WO2020063520A1 - 一种基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法及其在基因编辑中的应用 - Google Patents
一种基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法及其在基因编辑中的应用 Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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Definitions
- the invention belongs to the technical field of molecular biology. More specifically, it relates to a method for detecting off-target effects of an adenine single base editing system (ABE) based on whole genome sequencing and its application in gene editing.
- ABE adenine single base editing system
- the CRISPR / Cas9 system is a new artificial nuclease technology, which is a complex composed of gRNA (guide RNA) and Cas9 protein.
- gRNA guide RNA
- Cas9 protein With the help of the PAM (Protospacer adjacent motif) sequence at the 3 'end of the target site, the gRNA-Cas9 protein complex binds to the target DNA through 20 bases at the 5' end of the gRNA, thereby recruiting the endonuclease Cas9 to the target At the site, the target DNA is cut, thereby editing the target gene.
- PAM Protospacer adjacent motif
- the ABE system is composed of two components: TadA: TadA *: Cas9 fusion protein and gRNA. Under the guidance of gRNA, the TadA: TadA *: Cas9 fusion protein can bind to the target site on the DNA.
- the DNA strand complementary to the gRNA will be cut by Cas9 nuclease, instead of the A base at positions 4-9 on the complementary strand.
- the base is catalyzed by adenine deaminase, TadA protein, to form a base.
- the ABE system is more efficient. Because the ABE system can achieve A to G base substitution without inducing DNA double-strand breaks, it is also more secure than CRISPR / Cas9 nucleases.
- the purpose of the present invention is to provide a method for detecting off-target effects of adenine single base editing system based on whole-genome sequencing and its application in gene editing.
- the present invention detects off-target effects of the ABE system through techniques such as gene synthesis, molecular cloning, protein expression purification, in vitro transcription, nucleic acid purification, whole genome sequencing, PCR product deep sequencing, and bioinformatics analysis. At the same time, we verified the validity and sensitivity of the detection method by combining cell transfection and deep sequencing of PCR products.
- the present invention provides a method for detecting off-target effects of an adenine single base editing system based on whole genome sequencing, which is characterized by including the following steps:
- the TadA: TadA *: Cas9 and gRNA complex cut the DNA strand to be tested complementary to the gRNA, and at the same time, convert adenine on the non-complementary strand to hypoxanthine;
- the present invention provides a method for detecting off-target effects of the adenine single base editing system based on whole genome sequencing (EndoV-seq); the EndoV-seq first uses in vitro purified TadA: TadA *: Cas9 fusion protein and gRNA co-process genomic DNA; the complex of TadA: TadA *: Cas9 and gRNA will cut the DNA strand complementary to gRNA, and simultaneously convert A on non-complementary strand to I; then, use Endonuclease V (EnodV) cuts genomic DNA containing I bases, causing DNA double-strand breaks. Finally, whole-genome sequencing combined with bioinformatics analysis is used to detect DNA double-strand breaks, thereby exploring the off-target effects of the ABE system . We call this method EndoV-seq.
- the TadA: TadA *: Cas9 fusion protein includes an effector protein domain and an adenosine deaminase domain of the CRISPR / Cas system.
- the TadA: TadA *: Cas9 fusion protein includes an effector protein domain, a linker polypeptide, and an adenosine deaminase domain of the CRISPR / Cas system.
- TadA Cas9 fusion protein of the present invention is a fusion of Cas9 effector protein and adenosine deaminase (referred to as TadA protein), and those skilled in the art can use as needed
- One or more linking polypeptides link a Cas9 effector protein domain with one or more TadA proteins to obtain a fusion protein.
- the TadA proteins are all repeated once.
- linking sequence of the N-terminus and C-terminus of the Cas9 effector protein and the TadA protein is conventional technology in the art, and the linking polypeptide includes, but is not limited to, conventional linking polypeptide fragments in the art, such as GS linker.
- the gRNA sequence designed by the present invention includes:
- HBG GTGGGGAAGGGGCCCCCAAG AGG , where the underlined is the PAM sequence
- VEGFA3 GGTGAGTGAGTGTGTGCGTG TGG , where the underlined is the PAM sequence.
- TadA * Cas9 fusion proteins
- TadA is an abbreviation of adenosine deaminase
- TadA * is an abbreviation of TadA mutant
- Cas9 is a Cas9 effector protein of the CRISPR / Cas system.
- the Cas9 effector protein includes, but is not limited to, Cas protein Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 ( SaCas9), Lachnospiraceae Cpf1 (LbCpf1), Acidaminococcus Cpf1 (AsCpf1), Streptococcus thermomophilus Cas9 (StCas9), and Neisseria meningitidis Cas9 (NmCas9) and Francisella Cpf1 (FnCp).
- SpCas9 Cas protein Streptococcus pyogenes Cas9
- SaCas9 Staphylococcus aureus Cas9
- LbCpf1 Lachnospiraceae Cpf1
- AsCpf1 Acidaminococcus Cpf1
- Streptococcus thermomophilus Cas9 StCas9
- TadA * Cas9 fusion protein
- SEQ ID NO.1 the amino acid sequence of the adenosine deaminase TadA protein is shown in SEQ ID NO.1.
- the amino acid sequence of the TadA: TadA *: Cas9 fusion protein is shown in SEQ ID No. 2 or at least 80% and 85% of the amino acid shown in SEQ ID NO. 2 , 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical sequences.
- the TadA: TadA *: Cas9 fusion protein is prepared by synthesizing a synthetic TadA: TadA *: Cas9 fusion protein prokaryotic expression vector (pET42b-ABE7.10, SEQ ID ID NO .3) The vector is expressed in E. coli and the TadA: TadA *: Cas9 fusion protein is purified.
- the preparation process includes step (1) transforming pET42b-ABE7.10 into BL21Star TM (DE3) E. coli (Thermo Fisher) competence.
- the method for purifying and preserving the TadA: TadA *: Cas9 fusion protein in step (3) is: 4000 rpm, 10 minutes to collect the induced bacterial cells, and add 10 ml of lysate (100mM Tris-HCl, pH 8.0, 1M NaCl, 20 % Glycerol, 5 mM TCEP (Sigma-Aldrich), 0.4 mM PMSF (Sigma-Aldrich), protease inhibitor (Roche) and 20 mM Imidazole (Sigma-Aldrich)). Ultrasound (5min total, 2s on, 5s off) initially disrupted the cells.
