WO2021175287A1 - 检测单碱基编辑系统随机脱靶效应的方法 - Google Patents

检测单碱基编辑系统随机脱靶效应的方法 Download PDF

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WO2021175287A1
WO2021175287A1 PCT/CN2021/079082 CN2021079082W WO2021175287A1 WO 2021175287 A1 WO2021175287 A1 WO 2021175287A1 CN 2021079082 W CN2021079082 W CN 2021079082W WO 2021175287 A1 WO2021175287 A1 WO 2021175287A1
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crispr
effector protein
detection
crispr effector
base
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高彩霞
靳帅
朱子旭
费宏源
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Institute of Genetics and Developmental Biology of CAS
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Priority to BR112022017676A priority patent/BR112022017676A2/pt
Priority to CN202180019218.XA priority patent/CN115279922B/zh
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    • C12Q1/6869—Methods for sequencing
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12Q2600/00—Oligonucleotides characterized by their use
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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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Abstract

本发明属于基因编辑领域。本发明提供了快速高通量检测单碱基编辑系统的全基因组范围内随机脱靶效应的方法和工具。

Description

检测单碱基编辑系统随机脱靶效应的方法 技术领域
本发明属于基因编辑领域。具体而言，本发明涉及快速高通量检测单碱基编辑系统的全基因组范围内随机脱靶效应的方法。
发明背景
基因组编辑技术是基于人工核酸酶对基因组进行靶向修饰的基因工程技术，在农业和医学研究中发挥着越来越强大的作用。成簇的规律间隔的短回文重复序列及其相关系统(Clustered regularly interspaced short palindromic repeats/CRISPR associated,CRISPR)是目前使用最广泛的基因组编辑工具，在人工设计的向导RNA的导向作用下，Cas蛋白可以靶向基因组中的任意位置。
单碱基编辑系统是基于CRISPR系统开发的新型基因编辑技术，分为胞嘧啶单碱基编辑系统和腺嘌呤单碱基编辑系统，分别将胞嘧啶脱氨酶与腺嘌呤脱氨酶与Cas9单链切口酶融合，在向导RNA的靶向作用下，Cas9单链切口酶产生一个单链DNA区域，因此脱氨酶可以高效地分别将靶向位置的单链DNA上的C或A核苷酸脱去氨基，变为U碱基和I碱基，进而在细胞自身修复的过程中被修复为T碱基或G碱基。但是，胞嘧啶单碱基编辑系统被发现在基因组范围内会产生不可预测的脱靶现象，这很可能是由于胞嘧啶脱氨酶在基因组中过量表达，在基因组中的高转录活跃区产生的随机脱氨现象导致的，基因组范围的脱靶现象极大地影响了胞嘧啶单碱基编辑系统的使用。
迄今为止，评价单碱基编辑系统脱靶效应的唯一方法是使用全基因组测序技术，将经过单碱基编辑的大量细胞或者生物个体进行测序，统计全基因组范围的点突变数量，以评价该单碱基编辑系统在基因组范围内的随机脱靶效应，但是该方法花费高昂，实验周期长，无法高通量地检测多种单碱基编辑系统在基因组范围内的随机脱靶效应。
本发明仍需简便的、低成本的检测单碱基编辑系统的随机脱靶效应的方法。
发明简述
本发明人发现在细胞中与待鉴定的单碱基编辑器共转染一个与所述单碱基编辑系统正交的可以产生单链区域的其他CRISPR系统，可以在基因组内产生一个长时间稳定的单链区域，使得可以随机作用于单链DNA区域的单碱基编辑器在该单链区域的目标碱基上脱氨，通过扩增子高通量测序即可高效简便地检测该单碱基编辑系统的随机脱靶效应，该方法称为Trans-ssDNA amplicon deep sequencing(TA-AS)方法。
附图简述
图1、正交系统检测载体示意图。
图2、水稻原生质体转化验证TA-AS系统。
图3、BE3载体示意图。
图4、TA-AS方法检测不同单碱基编辑系统的脱靶效应。
图5、全基因组测序检测五种单碱基编辑系统的脱靶效应。
图6、全基因组测序结果与TA-AS结果的回归性分析。
发明详述
在一方面，本发明提供了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，并对扩增子进行测序；和
d)确定所述至少一个检测靶位点处的核苷酸突变。
