WO2021175287A1 - 检测单碱基编辑系统随机脱靶效应的方法 - Google Patents
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- C12N2310/00—Structure or type of the nucleic acid
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Definitions
- the invention belongs to the field of gene editing. Specifically, the present invention relates to a method for rapid and high-throughput detection of random off-target effects in a genome-wide range of a single-base editing system.
- Genome editing technology is a genetic engineering technology based on the targeted modification of the genome by artificial nucleases, and it is playing an increasingly powerful role in agricultural and medical research.
- Clustered regularly spaced short palindromic repeats and its related system are currently the most widely used genome editing tools.
- Cas The protein can be targeted to any location in the genome.
- the single-base editing system is a new gene editing technology developed based on the CRISPR system. It is divided into a cytosine single-base editing system and a adenine single-base editing system. Strand nickase fusion, under the targeting action of the guide RNA, the Cas9 single-stranded nickase produces a single-stranded DNA region, so the deaminase can efficiently separate the C or A nucleotides on the single-stranded DNA at the target position. Deamino groups become U bases and I bases, which are then restored to T bases or G bases in the process of cell repair.
- the cytosine single-base editing system has been found to produce unpredictable off-target phenomena in the genome.
- the present invention still needs a simple and low-cost method for detecting random off-target effects of a single-base editing system.
- the single-stranded region allows the single-base editor that can randomly act on the single-stranded DNA region to deaminate the target base in the single-stranded region, and the single-base can be detected efficiently and simply by high-throughput sequencing of amplicons Based on the random off-target effect of the editing system, this method is called the Trans-ssDNA amplifier deep sequencing (TA-AS) method.
- TA-AS Trans-ssDNA amplifier deep sequencing
- Figure 1 Schematic diagram of orthogonal system detection carrier.
- FIG. 1 Schematic diagram of BE3 carrier.
- the present invention provides a method for detecting random off-target effects of a base editing system, the method comprising:
- a detection CRISPR system that targets at least one detection target site in the genome is introduced into a cell or organism, and the detection CRISPR system can form a single-stranded DNA region at the at least one detection target site, and its guide RNA and The guide RNA of the base editing system to be tested is incompatible;
- the detection of a nucleotide mutation in the at least one detection target site indicates that the base editing system to be detected is off-target.
- the amount of nucleotide mutations detected in the at least one detection target site represents the degree of off-target, and the more nucleotide mutations detected, the higher the degree of off-target.
- the base editing system to be tested may include a base editor to be tested (base editor) or an expression construct containing its coding sequence, and/or its corresponding guide RNA (gRNA) or an expression containing its coding sequence Construct.
- the base editing system to be tested in step a) only includes the base editor to be tested or an expression construct containing its coding sequence.
- base editor refers to a fusion protein comprising a CRISPR effector protein and a deaminase. According to the different deaminase, the base editor can be divided into cytosine base editor and adenine base editor. In some preferred embodiments, the base editing system to be tested of the present invention includes a cytosine base editor.
- the cytosine base editor is usually a fusion protein containing CRISPR effector protein and cytosine deaminase.
- the cytosine deaminase in the base editor can deaminate the cytidine of the single-stranded DNA produced in the formation of the CRIPR effector protein-guide RNA-target DNA complex into U, and then realize C to U through base mismatch repair. Base substitution of T.
- the cytosine base editor further comprises uracil DNA glycosylase inhibitor (UGI).
- UMI Uracil DNA glycosylase inhibitor
- BER base excision repair
- the inclusion of Uracil DNA Glycosylase Inhibitor (UGI) in the cytosine base editor will increase the efficiency of C to T base editing.
- cytosine deaminase examples include, but are not limited to, for example, APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, CDA1, human APOBEC3A deaminase, or functional variants thereof.
- the cytosine deaminase is human APOBEC3A or a functional variant thereof.
- the cytosine deaminase is APOBEC1 or a functional variant thereof.
- the cytosine deaminase includes the amino acid sequence of SEQ ID NO: 7-10.
- the method of the present invention can be used to test the off-target effects of base editors containing various cytosine deaminase variants.
- CRISPR effector protein generally refers to the nuclease present in the naturally-occurring CRISPR system, as well as its modified form, its variant, its catalytically active fragment, and the like.
- the term covers any effector protein based on the CRISPR system that can achieve gene targeting (such as gene editing, gene targeted regulation, etc.) in cells.
- Cas9 nuclease examples include Cas9 nuclease or variants thereof.