- lysate 100mM Tris-HCl, pH 8.0, 1M NaCl, 20 % Glycerol, 5 mM TCEP (Sigma-Aldrich), 0.4 mM PMSF (Sigma-Aldrich), protease inhibitor (Roche) and 20 mM Imidazole (Sigma-A
- the mixed solution was poured into a chromatography column, and then Ni-NTA agarose resin (GE Healthcare) was washed with 40 ml of washing solution (100 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 20% glycerol, 5 mM TCEP, 20 mM imidazole). Then, the protein was eluted from the Ni column with an eluent (100 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 20% glycerol, 5 mM TCEP, 270 mM Imidazole). The eluted protein was passed through 5 ml cation exchange chromatography (Hi-Trap HP SP cation exchange column, GE Healthcare).
- the method for preparing the gRNA includes (1) chemically synthesizing gRNA; (2) synthesizing gRNA in vitro.
- the reaction system is a solution reaction system, and the solution reaction system further includes the TadA: TadA *: Cas9 fusion protein to convert adenine on the non-complementary strand into A buffer component required for hypoxanthine.
- the step (3) includes:
- the step (3) further comprises: predicting that the adenine single base editing system is in a cell (including a human cell, an animal cell, a plant cell, etc.) or a body (including a human, animal, Off-target effects in plants, etc.).
- the present invention provides a kit for detecting off-target effects of an adenine single base editing system based on whole genome sequencing, including a gRNA sequence or a TadA: TadA *: Cas9 fusion provided in the first aspect to target DNA Protein, EndoV nuclease.
- the present invention provides an application of a method for detecting off-target effects of an adenine single base editing system based on whole genome sequencing in detecting off-target effects of an adenine single base editing system.
- the present invention utilizes TadA: TadA *: Cas9 fusion protein and gRNA purified in vitro, and uses a fusion protein-gRNA complex to treat genomic DNA.
- the treated genomic DNA was then purified using a nucleic acid purification kit.
- the purified genomic DNA was digested with endonuclease V. After the digestion is completed, the genomic DNA is purified and subjected to whole genome sequencing to detect the off-target effect of the whole genome of the ABE system.
- the detection method (EndoV-seq) and the detection result are also within the protection scope of the present invention.
- gRNAs with high efficiency and specificity can be selected based on the detection results of EndoV-seq. EndoV-seq as a preferred method for gRNA is also within the scope of the present invention.
- the invention will promote the clinical application of precision gene editing therapy using the ABE system as a tool, the application of precision disease model construction, and the application of precision gene editing plants or crops.
- the detection limit of the method for the off-target efficiency of the intracellular adenine single base editing system can reach at least 0.13%.
- the method has a detection limit of at least 0.13% for the off-target efficiency of the adenine single base editing system at the HBG-OT9 site in the cell.
- EndoV-seq can also be used to detect the efficiency and off-target activity of other enzymes or chemical reagents capable of converting A bases to I bases.
- Enzymes include, but are not limited to, TadA adenine deaminase.
- the invention provides a method for detecting off-target effect of adenine single base editing system based on whole genome sequencing.
- the detection method can be used for detecting the off-target effect of adenine single base editing system, and promotes adenine single base editing system in disease genes.
- Applications such as therapy, model building, and gene-edited plant or crop cultivation have broad application prospects.
- Figure 1 shows the protein gel electrophoresis of Cas9 protein, BE3 protein, and TadA: TadA *: Cas9 protein; the first lane is the protein molecule Marker, the second lane is Cas9 protein, the third lane is BE3 protein, and the fourth lane is TadA: TadA *: Cas9 protein;
- Figure 2 shows the results of gRNA agarose gel electrophoresis. Both lanes are gRNA.
- FIG. 3 shows that TadA: TadA *: Cas9 protein-gRNA complex and EndoV co-treatment can cleave the target DNA molecule.
- FIG. 4 shows that EnodV-seq can detect DNA double-strand breaks at the target site.
- Figure 5 shows the genome-wide off-target effects of HBG and VEGFA3-mediated ABE systems detected by EndoV-seq.
- Circos plot shows the target sites and off-target sites detected by EndoV-seq. The red arrows indicate the target sites.
- Figure B shows the molecular pattern of off-target sites displayed by Weblog. The target sequence of gRNA is marked below, and PAM is marked with green letters.
- Figure 6 is the deep sequencing of PCR products to verify the off-target sites of HBG. Six of the 18 off-target sites can be verified and marked with *.
- Figure 7 shows the deep sequencing of PCR products to verify off-target sites of VEGFA3. Three of the 22 off-target sites can be verified and marked with an *.
- FIG. 8 is a schematic flowchart of a method for detecting off-target effects of an adenine single base editing system (EndoV-seq) based on whole genome sequencing of the present invention.
- EndoV-seq adenine single base editing system
- the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available. Experimental methods without specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.
- the present invention provides a system, method, kit, and application for detecting off-target effects of the adenine single base editing system based on whole genome sequencing.
- the method for detecting off-target effect of adenine single base editing system based on whole genome sequencing provided by the present invention adopts the kit for detecting off-target effect of adenine single base editing system based on whole genome sequencing provided by the present invention.
- the method includes: Not limited to one or more of the following steps:
- TadA * Cas9 fusion protein and preparation of gRNA
- TadA * Cas9 fusion protein
- a recombinant expression plasmid containing a gene encoding a TadA: TadA *: Cas9 fusion protein is prepared.
- the prokaryotic expression vector of the TadA: TadA *: Cas9 fusion protein is pET42b-ABE7.10 (SEQ ID ID NO. 3);
- Step (1) Transform pET42b-ABE7.10 into BL21 Star TM (DE3) E. coli (Thermo Fisher) competence.
- Step (2): Induce the expression of TadA: TadA *: Cas9 fusion protein: pick a single clone at 37 ° C and incubate it overnight at 1: 200 and inoculate 1L of LB medium containing 50 ⁇ g / ml kanamycin and incubate at 37 ° C to OD 600 0.7–0.8.
- the culture solution was placed in a refrigerator at 4 ° C for 1 hour, and a final concentration of 0.5mM IPTG was added at 18 ° C to induce protein expression for 14-16 hours.
- lysate 100 mM Tris-HCl, pH 8.0, 1 M NaCl, 20% glycerol, 5 mM TCEP (Sigma-Aldrich), 0.4 mM PMSF (Sigma-Aldrich), protease inhibitor (Roche) and 20 mM Imidazole (Sigma-Aldrich)).
- the mixed solution was poured into a chromatography column and 40 ml of washing solution (100 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 20% glycerol, 5 mM TCEP, 20 mM imidazole) was used to wash the Ni-NTA agarose resin (GE Healthcare).
