WO2021073173A1 - Technique for precise site-specific rna shearing in fish embryo - Google Patents

Technique for precise site-specific rna shearing in fish embryo Download PDF

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WO2021073173A1
WO2021073173A1 PCT/CN2020/100974 CN2020100974W WO2021073173A1 WO 2021073173 A1 WO2021073173 A1 WO 2021073173A1 CN 2020100974 W CN2020100974 W CN 2020100974W WO 2021073173 A1 WO2021073173 A1 WO 2021073173A1
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rna
mrna
sequence
casrx
embryos
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PCT/CN2020/100974
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French (fr)
Chinese (zh)
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何小镇
钟丽丽
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福州大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/40—Fish

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a technology for realizing precise point RNA shearing in fish embryos.
  • off-target Because of its key role in the endogenous process, it has a significant off-target effect (off-target). effect) and remains a challenging goal (Birming-Ham et al., 2006; Jackson et al., 2003). Therefore, the methods for directly studying the function of RNA are still limited.
  • RNA binding domains A key limitation in RNA engineering is the lack of RNA binding domains, which can easily be relocated and introduced into target cells.
  • the ms2-RNA binding domain recognizes an invariant 21-nucleotide (nt) RNA sequence (peabody, 1993) and therefore requires genome modification to label the desired transcript.
  • Pumilio homology domains have modular repeats, each protein module recognizes a single RNA base, but they can only target short 8nt RNA sequences (Cheong, Hall, 2006).
  • CRISPR-Cas systems can be reprogrammed to recognize 20-30 nt RNA, but their large size (about 1200aa) makes It is difficult to package into adeno-associated virus (AAV) for transmission in primary cells and in vivo.
  • AAV adeno-associated virus
  • CasRx can also be flexibly packaged into adeno-associated viruses.
  • Gene knockdown technology is required for gene function screening. Because siRNA technology does not work well in zebrafish, antisense oligonucleotides (Morpholino, MO) are mainly used for gene knockdown operations on zebrafish embryos. Technology (Nasevicius and Ekker, 2000). Although the MO technology works well, because it is easy to miss the target, it is generally necessary to design two specific MO sequences for knockdown of each gene. In addition, the company that synthesizes MO is not in China, which leads to a long order cycle. Moreover, besides off-target, MO technology has some defects such as high toxicity (Stainier et al., 2017; Van Gils and Vanakker, 2019). Therefore, the establishment of a new RNA interference technology in zebrafish is of great significance for the study of zebrafish gene function.
  • CasRx-mediated knockout has high efficiency and specificity.
  • CasRx can be flexibly packaged into adeno-associated virus.
  • CasRx is A programmable RNA binding module that can effectively locate cellular RNA.
  • CasRx can be used to efficiently cut off the corresponding RNA of the exogenous fluorescent protein to weaken the visual genetic marker; CasRx can also be used to efficiently knock down some phenotypes The RNA corresponding to the obvious or key gene causes a different phenotype or death.
  • a technique for realizing precise point RNA shearing in fish embryos including the following methods:
  • GFP wild-type CasRx sequence and exogenous fluorescent protein
  • sgRNA corresponding guide RNA
  • sgRNA endogenous gene guide RNA
  • CasRx sequence was added to nuclear localization sequence (nucleus localization sequence), and then SP6 promoter sequence was added to the upstream of the entire sequence, and capped by in vitro transcription kit (capped) and polyA tailed mRNA, purified and extracted RNA is stored at -80 degrees for later use; design primers for exogenous fluorescent protein (GFP, BFP) DNA to synthesize DNA, and then synthesize mRNA through in vitro transcription, and purify the extracted RNA in -80 degrees cryopreserved for later use; different sgRNAs are synthesized by DNA synthesis upstream of fixed sequence with T7 promoter and different downstream sequences that are partially complementary to the upstream, and then double-stranded DNA is obtained by PCR amplification and obtained by in vitro transcription kit , The purified and extracted RNA is frozen and stored at -80 degrees for later use.
  • GFP exogenous fluorescent protein
  • each component of RNA during microinjection of foreign genes the final mRNA concentration of each fluorescent protein in the mixture is 600 ng/ul; the final concentration of sgRNA is 100 ng/ul; CasRx mRNA The final concentration is 200 ng/ul.
