing.
- CRISPR–Cas technology uses the design of guide RNAs (gRNAs) to edit specific genes or regulate transcription.
- gRNAs guide RNAs
- the use of a single gRNA limits efficiency and biotechnological applications. Therefore, more and more studies are no longer using a single gRNA, but using multiple compound strategies for multi-site editing or transcriptional regulation.
- Multiplex CRISPR technology in which many gRNAs or Cas enzymes are expressed, promotes the application of bioengineering and greatly improves the scope and efficiency of gene editing and transcriptional regulation.
- Another approach is to use a promoter to transcribe all gRNAs into a single transcript, which are then processed to release individual gRNAs through different strategies that require each gRNA to be flanked by cleavable RNA sequences, Examples include self-cleaving ribozyme sequences (such as hammerhead ribozyme and HDV ribozyme), exogenous cleavage factor recognition sequences (such as Cys4), and endogenous RNA processing sequences (such as tRNA sequences and introns).
- self-cleaving ribozyme sequences such as hammerhead ribozyme and HDV ribozyme
- exogenous cleavage factor recognition sequences such as Cys4
- endogenous RNA processing sequences such as tRNA sequences and introns.
- the sgRNA expression cassette assembly cycle is long and the efficiency is low; the number of targeted targets in a single cell is small and the editing efficiency is low; when obtaining a multi-site edited single clone, it is necessary to screen a large number of clones, which is heavy workload and high cost .
- the purpose of the present invention is to propose a method for realizing polybase editing.
- the specific plan is as follows:
- the first aspect of the present invention provides a method for realizing polybase editing, comprising the following steps:
- Step 1 Design and synthesize gRNA arrays.
- Step 1 Design and synthesize gRNA arrays, assemble 2 to 15 gRNA arrays into expression vectors, and construct sgRNA-expressing vectors;
- the gRNA array contains five sgRNA expression cassettes connected in series, each of which contains a promoter, sgRNA and polyT in the 5' to 3' direction, and the sgRNA in the sgRNA expression cassette is a target gene site sgRNA;
- Step 2 Transfect the gRNA array into cells by the following method to achieve polybase editing
- gRNA arrays or their transcripts plasmids containing mCherry-inactivated eGFP reporter molecules, sgRNA plasmids that edit and activate eGFP, and base editors are co-transfected into cells;
- the vector expressing sgRNA or its transcription product is co-transfected into cells with the base editor.
- the second aspect of the present invention provides a method for realizing polybase editing, comprising the following steps:
- Step 1 Design and synthesize gRNA arrays.
- Step 1 Design and synthesize gRNA arrays, assemble 2 to 15 gRNA arrays into expression vectors, and construct sgRNA-expressing vectors;
- the gRNA array contains five sgRNA expression cassettes connected in series, each of which contains a promoter, sgRNA and polyT in the 5' to 3' direction, and the sgRNA in the sgRNA expression cassette is a target gene site sgRNA;
- Step 2 Transfect the gRNA array into cells with stable inducible base editors by the following method to achieve multi-base editing
- gRNA arrays or their transcripts 2–15 gRNA arrays or their transcripts, plasmids containing mCherry-inactivated eGFP reporter molecules, and sgRNA plasmids that edit and activate eGFP are co-transfected into cells with stable inducible base editors;
- the vector expressing sgRNA or its transcription product is transfected into a cell in which the inducible base editor is stable.
- the above-mentioned polybase editing method of the present invention also includes isolating and culturing the single clone of the transfected cells, performing Sanger sequencing and EditR analysis, selecting a single clone with high editing efficiency, and transfecting the gRNA array by method I or II.
- the vector for expressing sgRNA is obtained by assembling 10 gRNA arrays into the expression vector;
- the cell is a mammalian cell; preferably, the mammalian cell is a human mammalian cell; preferably, the mammalian cell is a human embryonic kidney cell; preferably, the mammalian cell is a human embryonic kidney cell;
- the mammalian cells are human embryonic kidney cells 293 .
- the promoter is hU6
- the vector expressing sgRNA expresses a reporter molecule; preferably, the reporter molecule is red fluorescent protein.
- the five sgRNA expression cassettes serially connected in series are synthesized by chemical methods.
- each transfection into 1 ⁇ 10 5 cells the transfection amount of each of the gRNA arrays is 200ng, and the plasmid containing the mCherry-inactivated eGFP reporter molecule
- the amount of transfection of the sgRNA plasmid that edits and activates eGFP is 10ng;
- the transfection amount of the sgRNA-expressing vector was 2 ⁇ g.
- the cells with stable inducible base editors are selected from monoclonal cells with high editing efficiency and stable with inducible base editors.
- the screening method for the stable cell monoclonal of the inducible base editor with high editing efficiency is:
- the original monoclonal corresponding to the transfected monoclonal with high editing efficiency is the monoclonal cell in which the inducible base editor with high editing efficiency is stable.
- the inducible base editor is a doxycycline-induced base editor; preferably, it is a doxycycline-induced cytosine base editor.
- the cells in which the inducible base editor is stable are selected from cells stably expressing PB-FNLS-BE3-NG1 or PB-evoAPOBEC1-BE4max-NG.
- the third aspect of the present invention provides the cells edited by the above polybase editing method.
- the fourth aspect of the present invention provides the vector for expressing sgRNA constructed by the above method.
- the fifth aspect of the present invention provides a base editing system, comprising 2 to 15 gRNA arrays or transcription products thereof, or the vector expressing sgRNA or transcription products thereof.
- the base editing system also includes a base editor
- the base editor is selected from an adenine base editor or a cytosine base editor;
- the base editor is a cytosine base editor.
- the sixth aspect of the present invention provides a kit for polybase editing, which includes the base editing system.
- the kit also includes a plasmid containing an mCherry-inactivated eGFP reporter molecule and an sgRNA plasmid that edits and activates eGFP.
- the seventh aspect of the present invention provides the application of the above polybase editing method in genome recoding, cell construction of polygenic genetic diseases or treatment of polygenic genetic diseases.
- the method for realizing multi-base editing provided by the present invention directly synthesizes 5 sgRNA expression cassettes and assembles from 5 sgRNA expression cassettes, which reduces the assembly time and increases the success rate; constructs multiple sgRNA expression cassettes containing 5 sgRNA expression cassettes transfected with gRNA arrays, multiple sites can be simultaneously targeted in a single cell, and the efficiency of screening multi-site edited cells is higher; through a small amount of single-clonal culture and screening, efficient multiple-site editing can be achieved in a single cell. Site editing.
- the present invention by transfecting gBlocks into cells with stable inducible base editors, under the induction of doxycycline, the stable and continuous expression of base editors can be achieved, which has a higher base yield than transient expression.
- Base editing efficiency As a preferred solution, the present invention can further improve base editing efficiency by screening stable cell clones with high editing efficiency inducible base editors, and further transfecting gBlocks into the screened high editing efficiency single clones.
- the present invention co-transfects mammalian cells with gBlocks, plasmids containing mCherry-inactivated eGFP reporter molecules, and sgRNA plasmids that edit and activate eGFP, and the amount of transfected reporter molecules is about
- the reporter molecule and the corresponding sgRNA were simultaneously transfected into single cells, more sgRNAs were transfected into single cells by gBlock to target gene loci.
- green fluorescence can be detected, and cells with red and green double fluorescence can be detected, which means that there are more sgRNAs transfected and edited.
- Enrichment of highly edited clones can be achieved by flow cytometric sorting.
- FIG. 1 is a schematic structural diagram of gBlock-YC1 and gBlockPC in Example 1.
- Figure 2 is the verification result of the base editing efficiency of the targeted locus in Example 1, wherein Figure 2-a is the editing efficiency of gBlock-PC, and Figure 2-b is the editing efficiency of gBlock-YC1; the dots represent individual biological replications , the bars represent the mean.
- FIG. 3 is a schematic diagram of the structure of doxycycline-induced cytidine deaminase piggyBac in Example 2, wherein, F, flag label; NLS, nuclear localization signal; cas9n-NG, Cas9D10A recognizes NG-PAM; APOBEC1, rat APOBEC1 ; evoAPOBEC1, evolved rat APOBEC1.
- Figure 4 is the verification result of the base editing efficiency of the targeted locus in Example 2, wherein, Figure 4-a is the editing efficiency of gBlock-PC, and Figure 4-b is the editing efficiency of gBlock-YC1; dots and triangles represent individual Biological replicates, bars represent mean values.