在一些实施方案中，在所述至少一个检测靶位点中检测出核苷酸突变表示所述待检测碱基编辑系统存在脱靶。在所述至少一个检测靶位点中检测出的核苷酸突变的量代表脱靶的程度，检测出核苷酸突变越多代表脱靶程度越高。
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(base editor)或包含其编码序列的表达构建体，和/或其相应的向导RNA(gRNA)或包含其编码序列的表达构建体。在一些实施方案中，步骤a)中所述待检测的碱基编辑系统只包含待检测的碱基编辑器(base editor)或包含其编码序列的表达构建体。
如本文所用，“碱基编辑器”是指包含CRISPR效应蛋白和脱氨酶的融合蛋白。根据脱氨酶的不同，碱基编辑器可以分为胞嘧啶碱基编辑器和腺嘌呤碱基编辑器。在一些优选实施方式中，本发明的待检测的碱基编辑系统包含胞嘧啶碱基编辑器。
胞嘧啶碱基编辑器通常是包含CRISPR效应蛋白和胞嘧啶脱氨酶的融合蛋白。碱基编辑器中的胞嘧啶脱氨酶能够将CRIPR效应蛋白-向导RNA-靶DNA复合物形成中产生的单链DNA的胞苷脱氨转换成U，再通过碱基错配修复实现C至T的碱基替换。在一些实施方案中，所述胞嘧啶碱基编辑器还包含尿嘧啶DNA糖基化酶抑制剂(UGI)。在细胞中，尿嘧啶DNA糖基化酶催化U从DNA上的去除并启动碱基切除修复(BER)，导致将U:G修复成C:G。因此，不受任何理论限制，在胞嘧啶碱基编辑器中包含尿嘧啶DNA糖基化酶抑制剂(UGI)将能够增加C至T碱基编辑的效率。
胞嘧啶脱氨酶的实例包括但不限于例如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。在一些实 施方式中，所述胞嘧啶脱氨酶是人APOBEC3A或其功能性变体。在一些实施方式中，所述胞嘧啶脱氨酶是APOBEC1或其功能性变体。在一些具体实施方式中，所述胞嘧啶脱氨酶包括SEQ ID NO:7-10的氨基酸序列。
然而，本发明的方法可用于测试包含各种胞嘧啶脱氨酶变体的碱基编辑器的脱靶效应。
如本文所用，术语“CRISPR效应蛋白”通常指在天然存在的CRISPR系统中存在的核酸酶，以及其修饰形式、其变体、其催化活性片段等。该术语涵盖基于CRISPR系统的能够在细胞内实现基因靶向(例如基因编辑、基因靶向调控等)的任何效应蛋白。
“CRISPR效应蛋白”的实例包括Cas9核酸酶或其变体。所述Cas9核酸酶可以是来自不同物种的Cas9核酸酶，例如来自化脓链球菌(S.pyogenes)的spCas9或衍生自金黄色葡萄球菌(S.aureus)的SaCas9。“Cas9核酸酶”和“Cas9”在本文中可互换使用，指的是包括Cas9蛋白或其片段(例如包含Cas9的活性DNA切割结构域和/或Cas9的gRNA结合结构域的蛋白)的RNA指导的核酸酶。Cas9是CRISPR/Cas(成簇的规律间隔的短回文重复序列及其相关系统)基因组编辑系统的组分，能在向导RNA的指导下靶向并切割DNA靶序列形成DNA双链断裂(DSB)。
“CRISPR效应蛋白”的实例还可以包括Cpf1核酸酶或其变体例如高特异性变体。所述Cpf1核酸酶可以是来自不同物种的Cpf1核酸酶，例如来自Francisella novicida U112、Acidaminococcus sp.BV3L6和Lachnospiraceae bacterium ND2006的Cpf1核酸酶。
在一些实施方案中，本发明的碱基编辑器的CRISPR效应蛋白是酸酶失活的CRISPR效应蛋白。在一些实施方案中，本发明的碱基编辑器的CRISPR效应蛋白是具有切口酶活性的CRISPR效应蛋白。在一些实施方案中，本发明的碱基编辑器的CRISPR效应蛋白是Cas9切口酶。在一些优选实施方案中，在一些实施方案中，本发明的碱基编辑器的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，所述nSpCas9包含SEQ ID NO:1所示氨基酸序列。在一些实施方案中，本发明的碱基编辑器的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。
本发明的检测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靶位点(检测靶位点)的向导RNA或包含其编码核苷酸序列的表达构建体。
在一些实施方案中，本发明的检测CRISPR系统的CRISPR效应蛋白是酸酶失活的CRISPR效应蛋白。在一些实施方案中，本发明的检测CRISPR系统的CRISPR效应蛋白是具有切口酶活性的CRISPR效应蛋白。在一些实施方案中，本发明的检测CRISPR系统的CRISPR效应蛋白是Cas9切口酶。在一些优选实施方案中，在一些实施方案中，本发明的检测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。在一些实施方案中，本发明的检测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。
检测CRISPR系统与待检测的碱基编辑系统的向导RNA不相容指的是检测CRISPR系统不能使用待检测的碱基编辑系统的向导RNA，待检测的碱基编辑系统也不能使用检测CRISPR系统的向导RNA。这取决于系统中使用的不同CRISPR效应蛋白。
在一些实施方案中，所述检测CRISPR系统中的CRISPR效应蛋白与所述待检测的碱基编辑器中的CRISPR效应蛋白来源不同，由此它们的向导RNA不相容。
在一些实施方案中，所述检测CRISPR系统中的CRISPR效应蛋白衍生自金黄色葡萄球菌(S.aureus)的SaCas9，其相应向导RNA包含SEQ ID NO:5所示支架序列。
在一些实施方案中，所述检测CRISPR系统中的CRISPR效应蛋白衍生自化脓链球菌(S.pyogenes)的SpCas9，其相应向导RNA包含SEQ ID NO:11所示支架序列。