  • Each embryo is injected approximately ⁇ 1nl;
  • RNA The dose of each component of RNA during microinjection of endogenous genes: the final concentration of sgRNA in the mixed solution is 100ng/ul; the final concentration of CasRx mRNA is 200 ng/ul. Each embryo is injected approximately ⁇ 1nl.
  • confocal was used to take multiple fluorescence images of the microinjected control group and each experimental group embryos, and then the control group and each experiment were taken.
  • the 30 embryos in the group were divided into 2 tubes of EP tubes (15 for each), and 200ul Trizol was added to the EP tubes, then frozen at -80 °C, and then RNA was extracted. Reverse transcription is performed with the RNA using a reverse transcription kit, and the obtained DNA is subjected to real-time fluorescent quantitative PCR.
  • the present invention uses CRISPR/CasRx-mediated RNA editing technology that can target RNA editing to act on zebrafish embryos.
  • CasRx-mediated knockout has high efficiency and specificity, and CasRx can be flexibly packaged into adeno-associated viruses.
  • CasRx is a programmable RNA binding module that can effectively locate cellular RNA.
  • CasRx can be used to efficiently cut off the corresponding RNA of exogenous fluorescent protein to weaken the visual genetic markers; CasRx can also be used to knock down some The RNA corresponding to the obvious type or key gene causes a different phenotype or death.
  • CasRx is a type of Cas13d.
  • the Cas13 RNA editing system is composed of Cas13 protein and a 64 to 66 nt length CRISPR RNA (crRNA).
  • the crRNA can recognize 22 to 30 nt specific RNA sequences through the spacer.
  • the Cas13 system as an RNA editing tool has the following major advantages: 1. Cas13 can identify all target RNAs by changing the sequence of the crRNA spacer; 2. Cas13 is different from Cas9 and Cpf1, and does not require specific sequence elements for the target sequence ( Such as the PAM site); 3.
  • the effective complex of Cas13 is the simplest one in the CRISPR-Cas system.
  • the system composed of Cas13-crRNA is easier to operate and deliver than the system of trimerization and multimerization; 4. Multiple targets CrRNAs of different target sequences can be delivered simultaneously. Efficient, easy to operate, and highly specific, it is destined that Cas13-mediated RNA editing technology can shine in the study of gene function (Kim, 2018).
  • the wild-type CasRx sequence is added to the nucleus localization sequence, and then the SP6 promoter sequence is added to the upstream of the entire sequence, and the capping is obtained by using an in vitro transcription kit.
  • (capped) and polyA tailed mRNA, purified and extracted RNA is stored at -80 degrees for later use; design primers for exogenous fluorescent protein (GFP, BFP) DNA to synthesize DNA, and then synthesize mRNA through in vitro transcription, and purify the extracted RNA in -80 degrees cryopreserved for later use; different sgRNAs are synthesized by DNA synthesis upstream of fixed sequence with T7 promoter and different downstream sequences that are partially complementary to the upstream, and then double-stranded DNA is obtained by PCR amplification and obtained by in vitro transcription kit , The purified and extracted RNA is frozen and stored at -80 degrees for later use.
  • GFP exogenous fluorescent protein
  • CRISPR/CasRx is a new type of RNA editing tool, and CasRx is currently the most efficient version reported. Although CRISPR/CasRx is very efficient in cells, there are no reports of gene knockdown in zebrafish embryos.
  • the present invention Compared with the traditional zebrafish embryo Morpholino knock-down technology, the present invention has convenient design and lower cost.
  • the design of guide RNA the method of primer pairing amplification is adopted, and the fixed forward primer is used to match the reverse direction of specific targeting.
  • the sequence can amplify the template DNA of the guide RNA, and provide purification and transcription to quickly obtain the target-specific guide RNA.
  • This component only requires fixed CasRx mRNA and specifically targeted guide RNA.
  • the component is simple, the dosage is appropriate, and the toxicity is small, which can reduce the toxicity of the sample.
  • the guide RNA sequence is short, the cost is low, the synthesis is convenient, and the toxicity is low. Multiple gRNAs can be added at the same time to achieve simultaneous knockdown of multiple genes.
  • CasRx-mediated RNA targeted cleavage has very high sequence specificity, because CasRx mRNA adopts WPRE and poly A tailed bi-stable measures, which can exist in the embryo for a long time and is stable, enough to ensure the target RNA during embryonic development The continuous cutting effect.
  • This study provides a simple method for analyzing the efficiency of targeted RNA cleavage.