- Fig. 5 is the protein level of cytosine base editor in evoAPOBEC1-BE4max-NG stably transfected cell monoclonal in Example 3, wherein anti-Cas9 (top) and anti-actin (bottom) are used.
- Figure 6 is the verification result of the base editing efficiency of the targeted locus in Example 3, where the values and error bars reflect the mean and standard deviation of four independent experiments.
- Fig. 7 is the evoAPOBEC1-BE4max-NG stable cell line introduced into the gBlocks pool in Example 4.
- FIG. 8 is a heat map of the mutation frequency of the targeted locus "C" based on the whole exon sequence analysis in Example 4.
- Fig. 9 is a flowchart of the construction of the integrated plasmid in Example 5.
- Figure 10 is an agarose gel electrophoresis image of the integrated plasmid in Example 5; wherein, the left side is the DNA ladder, and the rightmost empty vector is the control group; the arrows in lanes 5 and 7 are 22Kb.
- Figure 11 shows the basic quality indicators of single-cell RNA sequencing under three different delivery methods in Example 6; where a is the number of captured cells, b is the number of UMIs per unit, and c is the number of genes detected per cell .
- Figure 12 is the distribution analysis of target cells of different modified genes based on single-cell RNAseq in different delivery modes in Example 6; wherein, a, b, and c are the relationship between the number of edited gene sites and the number of cells in the three populations; d is the density map of the distribution of the number of edited gene loci detected by scRNAseq in the three populations, and the vertical line indicates the median value of the edited gene loci; e is the distribution of modified cells with different editing efficiencies for each gene locus Analysis, counting of different methods.
- Figure 13 is the single-cell sequencing analysis of the editing efficiency of sgRNA in different delivery methods in a single cell in Example 6; where, g is the editing efficiency of each sgRNA in a single cell; h is the RNA converted into a cell population based on single-cell RNA-Seq - Heatmap of target C editing efficiency in cell populations for the three delivery modes of Seq, with editing efficiency indicated in black intensity.
- Figure 14 is the monoclonal screening by Sanger sequencing in Example 7; where, a is the selection of 10 well-edited loci, the peak number of gBlocks is 3, and only one clone has all 10 gBlocks; b is 3 well-edited loci half of the clones did not have any editing, and 4 clones had all three editing sites; c was allelic editing of all target sites of each clone by Sanger sequencing and EditR; WT (wild type) - no allele editing; HZ (heterozygote) - partial allele editing; HM (homozygote) - all allele editing.
- Figure 15 is the analysis of genetic changes of highly modified HEK293T clones identified by WGS in Example 8; where a is the efficiency of converting TAG to TAA by heat map editing of target "C", followed by NC-negative control, clone 19 of Method 2 , clone 21 of method 3, and clone 19-1, 19-16, and 19-21 were obtained by second transfection using method 2 on the basis of clone 19.
- Figure 16 is the chromosomal distribution of exon snv in essential genes in Example 8; wherein, a-contains, b-does not contain the selected 50 essential gene targets; the X-axis represents each chromosome, and the y-axis represents the Chromosome counts, for better presentation, the number of exonic SNVs for essential genes on each chromosome is marked at the top of each bar.
- Rat APOBEC1 is present in the widely used CBE editors of BE3 and BE4, and the rAPOBEC1 enzyme induces DNA cytosine (C) deamination, which is guided by a Cas protein and gRNA complex to target specific sites.
- evoAPOBEC1 is an evolved APOBEC1.
- the 150 sgRNAs sequences targeting 152 gene loci used in the present invention are shown in Table 1, and the same gene name in Table 1 indicates that two positions are targeted, numbering 10, 12 and 13 gene loci
- the sgRNA sequences are identical.
- gBlock-YC1 A gBlock (i.e. gRNA array) containing 5 sgRNA expression cassettes was designed, named gBlock-YC1, and synthesized by a biological company.
- gBlock-YC1 carries sgRNAs of 5 loci (ORC3-1, ORC3-2, PTPA, PMSD13, NOP2-1).
- Each expression cassette contains hU6, sgRNA and polyT sequentially in the 5' to 3' direction.
- the sgRNA sequences of the five gene loci are shown in Table 1.
- five previously published sgRNAs (gBlock PC) were used as positive controls (Thuronyi, B.W. et al. Continuous evolution of base editors with expanded target compatibility and improved activity.
- gBlock-PC carries sgRNAs of 5 endogenous loci (HEK2, HEK3, HEK4, EMX1, RNF2).
- the backbone plasmid of gBlock-YC1 and gBlock-PC is puc57.
- the structures of gBlock-YC1 and gBlockPC are shown in Figure 1.
- HEK293T cells were transiently co-transfected with gBlock-YC1 and gBlockPC and base editor plasmid (evoAPOBEC1-BE4max-NG), respectively.
- Use Lipofectamine 3000 (Thermo Fisher Scientific cat#L3000015) for transfection. The transfection method is modified as follows after referring to the instruction manual: cells are seeded into a 48-well plate, 5 ⁇ 10 4 cells per well, and 250 ⁇ l of cell culture medium is added to culture 24h.
- HEK293T cells were seeded in 6-well plates, 5 ⁇ per well 105 cells were cultured for 24 hours, and then transfected according to the instructions of Lipofectamine 3000. 1 ⁇ g of super transposase plasmid (SBI System Biosciences cat#PB210PA-1) was used to transfect 4 ⁇ g of piggyBac targeting base editor plasmid. After 48h, the cells were selected with puromycin (2ug/ml). After 7-10 days of culture for polyclonal pool selection, or 5-7 days after clonal cell line selection, cells were sorted into single-cell 96-wells by flow cytometry. Puromycin was added regularly during long-term culture.
- Two doxycycline-induced CBE stable cell lines were transiently transfected with gBlock-PC and gBlock-YC1: the cells were seeded in 48-well poly(d-lysine) plates (Corning cat#354413), each well 1 ⁇ 10 5 cells were added and 300 ⁇ l of doxycycline (2 ⁇ g/ml) medium was added and cultured for 24 hours, and a system of 1 ⁇ g gBlock-PC or gBlock-YC1 and 2 ⁇ l Lipofectamine 3000 per well was used for transfection. After transfection, doxycycline was added to culture for 5 days, and the cells were collected for genomic DNA editing analysis.
- the editing efficiency of sgRNAs in gBlock-PC was about 60-70% in evoAPOBEC1-BE4max-NG stable cell line, slightly higher than 45-65% in FNLS-BE3-NG stable cell line.
- the editing efficiency of sgRNAs in gBlock-YC1 was approximately 30-75% in the evoAPOBEC1-BE4max-NG stable cell line, which was significantly higher than the 20-40% in the FNLS-BE3-NG stable cell line.
- the evoAPOBEC1-BE4max-NG stable cell line showed higher base editing efficiency.
- a preferred embodiment of the present invention uses the evoAPOBEC1-BE4max-NG stable cell line for gBlock transfection.
- gBlock-YC1 was transiently transferred into the resulting single clones, and four parallel experiments were set up. Seed the monoclonal cells in a 48-well poly(d-lysine) plate (Corning cat#354413), 1 ⁇ 105 cells per well, and add 300 ⁇ l doxycycline (2 ⁇ g/ml) medium for culture At 24 hours, the system of 1 ⁇ g gBlock-YC1 and 2 ⁇ l Lipofectamine 3000 per well was used for transfection. After transfection, doxycycline was added and cultured for 5 days, and the cells were collected for genomic DNA editing analysis.
- the targeted gene loci are numbered 1-152 in Table 1, and the sgRNA sequence is shown in Table 1.
- 10, 20 and 30 gBlocks pools were respectively co-transfected into clone 1 of the evoAPOBEC1-BE4max-NG stable cell line sorted out in Example 3, as shown in FIG. 7 .
- pools of 10, 20, and 30 gBlocks were delivered to stable cell lines cultured in doxycycline-containing medium or doxycycline-free medium, respectively.
- a heat map of the mutation frequency of the targeted locus "C” was obtained by whole exome sequencing (WES) analysis, as shown in Figure 8. Editing efficiency at most of the 52 loci was best when 10 gBlocks were delivered, compared to 20 gBlocks and 30 gBlocks.
- a preferred embodiment of the present invention delivers 10 gBlocks at a time.
- Each gBlock array contains 5 sgRNA expression cassettes in series. All gBlocks fragments include 5 sgRNA expression cassettes and are directly synthesized into the pUC57 cloning plasmid after containing IIS type BbsI restriction endonuclease sites at both ends. Two oligonucleotide chains SpeI-HF with BbsI restriction sites were annealed and then cloned into the destination vector for the expression of fluorescent protein (DsRed) driven by the CMV promoter.