在一些实施方案中，所述待检测碱基编辑器中的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)。在一些实施方案中，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)，所述待检测碱基编辑器中的CRISPR效应蛋白衍生自SaCas9，例如是nSaCas9(SEQ ID NO:2)。
在一些实施方案中，本发明的检测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。在一些实施方案中，本发明的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。
在一些实施方案中，所述细胞是真核细胞，例如哺乳动物细胞或植物细胞。或者，所述生物体是真核生物，例如哺乳动物或植物。
在另一方面，本发明还涉及用于本发明上述方法的试剂盒。所述试剂盒至少包含本发明的检测CRISPR系统，以及任选的所述检测CRISPR系统所靶向的靶位点的扩增引物。
实施例
实施例1、TA-AS系统的开发
据报道，许多CRISPR系统之间的向导RNA骨架是正交的，即CRISPR系统中的核酸酶只能与本系统的向导RNA形成蛋白-RNA复合体以行使功能。发明人以单碱基编辑系统使用nSpCas9(来源于酿脓链球杆菌的Cas9，经过D10A点突变的切口酶变体)为例，测试了与之正交的nSaCas9(金黄色葡萄球菌的Cas9，经过D10A点突变的切口酶变体)，dSaCas9(金黄色葡萄球菌的Cas9，经过D10A与N580A点突变的失活变体)与dLbCpf1(毛螺科菌的Cpf1蛋白，经过D832A点突变的失活变体)是否可以创造供胞嘧啶单碱基编辑系统产生脱靶的单链DNA区域。
1.1.靶标片段与载体构建
下表1为正交的CRISPR系统所使用的靶点，PAM序列用粗体标出，靶位点内的C碱基用下划线标识，OsCDC48-SaT1与OsNRT1.1B-SaT1靶点用于nSaCas9和dSaCas9系统的测试，OsEPSPS-Cpf1T1与OsPDS-Cfp1T1靶点用于LbCpf1系统的测试。
表1.
Figure PCTCN2021079082-appb-000001
本实验中所使用的待测试单碱基编辑系统为A3A-BE3系统，即碱基编辑器为人APOBEC3A脱氨酶、nSpCas9(酿脓链球杆菌)、UGI(尿嘧啶糖基化酶抑制剂)及NLS(核定位信号)组成的融合蛋白，其表达载体为pA3A-BE3，其靶点载体为pSp-sgRNA。其他三个CRISPR系统记为pnSaCas9与pSa-sgRNA，pdSaCas9和pSa-sgRNA靶点载体，pdLbCpf1与Lb-crRNA，载体结构如图1所示。
1.2.水稻原生质体转化验证TA-AS系统
分别将A3A-BE3载体与：pnSaCas9/pSa-sgRNA-OsCDC48-SaT1、pnSaCas9/pSa-sgRNA-OsNRT1.1B-SaT1、pdSaCas9/pSa-sgRNA-OsCDC48-SaT1、pdSaCas9/pSa-sgRNA-OsNRT1.1B-SaT1、pdLbCpf1/pLb-crRNA-OsEPSPS-Cpf1T1、pdLbCpf1/pLb-crRNA-OsPDS-Cfp1T1组合共转入水稻原生质体。
经过靶位点扩增子高通量测序，发现无编辑靶点的A3A-BE3在nSaCas9靶向的OsCDC48-SaT1与OsNRT1.1B-SaT1靶点上发生了高水平的C至T单碱基编辑现象，而在其他两组处理中未检测到明显的单碱基编辑现象，未处理组(Untreated)也未检测到单碱基编辑现象(图2)。这说明nSaCas9在植物中可以产生持续稳定的ssDNA区域，以供高通量检测胞嘧啶单碱基编辑系统的随机脱靶效应。
实施例2、靶位点扩增子测序检测现有的单碱基编辑系统的脱靶活性
利用TA-AS系统，对已报道的胞嘧啶单碱基编辑系统BE3、YEE-BE3、RK-BE3、A3A-BE3与eA3A-BE3系统进行了随机脱靶效应的分析。
2.1.靶标片段与载体构建
本实验中所涉及到的载体均为基于BE3单碱基编辑器骨架的单碱基编辑系统，将BE3载体中的rAPOBEC1脱氨酶替换为其他脱氨酶，以获得不同的单碱基编辑器，BE3的载体骨架如图3所示，RK、YEE表示来源于大鼠的rAPOBEC1脱氨酶的R33AK34A变体与W90YR126ER132E变体，eA3A表示人源的hAPOBEC3A的N57G变体。
本实验中所涉及到的靶点包括如下表2中的靶点，PAM序列用粗体标出，靶位点 内的C碱基用下划线标识，OsAAT1-T1，OsACTG-T1，OsEV-T1与OsCDC48-T1为胞嘧啶单碱基编辑系统所使用的靶位点，OsCDC48-SaT1，OsDEP1-SaT1，OsDEP1-SaT2与OsNRT1.1B-SaT1为nSaCas9所使用的的脱靶检测靶点。
表2.
Figure PCTCN2021079082-appb-000002
2.2.
水稻原生质体转化检测多个单碱基编辑系统的脱靶活性
本实验中，将不同的单碱基编辑系统载体、pnSaCas9载体与pSa-sgRNA三种载体共同转化入水稻原生质体细胞，检测不同单碱基编辑系统的脱靶效率，具体效率如图4。A3A-BE3系统展现出了最高的随机脱靶效应，BE3与eA3A其次，YEE与RK系统几乎不存在随机脱靶效应。
实施例3、植物个体全基因组测序验证TA-AS方法的准确性