- the protein was then eluted from the Ni column with an eluent (100 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 20% glycerol, 5 mM TCEP, 270 mM Imidazole).
- the eluted protein was passed through 5 ml cation exchange chromatography (Hi-Trap HP SP cation exchange column, GE Healthcare).
- FIG. 1 shows protein gel electrophoresis images of Cas9 protein, BE3 protein, and TadA: TadA *: Cas9 protein.
- gRNA is prepared by direct chemical synthesis or in vitro transcription.
- the preparation of gRNA by in vitro transcription includes the following steps: 1
- the gRNA transcription template DNA including the T7 promoter is obtained by PCR.
- the gRNA coding sequence is cloned into a transcription vector containing the T7 promoter, and then the vector is linearized to obtain a gRNA transcription template DNA containing the T7 promoter; 2 gRNA is transcribed in vitro;
- the method for in vitro transcription of gRNA is: using g7 transcription template DNA containing a T7 promoter as a template, and using MEGAshortscript T7 kit (Life Technologies) to produce gRNA.
- GRNA Qiagen
- GRNA was purified using an RNA purification kit, and the gRNA was eluted with nuclease-free water to obtain gRNA.
- the operating procedure of the in vitro transcription method of gRNA is as follows:
- Reaction was performed at 37 ° C for 2 hours. After the case, 1 ⁇ l of TURBO DNase was added to the reaction system, and the reaction was performed at 37 ° C for 15 minutes.
- Figure 2 shows the results of agarose gel electrophoresis of two gRNAs, HBG and VEGFA3.
- EndoV-seq detects single-base editing of ABE systems at target sites
- PCR reaction solution add 3 volumes of Buffer PCR-A and mix well, then transfer to the DNA preparation tube, place the DNA preparation tube in a 2ml centrifuge tube, centrifuge at 12,000g for 1min, and discard the filtrate.
- the PCR product was added to a 20 ⁇ l reaction system.
- This reaction system contains 2 ⁇ l 10 ⁇ NEBuffer 3,400nM TadA: TadA *: Cas9 fusion protein, 900nM gRNA and 200ng PCR product. Reaction at 37 ° C for 3h. Add RNase A and Proteinase K in order to remove gRNA and protein. Then purify again according to the above steps, take 100ng and 1 unit of Endo V (ThermoFisher) and mix 10 ⁇ l reaction system, react at 65 °C for 30min. 3% agarose assay. The test results are shown in Figure 3.
- EndoV digested TadA TadA *: Cas9 and gRNA-treated PCR products were able to cleave the PCR product containing the HEK293-2 target site.
- Cas9 protein was used as a positive control.
- the results in Figure 3 demonstrate that EndoV can be used to detect the deamination of the ABE system.
- EndoV-seq detects ABE system's off-target effects across the genome
- genomic DNA was mixed with 8 units of EndoV nuclease (ThermoFisher) in a 100 ⁇ l reaction system, and the reaction was performed at 65 ° C. for 3 hours.
- the genomic DNA was extracted with phenol-chloroform.
- sequenced Reads were aligned to a human reference genome using BWA software. Then use the online software (Digenome 2.0, http://www.rgenome.net/digenome-js/standalone) to score each locus of the genome and determine the cut score.
- the target sites and off-target sites were then amplified by PCR using the primers in Tables 2 and 3, and these PCR products were used for deep sequencing.
- Figure 6 through deep sequencing, we found that for HBG, 6 of the 18 off-target sites can be verified.
- VEGFA3, 3 of the 22 off-target sites could be verified. Therefore, the total verification rate of EndoV-seq is 22.5% (9/40), which indicates that EndoV-seq can effectively detect the off-target effect of the ABE system.
- HBG gRNA we found that the off-target efficiency of the HBG-OT9 locus in the cell is 0.13%, which is very close to the detection limit of the deep sequencing of PCR products 0.1% 1 (1.Tsai, SQet al.
- CIRCLE-seq a highly sensitive In vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nature methods 14,607-614 (2017).) shows that EndoV-seq has high sensitivity, and its sensitivity can reach at least 0.13%. The above results show that EndoV-seq can efficiently and sensitively detect off-target effects across the genome of the ABE system.
- the present invention provides a schematic flowchart of the method (EndoV-seq) for detecting the off-target effect of the adenine single base editing system based on whole genome sequencing, as shown in FIG.
- the embodiment of the present invention utilizes in vitro purified TadA: TadA *: Cas9 fusion protein, gRNA and genomic DNA to co-incubate.
- the complex of TadA: TadA *: Cas9 and gRNA will cut the DNA strand complementary to the gRNA, while converting A on the non-complementary strand to I (hypoxanthine, Inosine).
- endonuclease V Endonuclease V, EndoV
- whole-genome sequencing combined with bioinformatics analysis was used to detect off-target effects of the ABE system.
- a method based on whole-genome sequencing to detect the off-target effect of the Adenine single-base editing system can catalyze the efficient substitution of adenine (Adenine, A) to guanine (Guanine, G) at the target site.
- Adenine Adenine
- G guanine
- TadA * Cas9 fusion proteins to off-target sites that do not completely match the gRNA, resulting in off-target. It severely restricts the application of ABE system.
- EndoV-seq the first detection method capable of detecting off-target effects across the entire genome of the ABE system provided by the present invention, uses EndoV-seq to detect off-target sites of the ABE system in vitro, and combines in vivo experiments to verify. It is foreseeable that EndoV-seq will have broad application prospects in gene editing, especially in the field of gene editing therapy.