- DsRed fluorescent protein
- the constructed integrated plasmid contained 43 sgRNAs, and the plasmid was named 43-all-in-one.
- the ten gRNA arrays were delivered into the doxycycline-inducible evoAPOBEC1-BE4max-NG stably expressing cell line using the following 3 methods: Cells were plated in 48-well poly(d-lysine) plates (Corning cat#354413) In each well, 1 ⁇ 10 5 cells were added to 300 ⁇ l polytetracycline (2 ⁇ g/ml) for 24 hours, and the system of 21 ⁇ g plasmid and 3 ⁇ l Lipofectamine 3000 per well was used for transfection. After transfection, polytetracycline was added and cultured for 5 days, and the cells were collected for genomic DNA editing analysis.
- Method 1 10 gBlocks (200ng each), plasmid eGFP L202 Reporter (addgene #119129) (30ng) containing mCherry-inactivated eGFP reporter molecule, and 3ul Lipofectamine 3000.
- Method 2 10 gBlocks (200ng each), plasmid containing mCherry-inactivated eGFP reporter (eGFP L202 Reporter, addgene #119129 (30ng), eGFP L202 gRNA (addgene #119132) (10ng) and 3ul l Lipofectamine 3000 .
- a preferred embodiment of the present invention uses method 2 to deliver the gRNA array.
- gBlocks were transfected into highly modified clone 19 (from method 2) using method 2 and clones 19-1, 19 were selected from 22/96 clones -16 and 19-21, have higher editing (Sanger/EditR) in the selected locus compared to the original clone 19.
- method 2 in Example 6 is used to deliver ten gRNA arrays into cells, and then isolate and culture single clones from the transfected cell population, and again Method 2 in Example 6 was used to deliver ten gRNA arrays into highly modified single clones isolated and cultured.
- SNVs single nucleotide variations
- Indels insertions/deletions
- Ten gRNA arrays were delivered to clone 1 of the evoAPOBEC1-BE4max-NG stable cell line sorted out in Example 3 using method 2: the cells were seeded in 48-well poly(d-lysine) plates (Corning cat#354413 ), 1 ⁇ 10 5 cells per well, and 300 ⁇ l polytetracycline (2 ⁇ g/ml) were added to culture for 24 hours, and the system of 21 ⁇ g plasmid and 3 ⁇ l Lipofectamine 3000 per well was used for transfection. After transfection, polytetracycline was added and cultured for 5 days, and the cells were collected.
- Method 2 10 gBlocks (200ng each), plasmid containing mCherry-inactivated eGFP reporter (eGFP L202 Reporter, addgene #119129 (30ng), eGFP L202 gRNA (addgene #119132) (10ng) and 3ul l Lipofectamine 3000 .
- it further comprises isolating and culturing single clones from the transfected cell population, screening for high editing efficiency single clones, and again using method 2 to deliver the ten gRNA arrays to isolated and cultured highly modified single clones. Cloning. After transfection, polytetracycline was added and cultured for 5 days, and the cells were collected. Repeat this step according to the actual base editing situation.
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Abstract
提供了一种实现多碱基编辑的方法,将gRNA阵列通过2~15个gRNA阵列、含有mCherry-失活eGFP报告分子的质粒、编辑激活eGFP的sgRNA质粒与碱基编辑器共转染到细胞;或者,所述表达sgRNA的载体与碱基编辑器共转染到细胞。或者将gRNA阵列通过2~15个gRNA阵列、含有mCherry-失活eGFP报告分子的质粒、编辑激活eGFP的sgRNA质粒共转染到诱导型碱基编辑器稳定的细胞;或者所述表达sgRNA的载体转染到诱导型碱基编辑器稳定的细胞。从5个sgRNA表达盒开始组装,减少了组装时间;通过多个gRNA阵列转染,在单细胞内实现多个位点的同时靶向,筛选得到多位点编辑细胞的效率更高。
Description
本发明属于生物技术领域,具体涉及一种实现多碱基编辑的方法。
CRISPR–Cas技术通过设计导向RNA(gRNAs)进行特定基因的编辑或转录调控。尽管CRISPR–Cas技术具有普遍的实用性,但单个gRNA的使用限制了效率和生物技术的应用。因此,现在越来越多的研究不再采用单一的gRNA,而是使用多位点编辑或转录调控的多重复合策略。多重复合CRISPR技术,是许多gRNAs或Cas酶被表达,促进了生物工程应用,大大提高了基因编辑和转录调控的范围和效率。
目前,在单细胞中表达多个gRNA的方法主要有两种:一种方法是用单个RNA聚合酶启动子转录每个gRNA盒,然后通过Golden gate装配将多个gRNA表达盒克隆到单个质粒中。另一种方法是使用一个启动子将所有的gRNAs转录到一个转录本中,然后通过不同的策略进行处理以释放单个的gRNAs,这些策略要求每个gRNA的两侧都有可切割的RNA序列,例如自切割核酶序列(例如锤头状核酶和HDV核酶)、外源性切割因子识别序列(例如Cys4)和内源性RNA处理序列(例如tRNA序列和内含子)。
上述多重复合CRISPR技术,sgRNA表达盒组装周期长且效率低;单个细胞内靶向靶点数少且编辑效率低;获取多位点编辑的单克隆时,需要筛选大量克隆,工作量大、成本高。
发明内容
为了解决现有技术中的技术问题,本发明的目的是提出一种实现多碱基编辑的方法。具体方案如下:
本发明第一方面提供一种实现多碱基编辑的方法,包括如下步骤:
步骤1:设计并合成gRNA阵列;或,
步骤1:设计并合成gRNA阵列,将2~15个gRNA阵列组装到表达载体,构建得到表达sgRNA的载体;