利用植物全基因组测序进行全基因组脱靶效应的评估是目前最直接、最准确的检测方法。利用经过农杆菌介导转化不同单碱基编辑系统表达载体，分别获得了单碱基编辑系统BE3、YEE-BE3、RK-BE3、A3A-BE3、eA3A-BE3过表达的水稻T0代再生植物，同时以只经过农杆菌转化的植物作为对照组(Control)。对植物进行了全基因组测序，发现该五组过表达水稻基因组范围的的小片段插入和删除(Indel)数量无显著差异(图5a)，但在总的核苷酸变异数量(AllSNVs)上，BE3、A3A-BE3处理组与Control组有显著性的差异，与Control组相比，分别产生了额外的102与316个SNVs(图5b)。在CtoT的核苷酸变异数量(C至TSNV)上，BE3-BE3、A3A-BE3、eA3A-BE3与Control组有显著性的差异，分别产生了额外的69和243个C至T的SNVs(图5c)。与之相反，YEE-BE3与RK-BE3并没有检测到明显的脱靶现象(图5)。另外，将本实验中的五个单碱基编辑系统处理组的C至TSNV的平均值与图4中用TA-AS系统检测到的脱靶效应做相关性分析可以发现，TA-AS方法与全基因组测序结果有显著的相关性(图6)。对于这五个单碱基编辑系统，TA-AS方法与全基因组测序方法展现了相同的实验结果，这说明，该方法有较高的灵敏性与准确性，可以用来高通量、简便地检测单碱基编辑系统的随机脱靶效应。
序列表
SEQ ID NO:1 nSpCas9氨基酸序列
Figure PCTCN2021079082-appb-000003
SEQ ID NO:2 nSaCas9氨基酸序列
Figure PCTCN2021079082-appb-000004
Figure PCTCN2021079082-appb-000005
SEQ ID NO:3 dSaCas9氨基酸序列
Figure PCTCN2021079082-appb-000006
SEQ ID NO:4 dLbCpf1氨基酸序列
Figure PCTCN2021079082-appb-000007
Figure PCTCN2021079082-appb-000008
SEQ ID NO:5 Sa-sgRNA支架序列
Figure PCTCN2021079082-appb-000009
SEQ ID NO:6 Lb-crRNA支架序列
Figure PCTCN2021079082-appb-000010
SEQ ID NO:7 hA3A脱氨酶
Figure PCTCN2021079082-appb-000011
SEQ ID NO:8 rAPOBEC1-RK脱氨酶
Figure PCTCN2021079082-appb-000012
SEQ ID NO:9 rAPOBEC1-YEE脱氨酶
Figure PCTCN2021079082-appb-000013
SEQ ID NO:10 rAPOBEC1-eA3A脱氨酶
Figure PCTCN2021079082-appb-000014
SEQ ID NO:11 SpsgRNA支架序列
Figure PCTCN2021079082-appb-000015

Claims (20)

  1. 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。
  2. 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。
  3. 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。
  4. 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。
  5. 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。
  6. 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。
  7. 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。
  8. 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。
  9. 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。
  10. 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。
  11. 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。
  12. 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。
  13. 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。
  14. 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。
  15. 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。
  16. 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)。
  17. 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)。
  18. 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。
  19. 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。
  20. 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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