- the Cas9 nuclease may be a Cas9 nuclease from different species, such as spCas9 from S. pyogenes or SaCas9 derived from S. aureus.
- Cas9 nuclease and Cas9 are used interchangeably herein, and refer to RNA comprising Cas9 protein or fragments thereof (for example, a protein containing the active DNA cleavage domain of Cas9 and/or the gRNA binding domain of Cas9) Guided nuclease.
- Cas9 is a component of the CRISPR/Cas (clustered regularly spaced short palindrome repeats and related systems) genome editing system, which can target and cleave the DNA target sequence under the guidance of the guide RNA to form a DNA double-strand break (DSB) ).
- CRISPR/Cas clustered regularly spaced short palindrome repeats and related systems
- CRISPR effector proteins may also include Cpf1 nuclease or variants thereof such as highly specific variants.
- the Cpf1 nuclease may be Cpf1 nuclease from different species, for example, Cpf1 nuclease from Francisella novicida U112, Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006.
- the CRISPR effector protein of the base editor of the present invention is an acidase-inactivated CRISPR effector protein.
- the CRISPR effector protein of the base editor of the present invention is a CRISPR effector protein with nickase activity.
- the CRISPR effector protein of the base editor of the invention is a Cas9 nickase.
- the CRISPR effector protein of the base editor of the present invention is the nickase form (nSpCas9) of SpCas9 from S. pyogenes.
- the nSpCas9 includes the amino acid sequence shown in SEQ ID NO:1.
- the CRISPR effector protein of the base editor of the present invention is the nickase form (nSaCas9) of SaCas9 from S. aureus.
- nSaCas9 includes the amino acid sequence shown in SEQ ID NO: 2.
- the detection CRISPR system of the present invention may include a CRISPR effector protein or an expression construct containing its coding nucleotide sequence, and a guide RNA targeting at least one genomic target site (detection target site) or containing its coding nucleotide Sequence expression construct.
- the CRISPR effector protein of the detection CRISPR system of the present invention is a CRISPR effector protein in which oxidase is inactivated. In some embodiments, the CRISPR effector protein of the detection CRISPR system of the present invention is a CRISPR effector protein with nickase activity. In some embodiments, the CRISPR effector protein of the detection CRISPR system of the present invention is a Cas9 nickase. In some preferred embodiments, in some embodiments, the CRISPR effector protein of the detection CRISPR system of the present invention is the nickase form (nSpCas9) of SpCas9 from S. pyogenes.
- the nSpCas9 includes the amino acid sequence shown in SEQ ID NO:1.
- the CRISPR effector protein of the detection CRISPR system of the present invention is the nickase form (nSaCas9) of SaCas9 from S. aureus.
- the nSaCas9 includes the amino acid sequence shown in SEQ ID NO: 2.
- the guide RNA incompatibility between the detection CRISPR system and the base editing system to be tested means that the detection CRISPR system cannot use the guide RNA of the base editing system to be tested, and the base editing system to be tested cannot use the detection CRISPR system.
- Guide RNA This depends on the different CRISPR effector proteins used in the system.
- the source of the CRISPR effector protein in the detection CRISPR system and the CRISPR effector protein in the base editor to be detected are different, so their guide RNAs are incompatible.
- the CRISPR effector protein in the detection CRISPR system is derived from SaCas9 of Staphylococcus aureus (S. aureus), and its corresponding guide RNA includes the scaffold sequence shown in SEQ ID NO: 5.
- the CRISPR effector protein in the detection CRISPR system is derived from SpCas9 of S. pyogenes, and its corresponding guide RNA includes the scaffold sequence shown in SEQ ID NO: 11.
- the CRISPR effector protein in the base editor to be detected is derived from SpCas9, such as nSpCas9 (SEQ ID NO:1), and the CRISPR effector protein in the detection CRISPR system is derived from SaCas9, such as nSaCas9(SEQ ID NO: 2).
- the CRISPR effector protein in the detection CRISPR system is derived from SpCas9, such as nSpCas9 (SEQ ID NO:1)
- the CRISPR effector protein in the base editor to be detected is derived from SaCas9, such as nSaCas9(SEQ ID NO: 2).
- the detection CRISPR system of the present invention includes multiple guide RNAs targeting multiple genomic detection target sites or expression constructs containing nucleotide sequences encoding the guide RNAs.
- the base editing system to be tested of the present invention does not include a guide RNA or an expression construct thereof, or includes a guide RNA but targets a detection target site different from the detection CRISPR system.