- the base sequences of SEQ ID No. 4 and SEQ ID No. 5 according to the present invention are as follows (the base sequences of SEQ ID No. 4 and SEQ ID NO. 5 are the sequences of commercial plasmid vectors, so they are not written In subsequent sequence listings):
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Abstract
本发明提供了一种基于全基因组测序检测腺嘌呤单碱基编辑系统(Adenine base editor, ABE)脱靶效应的方法——EndoV-seq。所述腺嘌呤单碱基编辑系统由TadA:TadA*:Cas9的融合基因和gRNA两部分组分组成,其能催化靶位点处腺嘌呤(Adenine, A)至鸟嘌呤(Guanine, G)的高效置换。
Description
本申请要求了2018年9月30日提交中国专利局的,申请号201811160230.9,发明名称为“一种基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法及其在基因编辑中的应用”的中国专利申请的优先权,其全部内容通过引用结合在本申请中。
本发明属于分子生物学技术领域。更具体地,涉及是基于全基因组测序检测腺嘌呤单碱基编辑系统(Adenine base editor,ABE)脱靶效应的方法及其在基因编辑中的应用。
CRISPR/Cas9系统是一项新的人工核酸酶技术,其是由gRNA(guide RNA)和Cas9蛋白组成的复合物。在靶位点3’末端PAM(Protospacer adjacent motif)序列的帮助下,该gRNA-Cas9蛋白复合物通过gRNA 5’末端的20个碱基与靶DNA结合,从而将内切核酸酶Cas9招募到靶位点处,切割靶DNA,从而编辑靶基因。虽然,CRISPR/Cas9技术的出现,极大地提高了基因定点突变的效率,但目前依然无法满足临床基因治疗的需要。最近,基于CRISPR/Cas9技术,科学家开发了新一代的基因编辑系统——腺嘌呤单碱基编辑系统(Adenine base editor,ABE)。ABE系统是由TadA:TadA*:Cas9融合蛋白和gRNA两部分组分组成的。在gRNA的引导下,TadA:TadA*:Cas9融合蛋白能够与DNA上的靶位点结合,其中与gRNA互补的DNA链会被Cas9核酸酶切断,而非互补链上4-9位的A碱基则会被腺嘌呤脱氨酶——TadA蛋白——催化脱氨基形成I碱基。随着DNA的复制,I(次黄嘌呤,Inosine)碱基会被G(鸟嘌呤,Guanine)碱基替代,从而实现A至G的碱基置换。与CRISPR/Cas9核酸酶相比,ABE系统的效率更高。由于,ABE系统能在不诱导DNA双链断裂的情况下实现A至G的碱基置换,其安全性比CRISPR/Cas9核酸酶也更高。
大约48%的人致病单碱基突变可以通过A至G的碱基置换实现修复,从而最终实现遗传疾病的治疗,所以ABE系统在人类疾病基因治疗领域有着广泛的应用前景。但是,目前仍未有一项能够在全基因组范围内检测ABE系统脱靶效应的方法,这严重制约了ABE系统的应用。
发明内容
本发明的目的是针对上述ABE系统尚无全基因组范围内检测脱靶效应的方法,提供一种基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法及其在基因编辑中的应用。
在本发明一具体实施例中,本发明通过基因合成、分子克隆、蛋白表达纯化、体外转录、核酸纯化、全基因组测序、PCR产物深度测序、生物信息学分析等技术检测ABE系统的脱靶效应。同时,我们还结合细胞转染和PCR产物深度测序技术对该检测方法的有效性和灵敏度进行了验证。
本发明上述目的通过以下技术方案实现:
第一方面,本发明提供了一种基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法,其特征在于,包括如下步骤:
(1)、将TadA:TadA*:Cas9融合蛋白、一种或多种靶向待测DNA序列的gRNA以及基因组DNA共混后进行反应;其中,所述基因组DNA包含有待测DNA序列,在所述反应体系中,TadA:TadA*:Cas9和gRNA复合物切割与gRNA互补的待测DNA链,同时将非互补链上的腺嘌呤转变成次黄嘌呤;
(2)、在步骤(1)反应后的体系中加入内切核酸酶V切割包含次黄嘌呤DNA,造成DNA 双链断裂;
(3)、利用全基因组测序以及生物信息学分析检测腺嘌呤单碱基编辑系统的脱靶效应。
在本发明第一方面一具体实施例中,本发明提供了一种基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法(EndoV-seq);所述EndoV-seq首先利用体外纯化的TadA:TadA*:Cas9融合蛋白与gRNA共处理基因组DNA;TadA:TadA*:Cas9和gRNA的复合物将切割与gRNA互补的DNA链,同时将非互补链上的A转变成I;然后,利用内切核酸酶V(Endonuclease V,EnodV)切割包含I碱基的基因组DNA,造成DNA双链断裂;最后,利用全基因组测序结合生物信息学分析检测DNA双链断裂,从而探究ABE系统的脱靶效应。我们将这一方法命名为EndoV-seq。
在本发明第一方面一具体实施例中,所述TadA:TadA*:Cas9融合蛋白包括CRISPR/Cas系统的效应蛋白结构域、腺苷脱氨酶结构域。
在本发明第一方面一具体实施例中,所述TadA:TadA*:Cas9融合蛋白包括CRISPR/Cas系统的效应蛋白结构域、连接多肽、腺苷脱氨酶结构域。
本领域技术人员可以理解的是,本发明所述的TadA:TadA*:Cas9融合蛋白由Cas9效应蛋白与腺苷脱氨酶(简称TadA蛋白)融合而成,本领域技术人员可以根据需要,利用一条或多条连接多肽,将一个Cas9效应蛋白结构域与一个或多个TadA蛋白进行连接,获得融合蛋白,在本发明一具体实施例中,所述TadA蛋白均重复一次。可以理解的是,所述Cas9效应蛋白和TadA蛋白的N端和C端的连接顺序为本领域常规技术,连接多肽包括但不限于本领域常规的连接多肽片段,常见地,比如GS linker。
本领域技术人员可以理解的是,本领域技术人员可以根据具体需要,针对基因组DNA设计任意特异性靶向的gRNA,并对gRNA进行本领域熟知的修饰,以提高gRNA靶向特异性。在本发明一具体实施例中,本发明设计的gRNA序列包括:
HBG:GTGGGGAAGGGGCCCCCAAG
AGG,其中下划线标注的为PAM序列
VEGFA3:GGTGAGTGAGTGTGTGCGTG
TGG,其中下划线标注的为PAM序列。