所述gRNA阵列包含依次串联的5个sgRNA表达盒,每个所述sgRNA表达盒在5’至3’方向依次包含启动子、sgRNA和polyT,所述sgRNA表达盒中sgRNA为靶向基因位点的sgRNA;
步骤2:将所述gRNA阵列通过如下方法转染到细胞中,实现多碱基编辑;
I:2~15个gRNA阵列或其转录产物、含有mCherry-失活eGFP报告分子的质粒、编辑激活eGFP的sgRNA质粒与碱基编辑器共转染到细胞;
II:所述表达sgRNA的载体或其转录产物与碱基编辑器共转染到细胞。
本发明第二方面提供一种实现多碱基编辑的方法,包括如下步骤:
步骤1:设计并合成gRNA阵列;或,
步骤1:设计并合成gRNA阵列,将2~15个gRNA阵列组装到表达载体,构建得到表达sgRNA的载体;
所述gRNA阵列包含依次串联的5个sgRNA表达盒,每个所述sgRNA表达盒在5’至3’方向依次包含启动子、sgRNA和polyT,所述sgRNA表达盒中sgRNA为靶向基因位点的sgRNA;
步骤2:将所述gRNA阵列通过如下方法转染到诱导型碱基编辑器稳定的细胞中,实现多碱基编辑;
I:2~15个gRNA阵列或其转录产物、含有mCherry-失活eGFP报告分子的质粒与编辑激活eGFP的sgRNA质粒共转染到诱导型碱基编辑器稳定的细胞;
II:所述表达sgRNA的载体或其转录产物转染到诱导型碱基编辑器稳定的细胞。
本发明上述的多碱基编辑的方法,还包括分离培养转染后细胞的单克隆,进行Sanger测序和EditR分析,选择高编辑效率的单克隆,通过方法I或II进行gRNA阵列的转染。
本发明上述的多碱基编辑的方法,所述表达sgRNA的载体为将10个gRNA阵列组装到表达载体所得;
所述方法I中gRNA阵列为10个。
本发明上述的多碱基编辑的方法,所述细胞为哺乳动物细胞;优选地,所述哺乳动物细胞为人哺乳动物细胞;优选地,所述哺乳动物细胞为人胚胎肾细胞;优选地,所述哺乳动物细胞为人胚胎肾细胞293。
本发明上述的多碱基编辑的方法,所述启动子为hU6;
所述表达sgRNA的载体表达报告分子;优选地,所述报告分子为红色荧光蛋白。
本发明上述的多碱基编辑的方法,所述依次串联的5个sgRNA表达盒通过化学方法合成。
本发明上述的多碱基编辑的方法,I中每转染到1×10
5个细胞中,所述gRNA阵列每个的转染量为200ng,所述含有mCherry-失活eGFP报告分子的质粒的转染量为30ng,所述编辑激活eGFP的sgRNA质粒的转染量为10ng;
II中每转染到1×10
5个细胞中,所述表达sgRNA的载体的转染量为2μg。
本发明上述的多碱基编辑的方法,所述诱导型碱基编辑器稳定的细胞选自高编辑效率的诱导型碱基编辑器稳定的细胞单克隆。
进一步地,所述高编辑效率的诱导型碱基编辑器稳定的细胞单克隆的筛选方法为:
筛选诱导型碱基编辑器稳定的细胞单克隆,记为原始单克隆;
将1个gRNA阵列转染到筛选的原始单克隆中,筛选高编辑效率的转染后单克隆;
所述高编辑效率的转染后单克隆所对应的原始单克隆即为所述高编辑效率的诱导型碱基编辑器稳定的细胞单克隆。
本发明上述的多碱基编辑的方法,所述诱导型碱基编辑器为多西环素诱导的碱基编辑器;优选地,为多西环素诱导的胞嘧啶碱基编辑器。
本发明上述的多碱基编辑的方法,所述诱导型碱基编辑器稳定的细胞选自稳定表达PB-FNLS-BE3-NG1或PB-evoAPOBEC1-BE4max-NG的细胞。
本发明第三方面提供上述的多碱基编辑的方法所编辑得到的细胞。
本发明第四方面提供上述方法构建得到的表达sgRNA的载体。
本发明第五方面提供一种碱基编辑系统,包含2~15个所述的gRNA阵列或其转录产物,或者,所述的表达sgRNA的载体或其转录产物。
进一步地,所述碱基编辑系统还包含碱基编辑器;
所述碱基编辑器选自腺嘌呤碱基编辑器或胞嘧啶碱基编辑器;
优选地,所述碱基编辑器为胞嘧啶碱基编辑器。
本发明第六方面提供一种多碱基编辑的试剂盒,所述试剂盒包含所述的碱基编辑系统。
进一步地,所述试剂盒还包括含有mCherry-失活eGFP报告分子的质粒和编辑激活eGFP的sgRNA质粒。
本发明第七方面提供上述的多碱基编辑的方法在基因组重编码、多基因遗传病细胞构建或多基因遗传病治疗中的应用。
本发明的有益效果:
1、本发明提供的实现多碱基编辑的方法,直接合成5个sgRNA表达盒,从5个sgRNA表达盒开始组装,减少了组装时间,增加了成功率;构建多个含有5个sgRNA表达盒的gRNA阵列进行转染,在单细胞内实现多个位点的同时靶向,筛选得到多位点编辑细胞的效率更高;通过少量单克隆培养筛选,即可在单细胞内实现高效的多位点编辑。
2、本发明通过将gBlocks转染至诱导型碱基编辑器稳定的细胞,在多西环素的诱导下,可实现碱基编辑器稳定持续表达,与瞬时表达相比,具有更高得到碱基编辑效率。作为一个优选的方案,本发明通过筛选高编辑效率的诱导型碱基编辑器稳定的细胞单克隆,进一步将gBlocks转染至筛选的高编辑效率单克隆中,可以进一步提高碱基编辑效率。
3、作为一个优选的方案,本发明将gBlocks与含有mCherry-失活eGFP报告分子的质粒、编辑激活eGFP的sgRNA质粒共转染哺乳动物细胞中,转染的报告分子的量约是每个gBlock的十分之一,当报告分子和相应的sgRNA同时转染至单个细胞时,通过gBlock转染到单细胞的靶向基因位点的sgRNAs较多。当报告分子和相应的sgRNA同时在一个单细胞并发生单碱基编辑后,能检测到绿色荧光,红色与绿色双荧光的细胞,即说明转染进去的sgRNAs较多且发生了编辑。通过流式细胞分选即可实现高编辑克隆的富集。
图1为实施例1中gBlock-YC1和gBlockPC的结构示意图。
图2为实施例1中靶向基因座碱基编辑效率验证结果,其中,图2-a为gBlock-PC的编辑效率,图2-b为gBlock-YC1的编辑效率;点代表个体的生物复制,条代表平均值。
图3为实施例2中多西环素诱导的胞苷脱氨酶piggyBac结构示意图,其中,F,flag标签;NLS,核定位信号;cas9n-NG,Cas9D10A识别NG-PAM;APOBEC1,大鼠APOBEC1;evoAPOBEC1,进化的大鼠APOBEC1。
图4为实施例2中靶向基因座碱基编辑效率验证结果,其中,图4-a为gBlock-PC的编辑效率,图4-b为gBlock-YC1的编辑效率;点和三角形代表个体的生物复制,条形代表平均值。
图5为实施例3中evoAPOBEC1-BE4max-NG稳转细胞单克隆中胞嘧啶碱基编辑器的蛋白水平,其中,使用抗Cas9(上)和抗肌动蛋白(下)。
图6为实施例3中靶向基因座碱基编辑效率验证结果,其中,数值和误差线反映了四个独立实验的平均值和标准差。
图7为实施例4中gBlocks池导入evoAPOBEC1-BE4max-NG稳定细胞系。
图8为实施例4中基于外显子全序列分析的靶向基因座“C”突变频率的热图。
图9为实施例5中一体化质粒的构建流程图。
图10为实施例5中一体化质粒的琼脂糖凝胶电泳图;其中,左边是DNA梯,最右边的空向量为对照 组;泳道5和7的箭头为22Kb。
图11为实施例6中3种不同递送方法下单细胞RNA测序的基本质量指标;其中,a为捕获的细胞数,b为每个单元的UMI数,c为每个细胞检测到的基因数。
图12为实施例6中基于单细胞RNAseq的不同修饰基因靶细胞在不同传递方式群体中的分布分析;其中,a,b,c为3个群体中编辑基因位点数量与细胞数量的关系;d为scRNAseq在3个群体中检测到的编辑基因位点数量分布的密度图,垂直线表示编辑基因位点的中值;e为针对每个基因位点,对不同编辑效率的修饰细胞进行分布分析,不同方法的计数。
图13为实施例6中单细胞测序分析不同递送方式sgRNA在单细胞内的编辑效率;其中,g为单个细胞中每个sgRNA的编辑效率;h为基于单细胞RNA-Seq转化为细胞群体RNA-Seq的三种传递方式下细胞群体中目标C编辑效率的热图,编辑效率以黑色强度表示。
图14为实施例7中Sanger测序进行单克隆筛选;其中,a为选取10个经过良好编辑的基因座,gBlocks的峰值数为3,只有一个克隆拥有全部10个gBlocks;b为3个编辑良好的基因座进行筛选,一半的克隆没有任何编辑,4个克隆拥有全部的3个编辑位点;c为通过Sanger测序和EditR对每个克隆的所有靶位点进行等位基因编辑;WT(野生型)-无等位基因编辑;HZ(杂合子)-部分等位基因编辑;HM(纯合)-所有等位基因编辑。
图15为实施例8中WGS鉴定高度修饰HEK293T克隆的遗传变化分析;其中,a为目标“C”的热图编辑将TAG转换为TAA的效率,依次为NC-阴性对照,方法2的克隆19、方法3的克隆21、在克隆19的基础上利用方法2进行第二次转染得到克隆19-1、19-16、19-21,与亲本HEK293T的序列相比,在高度修饰的克隆中检测到的外显子SNV(SNV位于外显子和剪接位点)或其他SNV的数量;与亲本HEK293T的序列相比,克隆19、克隆21、克隆19-1、克隆19-16、克隆19-21的总snv数分别为23084、70356、35700、42595和31530;c-在必需基因中检测到的外显子SNV数量;d-不同类型SNV变化的分布;e-样本间检测到的C>T或G>T SNV的突变率;f-样本和染色体间检测到的C>T或G>T SNV的突变率;g-在高度修饰的克隆中检测到的外显子indels或其他indels的数量;h-在样本中检测到的indels的突变率;i-样本和染色体间检测到的indels突变率。