- the cell is a eukaryotic cell, such as a mammalian cell or a plant cell.
- the organism is a eukaryote, such as a mammal or a plant.
- the present invention also relates to a kit used in the above-mentioned method of the present invention.
- the kit at least includes the detection CRISPR system of the present invention, and optionally an amplification primer for detecting the target site targeted by the CRISPR system.
- the guide RNA backbones between many CRISPR systems are orthogonal, that is, the nuclease in the CRISPR system can only form a protein-RNA complex with the guide RNA of the system to perform functions.
- the inventor took the single-base editing system using nSpCas9 (Cas9 derived from Streptococcus pyogenes, a nickase variant with D10A point mutation) as an example, and tested the orthogonal nSaCas9 (Cas9 of Staphylococcus aureus, after D10A nickase variant with point mutation), dSaCas9 (Cas9 of Staphylococcus aureus, inactivated variant with D10A and N580A point mutations), and dLbCpf1 (Cpf1 protein of Lacetospiraceae, inactivated by D832A point mutation) Body) whether it is possible to create off-target single-stranded DNA regions for
- Table 1 below shows the targets used by the orthogonal CRISPR system.
- the PAM sequence is marked in bold, and the C base in the target site is underlined.
- the OsCDC48-SaT1 and OsNRT1.1B-SaT1 targets are used for nSaCas9 and dSaCas9 system test, OsEPSPS-Cpf1T1 and OsPDS-Cfp1T1 targets are used for LbCpf1 system test.
- the single-base editing system to be tested used in this experiment is the A3A-BE3 system, that is, the base editor is human APOBEC3A deaminase, nSpCas9 (Streptococcus pyogenes), UGI (uracil glycosylase inhibitor) and
- the fusion protein composed of NLS (nuclear localization signal), its expression vector is pA3A-BE3, and its target vector is pSp-sgRNA.
- the other three CRISPR systems are denoted as pnSaCas9 and pSa-sgRNA, pdSaCas9 and pSa-sgRNA target vector, pdLbCpf1 and Lb-crRNA, the vector structure is shown in Figure 1.
- SaT1, pdLbCpf1/pLb-crRNA-OsEPSPS-Cpf1T1, pdLbCpf1/pLb-crRNA-OsPDS-Cfp1T1 was co-transformed into rice protoplasts.
- the reported cytosine single-base editing systems BE3, YEE-BE3, RK-BE3, A3A-BE3 and eA3A-BE3 systems were analyzed for random off-target effects.
- the vectors involved in this experiment are all single-base editing systems based on the BE3 single-base editor backbone. Replace the rAPOBEC1 deaminase in the BE3 vector with other deaminases to obtain different single-base editors
- the vector backbone of BE3 is shown in Figure 3, RK and YEE represent the R33AK34A variant and W90YR126ER132E variant of rat-derived rAPOBEC1 deaminase, and eA3A represents the N57G variant of human-derived hAPOBEC3A.
- the targets involved in this experiment include the targets in Table 2 below.
- the PAM sequence is marked in bold, and the C base in the target site is underlined
- OsAAT1-T1, OsACTG-T1, OsEV-T1 and OsCDC48-T1 is the target site used by the cytosine single-base editing system
- OsCDC48-SaT1, OsDEP1-SaT1, OsDEP1-SaT2 and OsNRT1.1B-SaT1 are the off-target detection targets used by nSaCas9.