本领域技术人员可以理解的是,TadA:TadA*:Cas9融合蛋白中,TadA为腺苷脱氨酶的缩写,TadA*为TadA突变体的缩写,Cas9为CRISPR/Cas系统的Cas9效应蛋白。
进一步地,所述CRISPR/Cas系统的效应蛋白结构域中,所述Cas9效应蛋白包括但不限定于无切割活性或仅具有单链切割活性的Cas蛋白Streptococcus pyogenes Cas9(SpCas9),Staphylococcus aureus Cas9(SaCas9),Lachnospiraceae Cpf1(LbCpf1),Acidaminococcus Cpf1(AsCpf1),Streptococcus thermophilus Cas9(StCas9),and Neisseria meningitidis Cas9(NmCas9)和Francisella Cpf1(FnCpf1)等。
进一步地,所述的TadA:TadA*:Cas9融合蛋白中,腺苷脱氨酶TadA蛋白的氨基酸序列如SEQ ID NO.1所示。
在本发明第一方面一具体实施例中,所述TadA:TadA*:Cas9融合蛋白的氨基酸序列为SEQ ID NO.2所示或为与SEQ ID NO.2所示氨基酸至少80%、85%、90%、92%、95%、96%、97%、98%、99%或99.5%一致的序列。
在本发明第一方面一具体实施例中,所述TadA:TadA*:Cas9融合蛋白的制备方法是将合成的TadA:TadA*:Cas9融合蛋白原核表达载体(pET42b-ABE7.10,SEQ ID NO.3)载体在大肠杆菌中进行表达并纯化TadA:TadA*:Cas9融合蛋白。
更具体地,该制备过程包括步骤(1)将pET42b-ABE7.10转化到BL21Star
TM(DE3)E.coli(Thermo Fisher)感受态中。
更具体地,步骤(2)诱导TadA:TadA*:Cas9融合蛋白表达的方式是:挑取单克隆37℃过夜培养后1:200接种于1L含有50μg/ml卡那霉素的LB培养基中,37℃培养至OD
600=0.7–0.8。然后,将培养液放于4℃冰箱静置1h,加入终浓度0.5mM IPTG 18℃诱导蛋白表达14-16h。
更具体地,步骤(3)纯化及保存TadA:TadA*:Cas9融合蛋白的方式是:4000rpm,10min 收集诱导后的菌体,加入10ml裂解液(100mM Tris-HCl,pH 8.0,1M NaCl,20%甘油,5mM TCEP(Sigma-Aldrich),0.4mM PMSF(Sigma-Aldrich),蛋白酶抑制剂(Roche)and 20mM Imidazole(Sigma-Aldrich))。超声(5min total,2s on,5s off)初步破碎细胞。然后,15000rpm,4℃离心10min后收集上清液再次超声(5min total,2s on,5s off)。之后,15000rpm,4℃离心10min后收集上清液。上清液与Ni-NTA琼脂糖树脂(GE Healthcare)4℃孵育1.5h。混合液倒入层析柱,然后用40ml洗涤液(100mM Tris-HCl,pH 8.0,0.5M NaCl,20%甘油,5mM TCEP,20mM imidazole)清洗Ni-NTA琼脂糖树脂(GE Healthcare)。然后,用洗脱液(100mM Tris-HCl,pH 8.0,0.5M NaCl,20%甘油,5mM TCEP,270mM Imidazole)将蛋白从Ni柱洗脱。洗脱蛋白过5ml阳离子交换层析(Hi-Trap HP SP阳离子交换柱,GE Healthcare)。然后,用30kDa浓缩管浓缩(Millipore)至300μl。然后,将浓缩产物过0.22μm滤菌膜(Millipore)过滤除菌。用BCA试剂盒(Pierce Biotechnology)测蛋白浓度后暂存于4℃,如需长期保存,则将蛋白用液氮速冻后冻于-80℃保存。
更具体地,所述gRNA的制备方法是包括(1)化学合成gRNA;(2)体外转录合成gRNA。
在本发明第一方面一具体实施例中,所述反应体系为溶液反应体系,所述溶液反应体系还包含所述TadA:TadA*:Cas9融合蛋白将所述非互补链上的腺嘌呤转变成次黄嘌呤所需的缓冲液组分。
在本发明第一方面一具体实施例中,所述步骤(3)包括:
对步骤(2)酶切后的体系进行全基因组测序,获得全基因组测序结果;
对全基因组测序结果进行生物信息学分析,获得所述腺嘌呤单碱基编辑系统的脱靶数据;
进一步地,所述步骤(3)还包括:根据所述脱靶数据预测所述腺嘌呤单碱基编辑系统在细胞(包括人细胞、动物细胞、植物细胞等)或机体内(包括人、动物、植物等)的脱靶效应。
第二方面,本发明提供了一种基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的试剂盒,包括第一方面所提供的靶向目的DNA的gRNA序列或TadA:TadA*:Cas9融合蛋白、EndoV核酸酶。
第三方面,本发明提供了一种基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法在检测腺嘌呤单碱基编辑系统脱靶效应中的应用。
本发明利用体外纯化的TadA:TadA*:Cas9融合蛋白和gRNA,利用融合蛋白-gRNA复合物处理基因组DNA。然后,利用核酸纯化试剂盒纯化处理过的基因组DNA。再利用内切核酸酶V消化以上纯化的基因组DNA。消化完成后,再将该基因组DNA纯化,并进行全基因组测序检测ABE系统的全基因组脱靶效应。该检测方法(EndoV-seq)和检测结果也在本发明的保护范围之内。同时,还可以根据EndoV-seq的检测结果,优选出效率和特异性高的gRNA。EndoV-seq作为优选gRNA的方法也在本发明的保护范围之内。
另外,上述EndoV-seq在基因编辑中的应用也在本发明的保护范围之内。
本发明将促进以ABE系统为工具,进行的精准基因编辑治疗的临床应用,精准疾病模型构建的应用,精准基因编辑植物或作物的培育等应用。
在本发明第三方面一具体实施例中,所述方法对细胞内的腺嘌呤单碱基编辑系统脱靶效率的检测极限至少可达0.13%。
更具体地,所述所述方法对细胞内HBG-OT9位点的腺嘌呤单碱基编辑系统脱靶效率的检测极限至少可达0.13%。
第四方面,EndoV-seq还可以用于检测其他能够将A碱基转变成I碱基的酶或化学试剂的效率和脱靶活性。酶包括但不限定于TadA腺嘌呤脱氨酶。
本发明具有以下有益效果:
本发明提供了基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法,该检测方法可以用于腺嘌呤单碱基编辑系统脱靶效应的检测,促进腺嘌呤单碱基编辑系统在疾病基因治疗、模型构建、基因编辑植物或作物的培育等应用,具有十分广泛的应用前景。
图1为Cas9蛋白、BE3蛋白和TadA:TadA*:Cas9蛋白的蛋白凝胶电泳图;第一道泳道为蛋白分子Marker,第二道为Cas9蛋白,第三道为BE3蛋白,第四道为TadA:TadA*:Cas9蛋白;