  • Real-time PCR is used to quickly analyze the cleavage efficiency of the target RNA sequence by providing appropriate internal controls.
  • FIG. 1 Schematic diagram of the application of CasRx-mediated RNA editing system in zebrafish embryos.
  • Figure 2 The efficiency verification of CasRx-mediated RNA editing in cutting exogenous mRNA in zebrafish embryos;
  • a and B Representative images and efficiency statistics of CasRx cutting EGFP mRNA in zebrafish embryos;
  • C and D CasRx in zebrafish embryos Representative graph and efficiency statistics of cleavage of BFP mRNA in embryos.
  • Figure 3 The efficiency verification of CasRx-mediated RNA editing in cutting endogenous mRNA in zebrafish embryos; A: 24 h phenotype diagram; B: qPCR analysis diagram.
  • Target site in this application refers to any RNA sequence that is to be knocked down in the target nucleotide.
  • the RNA sequence near the target site can accommodate the recognition of the foreign sequence at the target site.
  • the target RNA sequence is a single-stranded RNA sequence, including, but not limited to, RNA sequences in cells and RNA sequences in viruses.
  • Exogenous RNA sequence in this application refers to exogenous fluorescent protein capped and tailed RNA.
  • the wild-type CasRx sequence was added to the nucleus localization sequence (sequence CCGCCACC), and then the SP6 promoter sequence (sequence CAPACGATTTAGGTGACACTATAGAA) was added to the upstream of the entire sequence, and capped using the in vitro transcription kit (capped) and polyA tailed mRNA, purified and extracted RNA is stored at -80 degrees for later use; design primers for exogenous fluorescent protein (GFP, BFP) DNA to synthesize DNA, and then synthesize mRNA through in vitro transcription, and purify the extracted RNA in -80 degrees cryopreserved for later use; different sgRNAs are synthesized by DNA synthesis upstream of fixed sequence with T7 promoter and different downstream sequences that are partially complementary to the upstream, and then double-stranded DNA is obtained by PCR amplification and obtained by in vitro transcription kit , The purified and extracted RNA is frozen and stored at -80 degrees for later use.
  • GFP ex
  • Sequence 1 CasRx template sequence>PSKII-SP6-kozak-NLS-CasRx-NLS-HA-WPRE-PSKII
  • Sequence 3 BFP template sequence>SP6-kozak-EGFP> SP6-vertebrate kozak-TagBFP -stop
  • CATACGATTTAGGTGACACTATAGAA SP6 promoter sequence
  • CCGCCACC nuclear localization sequence
  • Sequence 4 DNA template sequence for sgRNA synthesis primer synthesis sequence
  • the wild-type CasRx sequence was added to the nucleus localization sequence, and then the SP6 promoter sequence was added to the upstream of the entire sequence, and the in vitro transcription kit was used to obtain the capping (capped) and polyA-tailed mRNA, exogenous EGFP DNA sequence is obtained by in vitro transcription kits to obtain exogenous EGFP mRNA sequence, sgRNA has a T7 promoter sequence upstream of DNA synthesis and a downstream sequence that is partially complementary to the upstream.
  • the double-stranded DNA obtained by PCR amplification was obtained by using an in vitro transcription kit; then, it was frozen at -80°C for use. During the microinjection, single-cell embryos were mixed and injected at a certain ratio. The fluorescence images were taken with a confocal microscope for 12 hours and the embryos were frozen for qPCR analysis.
  • DNA template sequence for EGFP sgRNA synthesis DNA template sequence for EGFP sgRNA synthesis, primer synthesis sequence
  • the wild-type CasRx sequence was added to the nucleus localization sequence, and then the SP6 promoter sequence was added upstream of the entire sequence.
  • the in vitro transcription kit was used to obtain capped and polyA tailed mRNA, foreign BFP
  • the DNA sequence uses the in vitro transcription kit to obtain the exogenous BFP mRNA sequence.
  • the sgRNA is synthesized upstream with the T7 promoter sequence and the downstream sequence that is partially complementary to the upstream.
  • double-stranded DNA is obtained.
  • the in vitro transcription reagents are used. Obtained in a box; then freeze at -80°C for later use. During the microinjection, single-cell embryos were mixed and injected at a certain ratio.
  • the fluorescence images were taken with a confocal microscope for 12 hours and the embryos were frozen for qPCR analysis.