图16为实施例8中外显子snv在必需基因中的染色体分布;其中,a-含有,b-不含有在选定的50个必需基因靶点;X轴表示每个染色体,y轴表示该染色体的计数,为了更好的展示,每个染色体上必需基因的外显子SNV的数量被标记在每个条的顶部。
为了更清楚地理解本发明,现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中,各原始试剂材料均可商购获得,未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件,或按照仪器制造商所建议的条件。
CBE(Cytosine base editor),胞嘧啶碱基编辑器。大鼠APOBEC1(rAPOBEC1)存在于广泛使用的BE3和BE4的CBE编辑器中,rAPOBEC1酶诱导DNA胞嘧啶(C)脱氨,该酶由Cas蛋白和gRNA复合物引导靶向特定位点。evoAPOBEC1为进化的APOBEC1。
本发明中所利用的靶向152个基因位点的150个sgRNAs序列如表1所示,表1中相同基因名称表示 靶向的是两个位置,编号10、12和13的基因位点的sgRNA序列相同。
表1.靶向152个基因位点的150个sgRNAs
编号 | 基因名称(位置) | sgRNA序列 | SEQ ID NO |
1 | ORC3 | CCAAACCTAGCCTATTATCC | 1 |
2 | ORC3 | AGCTCTAATAAACCGAGCAC | 2 |
3 | PTPA | CCCTCCTAGCCCGACGTGAC | 3 |
4 | PSMD13 | GGCCCTAGGTGAGGATGTCA | 4 |
5 | NOP2 | CCATCTAAGATAGCAGCAGC | 5 |
6 | NOP2 | CCTAGCTACTTGGGAGTCTG | 6 |
7 | ANAPC5 | TCTCTAGAGATGGTTTATCA | 7 |
8 | KIAA0391 | AGAATCTCTATGTCTTTTGG | 8 |
9 | AQR | TTTGGCTACTTGGTCTCTTC | 9 |
10 | TBC1D3B | GATGCTTCTAGAAGCCTGGA | 10 |
11 | TBC1D3F | TTCGTCCCTAGCTCTGAAGG | 11 |
12 | TBC1D3C | GATGCTTCTAGAAGCCTGGA | 10 |
13 | TBC1D3 | GATGCTTCTAGAAGCCTGGA | 10 |
14 | BIRC5 | CCTTTCCTAAGACATTGCTA | 12 |
15 | MRPL12 | TGGAGGCTACTCCAGAACCA | 13 |
16 | NLGN4Y | GAAAAGCTATACTCTAGTGG | 14 |
17 | SRY | TGTCCTACAGCTTTGTCCAG | 15 |
18 | WDR3 | TTCAGTTCTAAGTCAACGTT | 16 |
19 | ECT2 | ATCTCCTAATTCTTCACAAA | 17 |
20 | RPL32 | TGCCTACTCATTTTCTTCAC | 18 |
21 | TFRC | ATGGTGGCTATCCACGATGG | 19 |
22 | POLR2B | ATAGCTAAACACTCATCATT | 20 |
23 | CDC23 | GCCAACTATGGCGTGACAGA | 21 |
24 | RIOK1 | TCATTCTATTTGCCTTTTTT | 22 |
25 | ORC3 | GCTTTCTAGCAGCCTCCCCA | 23 |
26 | MASTL | TTGTGCTACAGACTAAATCC | 24 |
27 | ATP2A2 | ACAACTAAAGTTCTGAGCTA | 25 |
28 | AURKA | GATTCCTAAGACTGTTTGCT | 26 |
29 | RBX1 | CTTTTCCTAGTGCCCATACC | 27 |
30 | LOC105373102 | CAAGGCTAAGTCCCACGTGC | 28 |
31 | CD99 | CAATCTTCTATTTCTCTAAA | 29 |
32 | ZBED1 | TCCTCGCTACAGGAAGCTGC | 30 |
33 | VAMP7 | TCTTTCCTATTTCTTCACAC | 31 |
34 | UTY | GAAACAGCTACAAAACCAGT | 32 |
35 | PPIE | GAGCTCTACGTCAGCTTCCA | 33 |
36 | NUDC | GGGCTAGTTGAATTTAGCCT | 34 |
37 | WDR77 | CCAATCTACTCAGTAACACT | 35 |
38 | SFPQ | CATCTAAAATCGGGGTTTTT | 36 |
39 | SFPQ | ACACACCTAAGTTGTGAAAA | 37 |
40 | NSL1 | CTCTCCTAAACTGCCCCTAG | 38 |
41 | RABGGTB | TGAATCTAGCTCACTAGCTC | 39 |
42 | ISG20L2 | ACTGCCACTAGTCTGTAGGG | 40 |
43 | DTL | TAGAATCTATAATTCTGTTG | 41 |
44 | MAGOH | AGTCTAGATTGGTTTAATCT | 42 |
45 | ZBTB8OS | GAAGCTAGGAGTTCAAGACT | 43 |
46 | TRNAU1AP | GCCTGGCTACATCATGGCAG | 44 |
47 | SNRPE | ATTTCTAGTTGGAGACACTT | 45 |
48 | MTOR | GCACTCTAGCCTGAACAGAG | 46 |
49 | POLR1A | GTAGCTGCTATCTCAGAGGC | 47 |
50 | ATL2 | TACTGTCTAATTTTTCTTCT | 48 |
51 | WDR33 | CTCCGTCTAAGGAGCTGGAA | 49 |
52 | UQCRC1 | TCCCGCCTAGAAGCGCAGCC | 50 |
53 | THOC7 | CCTGTCTATGGCTTAGGATC | 51 |
54 | PSMD6 | CTTTATCTATTTTGCAGTGT | 52 |
55 | RPN1 | CAGGGGCTACAGGGCATCCA | 53 |
56 | RUVBL1 | TGGTCATCTATTTCCAGGTG | 54 |
57 | FIP1L1 | CATGCCTATTCTGCAGGTGT | 55 |
58 | ETF1 | GACTACCTAGTAGTCATCAA | 56 |
59 | NSA2 | AGGCTAAGGCGGGCGGATCA | 57 |
60 | PRELID1 | AGACTGGCTACACAAACTGT | 58 |
61 | SRSF3 | GTCTTCTATTTCCTTTCATT | 59 |
62 | MDN1 | CTGTTCTATGGGTGGTCAGA | 60 |
63 | FARS2 | CACCTCTAGCATCTCAGCTC | 61 |
64 | RPL7L1 | CTGGGTCTAGTTCAGCTGAC | 62 |
65 | RARS2 | AAAGTCTAGAGGCAGAAGGC | 63 |
66 | VPS52 | CCAGCCTAGGTGACAGAGCA | 64 |
67 | WDR46 | GCCCCTAAAAGGCAAAGCTA | 65 |
68 | RFC2 | CTGCTCTAACTGGCCACCGG | 66 |
69 | TNPO3 | GTGAGCTATCGAAACAACCT | 67 |
70 | OGDH | CAGCATCTACGAGAAGTTCT | 68 |
71 | BUD31 | AGTCGACTAAGGCAGAATTT | 69 |
72 | NUP188 | CACTGCCCTATCTTTGCATA | 70 |
73 | SMC2 | CAAAATCTATTTTCCTTCCT | 71 |
74 | POLR1E | GCGTCTAGGTAATCTTCCTC | 72 |
75 | MED22 | CAGCGCTATTTATACCTGGA | 73 |
76 | MED27 | TGGGGGCTACTGCCGGCAGG | 74 |
77 | IARS | ACATGCTAGAAGTCTGCTGT | 75 |
78 | POLR3A | TTTGGACTATGTGACAAGGG | 76 |
79 | PDCD11 | TGCCACTAGTCCTCTAGCAC | 77 |
80 | PRPF19 | GGCCTACAGGCTGTAGAACT | 78 |
81 | NAT10 | TTCACTATTTCTTCCGCTTC | 79 |