图2为gRNA琼脂糖凝胶电泳结果,两条泳道均为gRNA。
图3为TadA:TadA*:Cas9蛋白-gRNA复合物和EndoV共处理能够切开靶DNA分子。
图4为EnodV-seq能够检测到靶位点处的DNA双链断裂。
图5为EndoV-seq检测HBG和VEGFA3两条gRNA介导的ABE系统的全基因组范围内的脱靶效应。A图是Circos plot展示EndoV-seq检测到的靶位点及脱靶位点,红色箭头指示的是靶位点。B图是Weblog展示的脱靶位点的分子模式。下方标注的是gRNA的靶序列,其中PAM标注为绿色字母。
图6为PCR产物深度测序验证HBG的脱靶位点,18个脱靶位点中有6个位点能被验证,并用*号标出。
图7为PCR产物深度测序验证VEGFA3的脱靶位点,22个脱靶位点中有3个位点能被验证,并用*号标出。
图8为本发明基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法(EndoV-seq)的流程示意图。
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。
除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明,以下实施例所用试剂和材料均为市购。未注明具体条件的实验方法,通常按照常规条件,或制造厂商所建议条件实施。
本发明一具体实施方式中,本发明提供了一种基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的系统、方法、试剂盒及其应用。
本发明提供的基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法,采用了本发明提供的基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应检测试剂盒,所述方法包括但不限于如下步骤的一个或多个步骤:
TadA:TadA*:Cas9融合蛋白的表达纯化和gRNA的制备
1、TadA:TadA*:Cas9融合蛋白的表达纯化
制备含编码TadA:TadA*:Cas9融合蛋白基因的重组表达质粒,本实施例中,所述TadA:TadA*:Cas9融合蛋白的原核表达载体为pET42b-ABE7.10(SEQ ID NO.3);
步骤(1)、将pET42b-ABE7.10转化到BL21 Star
TM(DE3)E.coli(Thermo Fisher)感受态中。
步骤(2)、诱导TadA:TadA*:Cas9融合蛋白表达:挑取单克隆37℃过夜培养后1:200接种于1L含有50μg/ml卡那霉素的LB培养基中,37℃培养至OD
600=0.7–0.8。培养液放于4℃冰箱静置1h,加入终浓度0.5mM IPTG 18℃诱导蛋白表达14-16h。
步骤(3)、纯化及保存TadA:TadA*:Cas9融合蛋白:4℃收集菌体并纯化蛋白。4000rpm,10min收集诱导后的菌体,加入10ml裂解液(100mM Tris-HCl,pH 8.0,1M NaCl,20%甘油,5mM TCEP(Sigma-Aldrich),0.4mM PMSF(Sigma-Aldrich),蛋白酶抑制剂(Roche)and 20mM Imidazole(Sigma-Aldrich))。超声(5min total,2s on,5s off)初步破碎细胞,15000rpm,4℃离心10min后收集上清液再次超声(5min total,2s on,5s off),15000rpm,4℃离心10min后收集上清液。上清液与Ni-NTA琼脂糖树脂(GE Healthcare)4℃孵育1.5h。混合液倒入层析柱,40ml洗涤液(100mM Tris-HCl,pH 8.0,0.5M NaCl,20%甘油,5mM TCEP,20mM imidazole)清洗Ni-NTA琼脂糖树脂(GE Healthcare)。然后用洗脱液(100mM Tris-HCl,pH 8.0,0.5M NaCl,20%甘油,5mM TCEP,270mM Imidazole)将蛋白从Ni柱洗脱。洗脱蛋白过5ml阳离子交换层析(Hi-Trap HP SP阳离子交换柱,GE Healthcare)。然后用30kDa浓缩管浓缩(Millipore)至300μl。然后将浓缩产物过0.22μm滤菌膜(Millipore)过滤除菌。用BCA试剂盒(Pierce Biotechnology)测蛋白浓度后暂存于4℃,如需长期保存,则将蛋白用液氮速冻后冻于-80℃保存。
蛋白表达检测结果如附图1,图1显示了Cas9蛋白、BE3蛋白和TadA:TadA*:Cas9蛋白的蛋白凝胶电泳图。
2、gRNA的制备
本发明实施例通过化学直接合成或者通过体外转录制备gRNA,其中,体外转录制备gRNA包括以下步骤①通过PCR的方式获得包含T7启动子的gRNA转录模板DNA。或者将gRNA编码序列克隆到包含T7启动子的转录载体中,然后将该载体线性化而获得包含T7启动子的gRNA转录模板DNA;②体外转录gRNA;
体外转录gRNA的方法是:以包含T7启动子的gRNA转录模板DNA为模板,用MEGAshortscript T7 kit(Life Technologies)转录生产gRNA。再用RNA纯化试剂盒纯化gRNA(Qiagen),并用不含核酸酶的水洗脱gRNA,即可获得gRNA。
具体地,gRNA体外转录方法的操作程序如下:
1)以gRNA转录模板DNA为模板,利用MEGAshortscript T7 kit(Life Technologies),按照如下表1所示体系配制反应体系。
表1反应体系
成分 | 用量 |
T7 10×反应液 | 2μl |
T7 ATP溶液 | 2μl |
T7 CTP溶液 | 2μl |
T7 GTP溶液 | 2μl |
T7 UTP溶液 | 2μl |
模板DNA | 1μg |
T7 RNA转录酶 | 2μl |
ddH 2O | 加水至20μl |
37℃反应2h,案后往反应体系中加1μl TURBO DNase,37℃反应15min。
2)gRNA的纯化,用Qiagen的RNaeasy Kit纯化,按照如下步骤进行:
a.加ddH
2O使得起始RNA的体积为100μl,混匀。
b.加350μl Binding Solution Concentrate到RNA样品中,并混匀。
c.加250μl 100%乙醇,并混匀。
d.将样品转移到柱子中,12000g离心15s。
e.用500μl Wash Solution洗两次,12000g离心15s。
f.加50μl ddH
2O将RNA从柱子上洗脱下来。
3)结果如图2所示,图2显示了HBG和VEGFA3两条gRNA的琼脂糖凝胶电泳结果。
EndoV-seq检测ABE系统在靶位点的单碱基编辑