  • the wild-type CasRx sequence was added to the nucleus localization sequence, and then the SP6 promoter sequence was added to the upstream of the entire sequence, and the in vitro transcription kit was used to obtain the capping (capped) and polyA tailed mRNA, sgRNA through DNA synthesis upstream with a T7 promoter sequence and a downstream sequence partially complementary to the upstream, after PCR amplification to obtain double-stranded DNA, using in vitro transcription kit to obtain; Freeze at -80°C for later use.
  • single-cell embryos were mixed and injected at a certain ratio.
  • the phenotype map was taken by a 24-hour inverted microscope and the embryos were frozen for qPCR analysis.
  • Wild-type CasRx was added to the nuclear localization sequence (nucleus localization sequence), and then the SP6 promoter sequence was added to the upstream of the entire sequence, and the in vitro transcription kit was used to obtain the capping (capped) and polyA tailed mRNA, sgRNA through DNA synthesis upstream with a T7 promoter sequence and a downstream sequence partially complementary to the upstream, after PCR amplification to obtain double-stranded DNA, using in vitro transcription kit to obtain; Freeze at -80°C for later use.
  • single-cell embryos were mixed and injected at a certain ratio. The phenotype map was taken by a 24-hour inverted microscope and the embryos were frozen for qPCR analysis.
  • microinjection is used in this example to introduce the mRNA composition of the present invention into zebrafish fertilized eggs.
  • the final mRNA concentration of each fluorescent protein in the composition is 600 ng/ul; the final concentration of sgRNA is 100 ng/ul; CasRx mRNA 200 ng/ul.
  • Each embryo is injected approximately ⁇ 1nl at the time of injection.

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Abstract

Provided is a technique for precise site-specific RNA shearing in a fish embryo, employing a CasRx-mediated RNA editing technique so that only CasRx mRNA and a specific targeting guide RNA need to be provided in order to achieve precise site-specific RNA knockdown in a fish embryo, bringing various advantages such as high efficiency, high specificity, convenience, and low costs. The present invention overcomes the low efficiency of existing siRNA technology and the problems of high costs and off-target effects of Morpholino-mediated gene knockdown technology.

Description

一种在鱼类胚胎中实现精确定点RNA剪切的技术A technology to achieve precise point RNA shearing in fish embryos 技术领域Technical field
本发明属于生物技术领域，具体涉及一种在鱼类胚胎中实现精确定点RNA剪切的技术。The invention belongs to the field of biotechnology, and specifically relates to a technology for realizing precise point RNA shearing in fish embryos.
背景技术Background technique
随着全基因组测序技术的不断发展完善以及大型基因组注释项目的实施，生物科学的研究进入后基因组时代。在后基因组时代，基因组研究的重心将转向基因功能，即由测定基因的DNA 序列、解释生命的所有遗传信息转移到从分子整体水平对生物学功能的研究上，在分子层面上探索人类健康和疾病的奥秘(Peltonen and McKusick, 2001)。科研人员们已经开始通过各种尝试，想要将基因组研究的成果尽早地运用到基础科学研究的各个领域，以及个性化医疗（personalized medicine）工作当中(Chan and Ginsburg, 2011)。不过面对海量枯燥的基因组信息，科研人员们该如何将这些数据转换成有意义的基因组功能？解决这个问题的一个关键在于尽快开发出一种高效率的、可靠的方法，来帮助科研人员研究基因型（genotype）对表型(phenotype）的影响作用。With the continuous development and improvement of whole-genome sequencing technology and the implementation of large-scale genome annotation projects, biological science research has entered the post-genome era. In the post-genome era, the focus of genome research will shift to gene function, that is, from determining the DNA sequence of genes and explaining all the genetic information of life to the study of biological functions from the overall molecular level, exploring human health and human health at the molecular level. The mystery of disease (Peltonen and McKusick, 2001). Researchers have begun to use various attempts to apply the results of genome research to various fields of basic scientific research as soon as possible, as well as personalized medicine (personalized medicine). medicine) at work (Chan and Ginsburg, 2011). However, in the face of massive boring genome information, how should researchers convert this data into meaningful genome functions? A key to solving this problem is to develop an efficient and reliable method as soon as possible to help researchers study the effect of genotype on phenotype.