- Example 3 Whole-genome sequencing of individual plants verifies the accuracy of the TA-AS method
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- 一种检测碱基编辑系统的随机脱靶效应的方法,所述方法包括:a)向细胞或生物体导入待检测的碱基编辑系统;b)向细胞或生物体导入靶向基因组中至少一个检测靶位点的检测CRISPR系统,所述检测CRISPR系统能够在所述至少一个检测靶位点处形成单链DNA区域,且其向导RNA与所述待检测的碱基编辑系统的向导RNA不相容;c)从所述细胞或生物体提取核酸并扩增所述至少一个检测靶位点的序列,对扩增子进行测序;和d)确定所述至少一个检测靶位点处的核苷酸突变。
- 权利要求1的方法,其中所述待检测的碱基编辑系统可以包含待检测的碱基编辑器或包含其编码序列的表达构建体,和/或其相应的向导RNA或包含其编码序列的表达构建体。
- 权利要求1的方法,其中所述待检测的碱基编辑系统包含胞嘧啶碱基编辑器。
- 权利要求3的方法,其中所述胞嘧啶碱基编辑器是包含CRISPR效应蛋白和胞嘧啶脱氨酶的融合蛋白。
- 权利要求4的方法,其中所述胞嘧啶脱氨酶选自APOBEC1脱氨酶、激活诱导的胞苷脱氨酶(AID)、APOBEC3G、CDA1、人APOBEC3A脱氨酶,或它们的功能性变体,例如,所述胞嘧啶脱氨酶包括SEQ ID NO:7-10的氨基酸序列。
- 权利要求4的方法,其中所述碱基编辑器的CRISPR效应蛋白是酸酶失活的CRISPR效应蛋白,例如是具有切口酶活性的CRISPR效应蛋白。
- 权利要求4的方法,其中所述碱基编辑器的CRISPR效应蛋白是Cas9切口酶。
- 权利要求4的方法,其中所述碱基编辑器的CRISPR效应蛋白是来自化脓链球菌(S.pyogenes)的SpCas9的切口酶形式(nSpCas9),例如其包含SEQ ID NO:1所示氨基酸序列。
- 权利要求4的方法,其中所述碱基编辑器的CRISPR效应蛋白是来自金黄色葡萄球菌(S.aureus)的SaCas9的切口酶形式(nSaCas9),例如,所述nSaCas9包含SEQ ID NO:2所示氨基酸序列。
- 权利要求1-9中任一项的方法,其中所述检测CRISPR系统可以包括CRISPR效应蛋白或包含其编码核苷酸序列的表达构建体,以及,靶向至少一个基因组检测靶位点的相应的向导RNA或包含其编码核苷酸序列的表达构建体。
- 权利要求10的方法,其中所述检测CRISPR系统的CRISPR效应蛋白是酸酶失活的CRISPR效应蛋白,例如是具有切口酶活性的CRISPR效应蛋白。
- 权利要求10的方法,其中所述检测CRISPR系统的CRISPR效应蛋白是Cas9切口酶。
- 权利要求10的方法,其中所述检测CRISPR系统的CRISPR效应蛋白是来自化 脓链球菌(S.pyogenes)的SpCas9的切口酶形式(nSpCas9),例如,所述nSpCas9包含SEQ ID NO:1所示氨基酸序列。
- 权利要求10的方法,其中所述检测CRISPR系统的CRISPR效应蛋白是来自金黄色葡萄球菌(S.aureus)的SaCas9的切口酶形式(nSaCas9),例如,所述nSaCas9包含SEQ ID NO:2所示氨基酸序列。
- 权利要求1-14中任一项的方法,所述检测CRISPR系统中的CRISPR效应蛋白与所述碱基编辑器中的CRISPR效应蛋白来源不同,由此它们的向导RNA不相容。
- 权利要求1-15中任一项的方法,所述碱基编辑器中的CRISPR效应蛋白衍生自SpCas9,例如是nSpCas9(SEQ ID NO:1),所述检测CRISPR系统中的CRISPR效应蛋白衍生自SaCas9,例如是nSaCas9(SEQ ID NO:2)。
- 权利要求1-15中任一项的方法,所述检测CRISPR系统中的CRISPR效应蛋白衍生自SpCas9,例如是nSpCas9(SEQ ID NO:1),所述碱基编辑器中的CRISPR效应蛋白衍生自SaCas9,例如是nSaCas9(SEQ ID NO:2)。
- 权利要求1-17中任一项的方法,所述检测CRISPR系统包含靶向多个基因组检测靶位点的多种向导RNA或包含所述向导RNA编码核苷酸序列的表达构建体。
- 权利要求1-18中任一项的方法,所述待检测的碱基编辑系统不包含向导RNA或其表达构建体,或者包含向导RNA但靶向不同于所述检测CRISPR系统的检测靶位点。
- 权利要求1-19中任一项的方法,所述细胞是真核细胞,例如哺乳动物细胞或植物细胞;所述生物体是真核生物,例如哺乳动物或植物。
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BR112022017676A BR112022017676A2 (pt) | 2020-03-04 | 2021-03-04 | Método para detectar o efeito fora do alvo aleatório do sistema de edição de base única |
EP21763688.5A EP4116430A4 (en) | 2020-03-04 | 2021-03-04 | METHOD FOR DETECTING THE RANDOM OFF-TARGET EFFECT OF A SINGLE BASE MACHINING SYSTEM |
CN202180019218.XA CN115279922A (zh) | 2020-03-04 | 2021-03-04 | 检测单碱基编辑系统随机脱靶效应的方法 |
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