82 | NARS2 | CCAGCTATAAAAGGCATGAA | 80 |
83 | SSRP1 | CGTTTCTACTCATCGGATCC | 81 |
84 | PSMC3 | GTGTGCCCTAGGCGTAGTAT | 82 |
85 | MRPL16 | ACACTCACTACACACGTTTG | 83 |
86 | DDB1 | TTGGCTAATGGATCCGAGTT | 84 |
87 | SF1 | CAAGTCTAGTTCTGTGGTGG | 85 |
88 | HINFP | TCAGCTCTACACTCTCGTAG | 86 |
89 | CLP1 | TGATCTCTACTTCAGATCCA | 87 |
90 | INTS5 | AAGGCTACGTCCCCTGTCGA | 88 |
91 | NCAPD2 | GACTTCCTAGGATCTGTGCC | 89 |
92 | RFC5 | AAGCAGGCTACCTTCTCCAC | 90 |
93 | POLE | GCTGGCTAATGGCCCAGCTG | 91 |
94 | POLE | GCCTTCCCTACACCCACCCT | 92 |
95 | DDX51 | CCCCAGCCTAGGCCGCCCTC | 93 |
96 | DDX51 | AAGAGCCTAGGCAGAGAGAA | 94 |
97 | RFC3 | CTTCTACTGGGATACAGCCT | 95 |
98 | POLE2 | GATTAACTACATTCTTACAG | 96 |
99 | PABPN1 | GCCCATCTATCCTGACCTGT | 97 |
100 | DLST | TTCCTCCTAAAGATCCAGGA | 98 |
101 | WARS | GAGTGCTACTGAAAGTCGAA | 99 |
102 | MFAP1 | TTGGACCCTAGGTAGTTTTC | 100 |
103 | GTF3C1 | GTCCTAGAGGTGGATCCACT | 101 |
104 | COG4 | CAGCTACAGGCGCAGCCTCT | 102 |
105 | NUBP1 | CTGTAGGCTAACGTGGCTGG | 103 |
106 | GINS2 | TTCTCTAGAAGTCCTGAGAC | 104 |
107 | RPS15A | ATCCCTAGAAAAAGAATCCC | 105 |
108 | RPS2 | AAACCCTATGTTGTAGCCAC | 106 |
109 | DCTN5 | AGCTCTAAGGAGCTTGAAGA | 107 |
110 | DCTN5 | AGATGCTAGACTTGCGTCAG | 108 |
111 | ATP6V0C | GAGGGTCTACTTTGTGGAGA | 109 |
112 | SMG6 | GTCTTCTACTCCAAAAACTC | 110 |
113 | PSMD11 | CTCACCTATGTCAGTTTCTT | 111 |
114 | SUPT6H | GGCCCCCTACCGATCCATCT | 112 |
115 | RPL27 | GCATCTAAAACCGCAGTTTC | 113 |
116 | VPS25 | TCCCTGCTAGAAGAACTTGA | 114 |
117 | MRPL10 | GCTGGCTACGAGTCCGGAAC | 115 |
118 | U2AF2 | CCGCCTCTACCAGAAGTCCC | 116 |
119 | DNM2 | GAGGCCTAGTCGAGCAGGGA | 117 |
120 | FBXO17 | TCGCTAGGACAGACGGATCC | 118 |
121 | CLASRP | TCTGCCTAATGTCGGTAATG | 119 |
122 | RPS16 | GTCAGCTACCAGCAGGGTCC | 120 |
123 | MRPL4 | GTGATTCTAACAGCGGAGCC | 121 |
124 | MRPL4 | TGTGGTCTAGTGTGACTTTG | 122 |
125 | RPS19 | TTGTTCTAATGCTTCTTGTT | 123 |
126 | RPL18A | TGCACCTAGAAGAAGGTGTT | 124 |
127 | ELL | GCGGCTAGGGCCAAGCCTGC | 125 |
128 | SNRPD2 | CGGCCCCTACTTGCCGGCGA | 126 |
129 | DOHH | GGGGCCCTAGGAGGGGGCCC | 127 |
130 | UBE2M | GCCAACCCTATTTCAGGCAG | 128 |
131 | ZC3H4 | GGACACTACTGGCAAAAGGG | 129 |
132 | SAE1 | ATGGACTAGTGTCTCGGCTT | 130 |
133 | LENG8 | GGTCTCTATGGTGGGAGCAC | 131 |
134 | EEF2 | GGCCGCCTACAATTTGTCCA | 132 |
135 | UBL5 | TTCTCATCTATTGATAATAA | 133 |
136 | RAE1 | AGCCACTACTTCTTATTCCT | 134 |
137 | TTI1 | AGGCTCTAAGCACTGCCAGG | 135 |
138 | ZNF335 | AGGTTCTAGGAGAAGATGGA | 136 |
139 | NFS1 | CTTCTAGTGTTGGGTCCACT | 137 |
140 | SON | ATTTGCTACCACCAAAATCT | 138 |
141 | SF3A1 | TCTTGTCTACTTCTTCCTCC | 139 |
142 | PPIL2 | CTGCTGCTACCAGGAGCTGA | 140 |
143 | PPIL2 | ACCTCTAGTGGTCATCAGGC | 141 |
144 | EP300 | TGTCTCTAGTGTATGTCTAG | 142 |
145 | RANGAP1 | TGAGTCTAGACCTTGTACAG | 143 |
146 | POLR3H | GGGCTAGTTGCTGGTCCACC | 144 |
147 | ADSL | CAACTCTACAGACATAATTC | 145 |
148 | SMC1A | ATACTGCTACTGCTCATTGG | 146 |
149 | PGK1 | AAGTACTAAATATTGCTGAG | 147 |
150 | RBMX | TTATCTACTGTGAATCAATC | 148 |
151 | RBMX | TTGTTTCTAGTATCTGCTTC | 149 |
152 | SKI | GGAATCTACGGCTCCAGCTC | 150 |
实施例1
1、gRNA阵列的合成
设计包含5个sgRNA表达盒的gBlock(即gRNA阵列),命名为gBlock-YC1,并由生物公司合成。gBlock-YC1携带5个基因位点(ORC3-1、ORC3-2、PTPA、PMSD13、NOP2-1)的sgRNA。每个表达盒在5’至3’方向依次包含hU6、sgRNA和polyT。5个基因位点的sgRNA的序列如表1。同时,以5个先前发表的sgRNAs(gBlock PC)作为阳性对照(Thuronyi,B.W.et al.Continuous evolution of base editors with expanded target compatibility and improved activity.Nat Biotechnol 37,1070-1079(2019))。gBlock-PC携带5个内源性位点(HEK2、HEK3、HEK4、EMX1、RNF2)的sgRNA。gBlock-YC1和gBlock-PC的骨架质粒为puc57。gBlock-YC1和gBlockPC的结构如图1所示。
2、转染HEK293T细胞
将gBlock-YC1和gBlockPC分别与碱基编辑器质粒(evoAPOBEC1-BE4max-NG)瞬时共转染HEK293T细胞。使用Lipofectamine 3000(Thermo Fisher Scientific cat#L3000015)进行转染,转染方法参考使用说明书后做如下修改:将细胞接种至48孔板中,每孔5×10
4个细胞,加入250μl细胞培养液培养24h。对于 单个gBlock质粒和碱基编辑器质粒,每孔共使用1ug DNA(碱基编辑器质粒750ng,单个gBlock质粒250ng)和Lipofectamine 3000 2μl的体系经行转染。
对靶向基因座进行Sanger测序和EditR分析,获得C-to-T转换的频率(%),如图2。gBlock-PC和gBlock-YC1所靶向的基因座的编辑效率分别为40%-50%和20%-50%。表明gBlock-YC1可以保持较高的碱基编辑效率。
实施例2
1、构建多西环素诱导的CBE稳定细胞系