通过技术进步，细胞功能和疾病转录组变化的绘图已经从微阵列（Schena et al，1995）转化为下一代测序和单细胞研究（Shendure et al ，2017）。然而，询问个体转录动力学的功能，并在观察到的转录变化和细胞表型之间建立因果关系，需要有能力主动控制或调节所需的转录。CRISPR-Cas9等DNA工程技术（Doudna Charpentier，2014；Hsu，2014）使研究人员能够解剖特定遗传元素的功能或纠正致病突变。然而，用于研究和操纵RNA的简单和可扩展的工具明显落后于它们的DNA对应物。现有的RNA干扰技术能够分解或抑制所需的转录物，由于其在内源性过程中的关键作用，因此具有显著的脱靶效应（off-target effect），并仍然是具有挑战性的目标（Birming-Ham等人，2006年；Jackson等人，2003年）。因此，直接研究RNA功能作用的方法仍然有限。Through technological advancement, the mapping of cell function and disease transcriptome changes has changed from microarrays (Schena et al. al, 1995) transformed into next-generation sequencing and single-cell research (Shendure et al, 2017). However, to inquire about the function of individual transcription dynamics and to establish a causal relationship between the observed transcriptional changes and the cell phenotype requires the ability to actively control or regulate the desired transcription. DNA engineering technologies such as CRISPR-Cas9 (Doudna Charpentier, 2014; Hsu, 2014) enable researchers to dissect the function of specific genetic elements or correct disease-causing mutations. However, the simple and scalable tools for researching and manipulating RNA lag significantly behind their DNA counterparts. The existing RNA interference technology can decompose or inhibit the required transcripts. Because of its key role in the endogenous process, it has a significant off-target effect (off-target). effect) and remains a challenging goal (Birming-Ham et al., 2006; Jackson et al., 2003). Therefore, the methods for directly studying the function of RNA are still limited.
技术问题technical problem
RNA工程中的一个关键限制是缺乏RNA结合域，这种结合域很容易被重定位并引入靶细胞。例如，ms2-RNA结合域识别一个不变的21核苷酸（nt）RNA序列（peabody，1993），因此需要基因组修饰来标记所需的转录物。Pumilio同源域具有模块化重复，每个蛋白质模块识别一个单独的RNA碱基，但它们只能针对短的8nt RNA序列（Cheong，Hall，2006）。虽然以前的特征化II型（Batra et al.，2017；O'Connell et al.，2014）和VI型（Abudayyeh et al.，2016；East Seletsky et al.，2016）CRISPR-Cas 系统可以重新编程以识别20-30 nt RNA，但它们的大尺寸（约1200aa）使得很难包装成腺相关病毒（AAV），用于初级细胞和体内传递。作为最紧凑的单细胞效应器Cas酶之一，CasRx也可以被灵活地包装成腺相关病毒。A key limitation in RNA engineering is the lack of RNA binding domains, which can easily be relocated and introduced into target cells. For example, the ms2-RNA binding domain recognizes an invariant 21-nucleotide (nt) RNA sequence (peabody, 1993) and therefore requires genome modification to label the desired transcript. Pumilio homology domains have modular repeats, each protein module recognizes a single RNA base, but they can only target short 8nt RNA sequences (Cheong, Hall, 2006). Although the previous characterization type II (Batra et al., 2017; O'Connell et al., 2014) and type VI (Abudayyeh et al., 2016; East Seletsky et al., 2016) CRISPR-Cas systems can be reprogrammed to recognize 20-30 nt RNA, but their large size (about 1200aa) makes It is difficult to package into adeno-associated virus (AAV) for transmission in primary cells and in vivo. As one of the most compact single-cell effector Cas enzymes, CasRx can also be flexibly packaged into adeno-associated viruses.
基因功能筛选的时候需要用到基因敲降技术（gene knockdown），由于siRNA技术在斑马鱼中效果不好，斑马鱼胚胎上进行基因knockdown操作时主要采用反义寡核苷酸（Morpholino, MO）技术(Nasevicius and Ekker, 2000)。MO技术虽然效果不错，但是因为容易脱靶，导致针对每个基因进行knockdown的时候一般都需要设计两条特异的MO序列，另外目前合成MO的公司并不在国内，导致订货周期偏长。而且，MO技术除了脱靶外还有毒性大等缺陷(Stainier et al., 2017; Van Gils and Vanakker, 2019)。因此，在斑马鱼中建立新的RNA干扰技术对于斑马鱼基因功能研究具有重要意义。Gene knockdown technology (gene knockdown) is required for gene function screening. Because siRNA technology does not work well in zebrafish, antisense oligonucleotides (Morpholino, MO) are mainly used for gene knockdown operations on zebrafish embryos. Technology (Nasevicius and Ekker, 2000). Although the MO technology works well, because it is easy to miss the target, it is generally necessary to design two specific MO sequences for knockdown of each gene. In addition, the company that synthesizes MO is not in China, which leads to a long order cycle. Moreover, besides off-target, MO technology has some defects such as high toxicity (Stainier et al., 2017; Van Gils and Vanakker, 2019). Therefore, the establishment of a new RNA interference technology in zebrafish is of great significance for the study of zebrafish gene function.