为了验证EndoV-seq是否能够用于检测ABE系统全基因组范围内的脱靶效应,我们利用了一条已经多次被验证能高效靶向靶位点的gRNA——HEK293-2,其靶序列为GAACACAAAGCATAGACTGC
GGG,其中下划线标注的为PAM序列。首先,我们通过PCR的方式扩增出了包含了HEK293-2位点的PCR产物,然后将该产物纯化,具体纯化方法如下。
按AxyPrep PCR cleanup kit的操作手册进行实验。
a.在PCR反应液中,加3个体积的Buffer PCR-A并混匀,然后转移到DNA制备管,将DNA制备管置于2ml离心管中,12,000g离心1min,将滤液弃掉。
b.将制备管放回2ml离心管中,加700μl Buffer W2,12000g离心1min,将滤液弃掉。
c.将制备管放回2ml离心管中,加400μl Buffer W2,12000g离心1min,弃滤液。
d.12,000g离心3min,使Buffer W2中的乙醇充分弃掉。
e.将制备管置于新的1.5ml离心管中,在制备管中央加25-30μl的无核酸酶的水,静置1min。12000g离心1min(无核酸酶的水用前先65℃预热)。
获得了纯化后的PCR产物之后,将PCR产物加入到20μl反应体系中。该反应体系中含有2μl 10×NEBuffer 3,400nM TadA:TadA*:Cas9融合蛋白,900nM gRNA and 200ng PCR产物。37℃反应3h。依次加入RNase A和Proteinase K移除gRNA及蛋白。然后再次按照如上步骤纯化,取100ng与1单位的Endo V(ThermoFisher)混合10μl反应体系,65℃反应30min。3%琼脂糖检测。检测结果如图3所示,用EndoV消化TadA:TadA*:Cas9和gRNA处理过的PCR产物,能够将包含HEK293-2靶位点的PCR产物切断。其中Cas9蛋白作为阳性对照。图3的结果说明EndoV能够用于检测ABE系统的脱氨基作用。
为了进一步检测EndoV-seq是否能够用于检测ABE系统的脱氨基作用,我们进一步用TadA:TadA*:Cas9融合蛋白和HEK293-2gRNA一起处理人HEK293T细胞的基因组DNA。首先,我们用基因组DNA提取试剂盒从HEK293T细胞中提取基因组DNA(DNeasy Blood & Tissue Kit,Qiagen),操作方法完全按照说明书进行。然后用TadA:TadA*:Cas9融合蛋白和HEK293-2gRNA一起处理人HEK293T细胞的基因组DNA。在500μl反应体系中我们加有50μl 10×NEBuffer 3,400nM ABE7.10,900nM gRNA and 10μg基因组DNA。37℃反应8h。8h后,向反应体系中加入RNase A和Proteinase K移除gRNA及蛋白。然后用苯酚/氯仿/异戊醇仿抽提基因组DNA,操作步骤如下。
a.向以上反应加入1体积的苯酚/氯仿/异戊醇剧烈混匀,室温下静置10分钟,待分层后12000rpm离心10分钟;
b.吸取上层水相,并记录其体积;
c.加入1/10体积的3M NaAc,加3倍体积冷的无水乙醇(-20℃冰箱),剧烈混匀。然后,冰上孵育15分钟;
d.离心(12000rpm,15分钟,4℃),用移液器尽可能除去乙醇;
e.加入0.5ml 70%乙醇洗DNA沉淀一次,12000rpm,离心2分钟,吸取并尽可能弃掉乙醇;
f.加入30μl水溶解DNA,然后用Nanodrop测定其浓度;
然后,取4μg基因组DNA与8单位的EndoV核酸酶(ThermoFisher)混合100μl反应体系,65℃反应3h,酚氯仿抽提基因组DNA。最后,取1μg基因组DNA做全基因组测序。然后用BWA软件将测序的Reads比对到人参考基因组。我们发现EndoV-seq确实能够检测到ABE系统介导的靶位点的修饰,结果如图4所示。
EndoV-seq检测ABE系统在全基因组范围内的脱靶效应
为了进一步探究EndoV-seq是否能够用于检测ABE系统全基因范围内的脱靶效应。我们进一步利用在实施例1中所转录出的HBG和VEGFA3两条gRNA,然后将其分别与TadA:TadA*:Cas9融合蛋白孵育。然后用该蛋白-RNA复合物处理HEK293-2基因组DNA。
在500μl反应体系中我们加有50μl 10×NEBuffer 3,400nM ABE7.10,900nM gRNA and 10μg基因组DNA。37℃反应8h。8h后,向反应体系中加入RNase A和Proteinase K移除gRNA及蛋白。然后用苯酚/氯仿/异戊醇仿抽提基因组DNA,操作步骤如下。
a.向以上反应加入1体积的苯酚/氯仿/异戊醇剧烈混匀,室温下静置10分钟,待分层后12000rpm离心10分钟;
b.吸取上层水相,并记录其体积;
c.加入1/10体积的3M NaAc,加3倍体积冷的无水乙醇(-20℃冰箱),剧烈混匀。然后,冰上孵育15分钟;
d.离心(12000rpm,15分钟,4℃),用移液器尽可能除去乙醇;
e.加入0.5ml 70%乙醇洗DNA沉淀一次,12000rpm,离心2分钟,吸取并尽可能弃掉乙醇;
f.加入30μl水溶解DNA,然后用Nanodrop测定其浓度;
然后,取4μg基因组DNA与8单位的EndoV核酸酶(ThermoFisher)混合100μl反应体系,65℃反应3h,酚氯仿抽提基因组DNA。最后,取1μg基因组DNA做全基因组测序。然后,用BWA软件将测序的Reads比对到人参考基因组。再利用在线软件(Digenome 2.0,http://www.rgenome.net/digenome-js/standalone)对基因组各个位点进行打分,确定其切割的分值。参考之前用Digenome-seq检测胞嘧啶单碱基编辑系统脱靶效应的研究,我们将分值大于0.1的位点定义为阳性脱靶位点。我们发现EndoV-seq能够检测到靶位点以及脱靶位点,研究结果如图5所示,EndoV-seq检测到的脱靶位点见表2和表3。
表2 EndoV-seq检测到的HBG gRNA介导的ABE系统的脱靶位点统计
表3 EndoV-seq检测到的VEGFA3gRNA介导的ABE系统的脱靶位点统计
为了进一步研究EndoV-seq的有效性和敏感度。我们将pcDNA3.1-ABE7.10载体(由广州艾基生物科技有限公司合成,SEQ ID NO.4)与表达HBG(或VEGFA3)gRNA的gRNA表达载体pUC19-SpCas9-gRNA(SEQ ID NO.5,本实验室构建)共转染到293T细胞中,48h后收集细胞。利用基因组DNA提取试剂盒提取基因组DNA(DNeasy Blood & Tissue Kit,Qiagen),操作方法完全按照说明书进行。然后利用表2和表3中的引物通过PCR扩增靶位点和脱靶位点,并将这些PCR产物用于深度测序。如图6,通过深度测序,我们发现,对于HBG来说,18个脱靶位点中有6个位点能被验证。而对于VEGFA3来说,22个脱靶位点中有3个位点能被验证。所以,EndoV-seq的总验证率为22.5%(9/40),说明EndoV-seq能够有效检测ABE系统的脱靶效应。对于HBG gRNA来说,我们发现HBG-OT9位点在细胞内的脱靶效率为0.13%,十分接近PCR产物深度测序的检测极限0.1%