利用PB转座子技术构建两个多西环素诱导的PB-FNLS-BE3-NG1和PB-evoAPOBEC1-BE4max-NG稳定表达的HEK293T细胞系:将HEK293T细胞接种于6孔板,每孔5×10
5个细胞,培养24h后,按照Lipofectamine 3000的使用说明书进行转染,用1μg超级转座酶质粒(SBI System Biosciences cat#PB210PA-1)转染4μg piggyBac靶向碱基编辑器质粒。48h后,细胞用嘌呤霉素(2ug/ml)进行筛选。多克隆池筛选后培养7-10天,或克隆细胞系筛选后5-7天,通过流式细胞术将细胞分选到单细胞96孔中。长期培养时定期加入嘌呤霉素。
多西环素诱导的胞苷脱氨酶piggyBac结构如图3所示。
2、转染多西环素诱导的CBE稳定细胞系
将gBlock-PC和gBlock-YC1分别瞬时转染两种多西环素诱导的CBE稳定细胞系:将细胞接种于48孔聚(d-赖氨酸)板(Corning cat#354413)中,每孔1×10
5个细胞,并加入并加入300μl含多西环素(2μg/ml)培养基培养24h,每孔1μg gBlock-PC或gBlock-YC1和2μl Lipofectamine 3000的体系进行转染。转染后,再加入多西环素培养5d,收集细胞进行基因组DNA编辑分析。
对靶向基因座进行Sanger测序和EditR分析,获得C-to-T转换的频率(%),如图4。gBlock-PC中sgRNAs的编辑效率在evoAPOBEC1-BE4max-NG稳定细胞系中约为60-70%,略高于在FNLS-BE3-NG稳定细胞系的45-65%。gBlock-YC1中sgRNAs的编辑效率在evoAPOBEC1-BE4max-NG稳定细胞系约为30-75%,显著高于在FNLS-BE3-NG稳定细胞系的20-40%。evoAPOBEC1-BE4max-NG稳定细胞系的碱基编辑效率更高。
为了获得更高的碱基编辑效率,本发明的一个优选实施方案采用evoAPOBEC1-BE4max-NG稳定细胞系进行gBlock的转染。
实施例3
1、从evoAPOBEC1-BE4max-NG稳定细胞系分选出单克隆
利用流式细胞仪从evoAPOBEC1-BE4max-NG稳定细胞系中分选出单克隆,得到克隆1,3,4,5,6,16,17,19,21,23,25,进行培养。在多西环素诱导5天后,进行蛋白质免疫印迹,进行了三次独立重复实验,每个克隆的胞嘧啶碱基编辑器的蛋白表达水平如图5,图5中免疫印迹图片是三个独立实验的代表。
2、转染单克隆
将gBlock-YC1瞬转到所得的单克隆中,设置四个平行实验。将单克隆细胞接种于48孔聚(d-赖氨酸)板(Corning cat#354413)中,每孔1×10
5个细胞,并加入300μl含多西环素(2μg/ml)培养基培养24h, 每孔1μg gBlock-YC1和2μl Lipofectamine 3000的体系进行转染。转染后,再加入强力霉素培养5d,收集细胞进行基因组DNA编辑分析。
对靶向基因座进行Sanger测序和EditR分析,获得C.G-to-T.A转换的频率(%),如图6。克隆1中5个基因位点的编辑效率在11个克隆中最高的。
实施例4
10个gBlocks:所靶向基因位点是表1中编号1-52,sgRNA序列如表1所示。
20个gBlocks:所靶向基因位点是表1中编号1-102,sgRNA序列如表1所示。
30个gBlocks:所靶向基因位点是表1中编号1-152,sgRNA序列如表1所示。
将10、20和30个gBlocks池分别共转染到实施例3分选出的evoAPOBEC1-BE4max-NG稳定细胞系的克隆1中,如图7。具体地将10、20和30个gBlocks池分别递送到含有多西环素的培养基的稳转细胞系内或者不含多西环素的培养基培养的稳转细胞系内。
将细胞接种于48孔聚(d-赖氨酸)板(Corning cat#354413)中,每孔1×10
5个细胞,并加入300μl含多西环素(2μg/ml)培养基,20mM p53抑制剂(Stem Cell Technologies cat#72062)和20ng/ml人源重组bFGF(Stem Cell Technologies cat#78003)培养24h,对于10个gBlocks池,每孔采用200ng/gBlocks和3ul Lipofectamine 3000的体系进行转染,20ng绿色荧光蛋白作为转染对照;对于20个gBlocks池,每孔采用150ng/gBlocks和3ul Lipofectamine 3000的体系进行转染,20ng绿色荧光蛋白作为转染对照;对于30个gBlocks池,每孔采用100ng/gBlocks和3ul Lipofectamine 3000的体系进行转染,20ng绿色荧光蛋白作为转染对照。转染后,再加入多西环素培养5d,收集细胞进行基因组DNA编辑分析。
通过全外显子测序(WES)分析,获得靶向基因座“C”突变频率的热图,如图8。与递送20个gBlocks和30个gBlocks相比,当递送10个gBlocks时,在52个基因位点中的多数位点的编辑效率是最好的。
为了获得更高的碱基编辑效率,本发明的一个优选实施方案一次递送10个gBlock。
实施例5
通过Golden gate assembly将10个gBlocks组装到含DsRed表达载体,如图9。
软件设计靶向基因位点的sgRNAs序列,串联并送商业公司合成多个gRNA阵列单元(gBlocks),每个gBlock阵列包含依次串联的5个sgRNA表达盒。所有gBlocks片段包括5个sgRNA表达框,并在两端含有IIS型BbsI限制性内酶酶切位点后,直接合成到PUC57克隆质粒中。两个具有BbsI酶切位点的寡核苷酸链SpeI-HF经退火后克隆到CMV启动子驱动荧光蛋白(DsRed)表达的目的载体中。用BbsI-HF分别酶切10gBlocks和目的质粒,用凝胶提取试剂盒(Zymo Research cat#11-301C)进行凝胶提取。用T4 DNA连接酶(NEB cat#M0202S)在16℃过夜,将gBlocks片段与质粒连接。连接反应完成后,将2μl反应混合物转化到大肠杆菌NEB Stable菌株。根据使用说明书,使用QIAprep spin纯化试剂盒(cat#27104)从菌液中分离质粒DNA。
通过琼脂糖凝胶电泳分析最终的一体化质粒中sgRNAs是否插入成功。选取九个质粒进行检测,九个质粒均用核酸内切酶spe1线性化,因为在多个sgRNAs插入位点的两侧都有一个SpeI位点,当多个sgRNAs在质粒中成功插入时,用SpeI酶切质粒后,在凝胶电泳上可以看到两条条带。一个片段长约为4479bp,另一个片段长约为22140bp。九个被检测的质粒中有两个具有正确的插入大小,sgRNAs插入成功。结果 如图10。
通过sanger测序验证多个sgRNAs的插入。由测序结果可知,构建的一体化质粒含43个sgRNA,该质粒命名为43-all-in-one。
实施例6
采用以下3种方法将十个gRNA阵列递送到多西环素诱导的evoAPOBEC1-BE4max-NG稳定表达细胞系中:将细胞接种于48孔聚(d-赖氨酸)板(Corning cat#354413)中,每孔1×10
5个细胞,并加入300μl多四环素(2μg/ml)培养24h,每孔21μg质粒和3μl Lipofectamine 3000的体系进行转染。转染后,再加入多四环素培养5d,收集细胞进行基因组DNA编辑分析。
方法1:10个gBlocks(每个200ng)、含有mCherry-失活eGFP报告分子的质粒eGFP L202 Reporter(addgene#119129)(30ng)和3ul Lipofectamine 3000。
方法2:10个gBlocks(每个200ng)、含有mCherry-失活eGFP报告分子的质粒(eGFP L202 Reporter,addgene#119129(30ng)、eGFP L202 gRNA(addgene#119132)(10ng)和3ul l Lipofectamine 3000。
方法3:2ug 43-all-in-one质粒和3ul Lipofectamine 3000。
10个gBlocks:所靶向基因位点是表1中编号1-52,sgRNA序列如表1所示。
从每种方法下分离约1000个单细胞,3种不同递送方法下单细胞RNA测序的基本质量指标如图11。利用CRISPResso2软件,比对上HEK293T细胞中47个基因位点的38个,并观察到三种方法中随着单细胞内编辑位点数量的增加,细胞的数量而减少。方法2中多基因位点同时编辑的细胞数量最多,绘制细胞的种群密度图,分析每个目标的编辑效率以及目标位置的编辑事件呈双峰分布(图12)。
同时,分析每个细胞中所有靶向位点的编辑效率和每个递送方法下中所有靶向位点的总编辑效率,如图13。结果表明,方法2是三种递送方法中编辑效率最高的。
为了获得更高的碱基编辑效率,本发明的一个优选实施方案采用方法2进行gRNA阵列的递送。
实施例7
分别从实施例6的方法2和方法3转染的细胞群体中分离培养了28/96和24/96个单克隆。