技术解决方案Technical solutions
本发明的目的在于提供一种在鱼类胚胎中实现精确定点RNA剪切的技术，CasRx介导的敲除具有较高的效率和特异性，CasRx可以被灵活地包装成腺相关病毒，CasRx是一种可编程的RNA结合模块，可以有效地定位细胞RNA，可利用CasRx高效剪切掉外源荧光蛋白相应的RNA，使其可视化的遗传标记减弱；也可利用CasRx高效敲降一些具有表型明显或关键基因所对应的RNA，使其出现不一样的表型或者死亡。The purpose of the present invention is to provide a technology for realizing precise point RNA shearing in fish embryos. CasRx-mediated knockout has high efficiency and specificity. CasRx can be flexibly packaged into adeno-associated virus. CasRx is A programmable RNA binding module that can effectively locate cellular RNA. CasRx can be used to efficiently cut off the corresponding RNA of the exogenous fluorescent protein to weaken the visual genetic marker; CasRx can also be used to efficiently knock down some phenotypes The RNA corresponding to the obvious or key gene causes a different phenotype or death.
为实现上述目的，本发明采用如下技术方案：In order to achieve the above objectives, the present invention adopts the following technical solutions:
一种在鱼类胚胎中实现精确定点RNA剪切的技术，包括以下方法：A technique for realizing precise point RNA shearing in fish embryos, including the following methods:
A、野生型CasRx序列及外源荧光蛋白（GFP、BFP）mRNA及相应的导向RNA（sgRNA）和内源基因的导向RNA（sgRNA）的设计和制备；A. Design and preparation of wild-type CasRx sequence and exogenous fluorescent protein (GFP, BFP) mRNA and corresponding guide RNA (sgRNA) and endogenous gene guide RNA (sgRNA);
B、显微注射时RNA各组分的剂量确定；B. Determine the dosage of each component of RNA during microinjection;
C、外源mRNA在斑马鱼胚胎中的切割效率检测方法；C. Detection method for cutting efficiency of exogenous mRNA in zebrafish embryos;
D、内源mRNA在斑马鱼胚胎中的切割效率检测方法；D. Detection method for cutting efficiency of endogenous mRNA in zebrafish embryos;
E、内源多个基因同时进行敲降的方法。E. The method of knocking down multiple endogenous genes at the same time.
具体包括以下方法：Specifically include the following methods:
（1）野生型CasRx序列及外源荧光蛋白（GFP、BFP）mRNA及相应的导向RNA（sgRNA）和内源基因的导向RNA（sgRNA）的设计和制备；(1) Design and preparation of wild-type CasRx sequence and exogenous fluorescent protein (GFP, BFP) mRNA and corresponding guide RNA (sgRNA) and endogenous gene guide RNA (sgRNA);
CasRx序列分别加入核定位序列 (nucleus localization sequence)，然后于整段序列之上游加入SP6启动子序列，利用体外转录的试剂盒得到加帽 (capped)及加polyA尾的mRNA，纯化提取的RNA于-80 度冻存备用；设计外源荧光蛋白（GFP、BFP）DNA的引物合成DNA,再经过体外转录合成mRNA，纯化提取的RNA于-80 度冻存备用 ；不同的sgRNA通过DNA合成上游带有T7启动子的固定序列及与上游存在部分互补的不同的下游序列，经过PCR扩增得到双链DNA，利用体外转录的试剂盒得到，纯化提取的RNA于-80 度冻存备用。CasRx sequence was added to nuclear localization sequence (nucleus localization sequence), and then SP6 promoter sequence was added to the upstream of the entire sequence, and capped by in vitro transcription kit (capped) and polyA tailed mRNA, purified and extracted RNA is stored at -80 degrees for later use; design primers for exogenous fluorescent protein (GFP, BFP) DNA to synthesize DNA, and then synthesize mRNA through in vitro transcription, and purify the extracted RNA in -80 degrees cryopreserved for later use; different sgRNAs are synthesized by DNA synthesis upstream of fixed sequence with T7 promoter and different downstream sequences that are partially complementary to the upstream, and then double-stranded DNA is obtained by PCR amplification and obtained by in vitro transcription kit , The purified and extracted RNA is frozen and stored at -80 degrees for later use.