1(1.Tsai,S.Q.et al.CIRCLE-seq:a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets.Nature methods 14,607-614(2017).),说明EndoV-seq具有很高的灵敏度,其灵敏度至少可达0.13%。以上结果说明,EndoV-seq能够高效、灵敏地检测ABE系统全基因组范围内的脱靶效应。
为进一步说明本发明的有益效果,本发明提供了本发明基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法(EndoV-seq)的流程示意图,如图8所示。
如图8,本发明实施例利用体外纯化的TadA:TadA*:Cas9融合蛋白、gRNA以及基因组DNA共孵育。在该反应体系中,TadA:TadA*:Cas9和gRNA的复合物将切割与gRNA互补的DNA链,同时将非互补链上的A转变成I(次黄嘌呤,Inosine)。然后,利用内切核酸酶V(Endonuclease V,EndoV)切割包含I碱基的基因组DNA,造成DNA双链断裂。最后,利用全基因组测序结合生物信息学分析检测ABE系统的脱靶效应。
基于全基因组测序检测腺嘌呤单碱基编辑系统(Adenine base editor,ABE)脱靶效应的方法,能催化靶位点处腺嘌呤(Adenine,A)至鸟嘌呤(Guanine,G)的高效置换,在人类疾病基因编辑治疗和疾病模型构建中有广泛的应用前景。但由于CRISPR/Cas9系统的特异性不高,易将TadA:TadA*:Cas9融合蛋白靶向到与gRNA不完全匹配的脱靶位点上,导致脱靶。严重制约了ABE系统的应用。为此,本发明提供的首个能够检测ABE系统全基因组范围内脱靶效应的检测方法EndoV-seq,利用EndoV-seq能在体外检测到ABE系统的脱靶位点,并结合体内实验进行验证。可以预见,EndoV-seq在基因编辑,尤其是基因编辑治疗领域,将具有广泛的应用前景。
本发明所述SEQ ID NO.4和SEQ ID NO.5的碱基序列分别如下(所述SEQ ID NO.4和SEQ ID NO.5的碱基序列为商业质粒载体的序列,故不写入后续序列表中):
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (15)
- 一种基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法,其特征在于,包括如下步骤:(1)、将TadA:TadA*:Cas9融合蛋白、一种或多种靶向待测DNA序列的gRNA以及基因组DNA共混后进行反应;其中,所述基因组DNA包含有待测DNA序列,在所述反应体系中,TadA:TadA*:Cas9和gRNA复合物切割与gRNA互补的待测DNA链,同时将非互补链上的腺嘌呤转变成次黄嘌呤;(2)、在步骤(1)反应后的体系中加入内切核酸酶V切割包含次黄嘌呤的DNA,造成DNA双链断裂;(3)、利用全基因组测序以及生物信息学分析检测腺嘌呤单碱基编辑系统的脱靶效应。
- 根据权利要求1所述的方法,其特征在于,所述TadA:TadA*:Cas9融合蛋白包括CRISPR/Cas系统的效应蛋白结构域、腺苷脱氨酶结构域。
- 根据权利要求1所述的方法,其特征在于,所述TadA:TadA*:Cas9融合蛋白包括CRISPR/Cas系统的效应蛋白结构域、连接多肽、腺苷脱氨酶结构域。
- 根据权利要求1所述方法,其特征在于,所述TadA:TadA*:Cas9融合蛋白包括CRISPR/Cas系统的效应蛋白结构域,所述CRISPR/Cas系统的效应蛋白结构域中,所述Cas9效应蛋白包括但不限定于无切割活性或仅具有单链切割活性的Cas蛋白,所述无切割活性或仅具有单链切割活性的Cas蛋白包括Streptococcus pyogenes Cas9,Staphylococcus aureus Cas9,Lachnospiraceae Cpf1,Acidaminococcus Cpf1,Streptococcus thermophilus Cas9,and Neisseria meningitidis Cas9,Francisella Cpf1中的一种或多种。
- 根据权利要1所述方法,其特征在于,所述的TadA:TadA*:Cas9融合蛋白中,所述TadA:TadA*:Cas9融合蛋白包括腺苷脱氨酶TadA蛋白,所述腺苷脱氨酶TadA蛋白的氨基酸序列如SEQ I D NO.1所示。
- 根据权利要求1所述的方法,其特征在于,所述TadA:TadA*:Cas9融合蛋白的氨基酸序列为SEQ I D NO.2所示或为与SEQ I D NO.2所示氨基酸至少80%、85%、90%、92%、95%、96%、97%、98%、99%或99.5%一致的序列。
- 根据权利要求1所述的方法,其特征在于,所述TadA:TadA*:Cas9融合蛋白是利用原核表达载体在细菌中表达并纯化获得的。
- 根据权利要求1所述的方法,其特征在于,所述反应体系为溶液反应体系,所述溶液反应体系还包含所述TadA:TadA*:Cas9融合蛋白将所述非互补链上的腺嘌呤转变成次黄嘌呤所需的缓冲液组分。
- 根据权利要求1所述的方法,其特征在于,所述步骤(3)包括:对步骤(2)酶切后的体系进行全基因组测序,获得全基因组测序结果;对全基因组测序结果进行生物信息学分析,获得所述腺嘌呤单碱基编辑系统的脱靶数据。
- 根据权利要求9所述的方法,其特征在于,所述步骤(3)还包括:根据所述脱靶数 据预测所述腺嘌呤单碱基编辑系统在细胞或机体内的脱靶效应。
- 根据权利要求10所述的方法,其特征在于,所述细胞包括人细胞、动物细胞或植物细胞。
- 根据权利要求10所述的方法,其特征在于,所述机体包括人机体、动物机体或植物机体。
- 一种基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的试剂盒,其特征在于,包括权利要求1提供的靶向目的DNA的gRNA序列或TadA:TadA*:Cas9融合蛋白、EndoV核酸酶。
- 如权利要求1所述一种基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法在检测腺嘌呤单碱基编辑系统脱靶效应中的应用。
- 如权利要求14所述的基于全基因组测序检测腺嘌呤单碱基编辑系统脱靶效应的方法在检测腺嘌呤单碱基编辑系统脱靶效应中的应用,其特征在于,所述方法对细胞内的腺嘌呤单碱基编辑系统脱靶效率的检测极限至少可达0.13%。
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