对于方法2的克隆,挑选了10个容易编辑的基因座(表1中PSMD13,ANAPC5,BIRC5,WDR3,MASTL,RBX1,PPIE,RABGGTB,SNRPE,UQCRC1),进行PCR扩增,然后进行Sanger测序和EditR分析,发现4个克隆没有被转入任何gBlocks和24个克隆分别被转入1-10个不同数目的gBlocks,其中克隆19被转入了所有10个gBlocks。
对于方法3的克隆,使用3个容易编辑的基因座(表1中PSMD13,ANAPC5,BIRC5)进行筛选,发现13个克隆3个位置都没被编辑,11个克隆分别有几个位点被编辑,其中克隆11、20、21和24在3个位点都有编辑。
对两个高度修饰的克隆:克隆19(来自方法2)和克隆21(来自方法3)的所有靶向基因座进行了Sanger测序。结果所示,在克隆19中,在33/47个基因组位点上发现了TAG到TAA的转变,其中9个位点为纯合位点,14/47个位点为未编辑位点;在克隆21中,发现了27/40个位点发生了TAG到TAA转变,其中10个位点为纯合位点,13/40个位点为未编辑位点(图14)。
为了确定编辑效率是否可以随着随后的转染轮而提高,使用方法2将gBlocks转染到高度修饰的克隆 19(来自方法2)中,并从22/96克隆中选择克隆19-1、19-16和19-21,与原始克隆19相比,在选择的基因座中有更高的编辑(Sanger/EditR)。
为了获得更高的碱基编辑效率,本发明的一个优选实施方案,采用实施例6中的方法2将十个gRNA阵列递送到细胞中,然后从转染的细胞群体中分离培养单克隆,再次采用实施例6中的方法2将十个gRNA阵列递送到分离培养的高度修饰的单克隆中。
实施例8
为了全面评估CBE全基因组TAG到TAA转化的靶向编辑和脱靶效率,对实施例7中高度修饰的克隆(19,21,19-1,19-16,19-21)和阴性对照(HEK293T细胞)进行30倍全基因组测序(WGS)。
在靶向编辑方面,在高度修饰的克隆中,有39/47个基因位点被比对上,其中28个位点有着较高编辑,克隆19-1、19-16、19-21在选择位点的编辑能力比克隆19有所提高,这一结果与实施例7的Sanger测序结果一致。
为了找出脱靶事件,分析高度修饰克隆(19,21,19-1,19-16,19-21)中的单核苷酸变异(SNVs)和插入/缺失(indels)。与对照组相比,减去靶向位置后,克隆19、克隆21、克隆19-1、克隆19-16、克隆19-21的SNVs分别为23084、70356、35700、42595和31530。进一步分析发现,277、805、419、470、358个SNVs分别位于外显子上,只有33、77、42、46、40个SNVs分别位于必需基因的外显子上。将SNVs分为不同的突变类型,发现C-to-T(G-to-A)转换是最常见的编辑(图15)。SNV突变率很低,但在每个克隆中都可以看到,并分布在每个染色体上。除SNVs外,在这些克隆中检测到的indels数分别为558、715、717、662、655,其中一小部分位于外显子,而没有在必需基因的外显子上。每个克隆和染色体的indel比率也都很低(图16)。
实施例9
采用方法2将十个gRNA阵列递送到实施例3分选出的evoAPOBEC1-BE4max-NG稳定细胞系的克隆1中:将细胞接种于48孔聚(d-赖氨酸)板(Corning cat#354413)中,每孔1×10
5个细胞,并加入300μl多四环素(2μg/ml)培养24h,每孔21μg质粒和3μl Lipofectamine 3000的体系进行转染。转染后,再加入多四环素培养5d,收集细胞。
方法2:10个gBlocks(每个200ng)、含有mCherry-失活eGFP报告分子的质粒(eGFP L202 Reporter,addgene#119129(30ng)、eGFP L202 gRNA(addgene#119132)(10ng)和3ul l Lipofectamine 3000。
在一个更优选的实施方案中,进一步包括从转染的细胞群体中分离培养单克隆,筛选高编辑效率的单克隆,再次采用方法2将这十个gRNA阵列递送到分离培养的高度修饰的单克隆中。转染后,再加入多四环素培养5d,收集细胞。根据实际的碱基编辑情况,重复该步骤。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (19)
- 一种实现多碱基编辑的方法,其特征在于,包括如下步骤:步骤1:设计并合成gRNA阵列;或,步骤1:设计并合成gRNA阵列,将2~15个gRNA阵列组装到表达载体,构建得到表达sgRNA的载体;所述gRNA阵列包含依次串联的5个sgRNA表达盒,每个所述sgRNA表达盒在5’至3’方向依次包含启动子、sgRNA和polyT,所述sgRNA表达盒中sgRNA为靶向基因位点的sgRNA;步骤2:将所述gRNA阵列通过如下方法转染到细胞中,实现多碱基编辑;I:2~15个gRNA阵列或其转录产物、含有mCherry-失活eGFP报告分子的质粒、编辑激活eGFP的sgRNA质粒与碱基编辑器共转染到细胞;II:所述表达sgRNA的载体或其转录产物与碱基编辑器共转染到细胞。
- 一种实现多碱基编辑的方法,其特征在于,包括如下步骤:步骤1:设计并合成gRNA阵列;或,步骤1:设计并合成gRNA阵列,将2~15个gRNA阵列组装到表达载体,构建得到表达sgRNA的载体;所述gRNA阵列包含依次串联的5个sgRNA表达盒,每个所述sgRNA表达盒在5’至3’方向依次包含启动子、sgRNA和polyT,所述sgRNA表达盒中sgRNA为靶向基因位点的sgRNA;步骤2:将所述gRNA阵列通过如下方法转染到诱导型碱基编辑器稳定的细胞中,实现多碱基编辑;I:2~15个gRNA阵列或其转录产物、含有mCherry-失活eGFP报告分子的质粒与编辑激活eGFP的sgRNA质粒共转染到诱导型碱基编辑器稳定的细胞;II:所述表达sgRNA的载体或其转录产物转染到诱导型碱基编辑器稳定的细胞。
- 根据权利要求1或2所述的多碱基编辑的方法,其特征在于,还包括分离培养转染后细胞的单克隆,进行Sanger测序和EditR分析,选择高编辑效率的单克隆,通过方法I或II进行gRNA阵列的转染。
- 根据权利要求1或2所述的多碱基编辑的方法,其特征在于,所述表达sgRNA的载体为将10个gRNA阵列组装到表达载体所得;所述方法I中gRNA阵列为10个。
- 根据权利要求1或2所述的多碱基编辑的方法,其特征在于,所述细胞为哺乳动物 细胞;优选地,所述哺乳动物细胞为人哺乳动物细胞;优选地,所述哺乳动物细胞为人胚胎肾细胞;优选地,所述哺乳动物细胞为人胚胎肾细胞293。
- 根据权利要求1或2所述的多碱基编辑的方法,其特征在于,所述启动子为hU6;所述表达sgRNA的载体表达报告分子;优选地,所述报告分子为红色荧光蛋白。
- 根据权利要求1或2所述的多碱基编辑的方法,其特征在于,所述依次串联的5个sgRNA表达盒通过化学方法合成。
- 根据权利要求1或2所述的多碱基编辑的方法,其特征在于,I中每转染到1×10 5个细胞中,所述gRNA阵列每个的转染量为200ng,所述含有mCherry-失活eGFP报告分子的质粒的转染量为30ng,所述编辑激活eGFP的sgRNA质粒的转染量为10ng;II中每转染到1×10 5个细胞中,所述表达sgRNA的载体的转染量为2μg。
- 根据权利要求2所述的多碱基编辑的方法,其特征在于,所述诱导型碱基编辑器稳定的细胞选自高编辑效率的诱导型碱基编辑器稳定的细胞单克隆。
- 根据权利要求9所述的多碱基编辑的方法,其特征在于,所述高编辑效率的诱导型碱基编辑器稳定的细胞单克隆的筛选方法为:筛选诱导型碱基编辑器稳定的细胞单克隆,记为原始单克隆;将1个gRNA阵列转染到筛选的原始单克隆中,筛选高编辑效率的转染后单克隆;所述高编辑效率的转染后单克隆所对应的原始单克隆即为所述高编辑效率的诱导型碱基编辑器稳定的细胞单克隆。
- 根据权利要求2所述的多碱基编辑的方法,其特征在于,所述诱导型碱基编辑器为多西环素诱导的碱基编辑器;优选地,为多西环素诱导的胞嘧啶碱基编辑器。
- 根据权利要求2所述的多碱基编辑的方法,其特征在于,所述诱导型碱基编辑器稳定的细胞选自稳定表达PB-FNLS-BE3-NG1或PB-evoAPOBEC1-BE4max-NG的细胞。
- 权利要求1或2所述的多碱基编辑的方法所编辑得到的细胞。
- 一种权利要求1或2所述方法构建得到的表达sgRNA的载体。
- 一种碱基编辑系统,其特征在于,包含2~15个权利要求1或2所述的gRNA阵列或其转录产物,或者,权利要求13所述的表达sgRNA的载体或其转录产物。
- 根据权利要求15所述的碱基编辑系统,其特征在于,所述碱基编辑系统还包含碱基编辑器;所述碱基编辑器选自腺嘌呤碱基编辑器或胞嘧啶碱基编辑器;优选地,所述碱基编辑器为胞嘧啶碱基编辑器。
- 一种多碱基编辑的试剂盒,其特征在于,所述试剂盒包含权利要求15所述的碱基编辑系统。
- 根据权利要求17所述的试剂盒,其特征在于,所述试剂盒还包括含有mCherry-失活eGFP报告分子的质粒和编辑激活eGFP的sgRNA质粒。
- 权利要求1或2所述的多碱基编辑的方法在基因组重编码、多基因遗传病细胞构建或多基因遗传病治疗中的应用。
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