（2）显微注射时混合液中RNA各组分的剂量确定；(2) Determine the dosage of each component of RNA in the mixed solution during microinjection;
a、外源基因时显微注射时RNA各组分的剂量：混合液中各荧光蛋白终mRNA浓度为600 ng/ul；sgRNA终浓度为100ng/ul；CasRx mRNA 终浓度为200 ng/ul。每个胚胎注射大约~1nl;a. The dosage of each component of RNA during microinjection of foreign genes: the final mRNA concentration of each fluorescent protein in the mixture is 600 ng/ul; the final concentration of sgRNA is 100 ng/ul; CasRx mRNA The final concentration is 200 ng/ul. Each embryo is injected approximately ~1nl;
b、内源基因时显微注射时RNA各组分的剂量：混合液中sgRNA终浓度为100ng/ul；CasRx mRNA终浓度为200 ng/ul。每个胚胎注射大约~1nl。b. The dose of each component of RNA during microinjection of endogenous genes: the final concentration of sgRNA in the mixed solution is 100ng/ul; the final concentration of CasRx mRNA is 200 ng/ul. Each embryo is injected approximately ~1nl.
（3）外源mRNA在斑马鱼胚胎中的切割效率检测方法；(3) Detection method for cutting efficiency of exogenous mRNA in zebrafish embryos;
斑马鱼胚胎单胞期注射相应实验组和对照组组分后，约12 h时运用共聚焦拍摄显微注射的对照组及各实验组胚胎的多张荧光图，之后分别取对照组及各实验组30颗胚胎分装2管EP管（各15颗），并往EP管中加200ul Trizol后冻于-80 ℃，之后提RNA。用所提的RNA利用逆转录试剂盒进行逆转录，得到的DNA进行实时荧光定量PCR。利用相应的软件LAS AF Lite、GraphPad Prism 5、ImageJ对共聚焦拍摄的荧光图和LightCycler® 96 SW 1.1、GraphPad Prism 5对定量PCR的图进行相应的分析。After zebrafish embryo single cell phase injection of the corresponding experimental group and control group components, at about 12 h, confocal was used to take multiple fluorescence images of the microinjected control group and each experimental group embryos, and then the control group and each experiment were taken. The 30 embryos in the group were divided into 2 tubes of EP tubes (15 for each), and 200ul Trizol was added to the EP tubes, then frozen at -80 ℃, and then RNA was extracted. Reverse transcription is performed with the RNA using a reverse transcription kit, and the obtained DNA is subjected to real-time fluorescent quantitative PCR. Use the corresponding software LAS AF Lite, GraphPad Prism 5, ImageJ's fluorescence image taken by confocal and LightCycler® 96 SW 1.1, GraphPad Prism 5 performs corresponding analysis on the graphs of quantitative PCR.
( 4 )内源mRNA在斑马鱼胚胎中的切割效率检测方法；(4) A method for detecting the cutting efficiency of endogenous mRNA in zebrafish embryos;
斑马鱼胚胎单胞期注射相应实验组和对照组组分后，在24h时利用荧光倒置显微镜对显微注射及未处理的WT分别进行表型拍照并在24 h时进行畸形统计，24 h之后取对照组及各实验组的畸形胚胎30颗胚胎分装3管EP管（各10颗），并往EP管中加200ul Trizol后冻于-80 ℃，之后提RNA。用所提的RNA利用逆转录试剂盒进行逆转录，得到的DNA进行实时荧光定量PCR。利用相应的软件LightCycler® 96 SW 1.1、GraphPad Prism 5对定量PCR的图进行相应的分析。After zebrafish embryo single cell stage injection of the corresponding experimental group and control group, the phenotype of the microinjected and untreated WT was taken with a fluorescent inverted microscope at 24 h, and abnormality statistics were performed at 24 h. After 24 h Take 30 embryos from the control group and each experimental group into 3 tubes of EP tubes (10 each), add 200ul Trizol to the EP tubes and freeze them at -80 ℃, and then extract RNA. Reverse transcription is performed with the RNA using a reverse transcription kit, and the obtained DNA is subjected to real-time fluorescent quantitative PCR. Use the corresponding software LightCycler® 96 SW 1.1, GraphPad Prism 5 Perform corresponding analysis on the graph of quantitative PCR.
( 5 )内源多个基因同时进行敲降的方法。(5) A method of knocking down multiple endogenous genes at the same time.
配置CasRx mRNA 终浓度为200 ng/ul，A 基因导向sgRNA终浓度为100ng/ul，B 基因导向sgRNA终浓度为100ng/ul, C 基因导向sgRNA终浓度为100ng/ul的混合液，将该组分同时注射到斑马鱼胚胎中，观察斑马鱼胚胎的发育情况。Configure the final concentration of CasRx mRNA at 200 ng/ul, the final concentration of A gene targeting sgRNA at 100ng/ul, the final concentration of B gene targeting sgRNA at 100ng/ul, and the final concentration of C gene targeting sgRNA at 100ng/ul. Inject into zebrafish embryos at the same time to observe the development of zebrafish embryos.
本发明利用可以RNA靶向编辑的CRISPR/CasRx介导的RNA编辑技术作用于斑马鱼胚胎，CasRx介导的敲除具有较高的效率和特异性，CasRx可以被灵活地包装成腺相关病毒，CasRx是一种可编程的RNA结合模块，可以有效地定位细胞RNA，可利用CasRx高效剪切掉外源荧光蛋白相应的RNA，使其可视化的遗传标记减弱；也可利用CasRx敲降一些具有表型明显或关键基因所对应的RNA，使其出现不一样的表型或者死亡。The present invention uses CRISPR/CasRx-mediated RNA editing technology that can target RNA editing to act on zebrafish embryos. CasRx-mediated knockout has high efficiency and specificity, and CasRx can be flexibly packaged into adeno-associated viruses. CasRx is a programmable RNA binding module that can effectively locate cellular RNA. CasRx can be used to efficiently cut off the corresponding RNA of exogenous fluorescent protein to weaken the visual genetic markers; CasRx can also be used to knock down some The RNA corresponding to the obvious type or key gene causes a different phenotype or death.
CasRx是Cas13d 的一种。Cas13 RNA编辑系统由Cas13蛋白以及一条64到66 nt长度的CRISPR RNA（crRNA）组成，crRNA能够通过间隔区识别22到30 nt特异RNA序列。Cas13系统作为RNA编辑工具具有以下几大优势：1、Cas13能够通过改变crRNA间隔区的序列从而识别所有的目标RNA；2、Cas13不同于Cas9以及Cpf1,对于目标序列并不需要特异的序列元件（如PAM位点）；3、Cas13的有效复合体是CRISPR–Cas系统中最简单的一种，Cas13–crRNA组成的系统比三聚获多聚的系统更容易操作和传递；4、多个针对不同靶序列的crRNAs可以同时进行传递。高效、易于操作且特异性高等特点，注定Cas13介导的RNA编辑技术能够在基因功能研究中大放异彩 (Kim, 2018)。CasRx is a type of Cas13d. The Cas13 RNA editing system is composed of Cas13 protein and a 64 to 66 nt length CRISPR RNA (crRNA). The crRNA can recognize 22 to 30 nt specific RNA sequences through the spacer. The Cas13 system as an RNA editing tool has the following major advantages: 1. Cas13 can identify all target RNAs by changing the sequence of the crRNA spacer; 2. Cas13 is different from Cas9 and Cpf1, and does not require specific sequence elements for the target sequence ( Such as the PAM site); 3. The effective complex of Cas13 is the simplest one in the CRISPR-Cas system. The system composed of Cas13-crRNA is easier to operate and deliver than the system of trimerization and multimerization; 4. Multiple targets CrRNAs of different target sequences can be delivered simultaneously. Efficient, easy to operate, and highly specific, it is destined that Cas13-mediated RNA editing technology can shine in the study of gene function (Kim, 2018).
本发明在具体的实施中，野生型CasRx序列分别加入核定位序列 (nucleus localization sequence)，然后于整段序列之上游加入SP6启动子序列，利用体外转录的试剂盒得到加帽 (capped)及加polyA尾的mRNA，纯化提取的RNA于-80 度冻存备用；设计外源荧光蛋白（GFP、BFP）DNA的引物合成DNA,再经过体外转录合成mRNA，纯化提取的RNA于-80 度冻存备用 ；不同的sgRNA通过DNA合成上游带有T7启动子的固定序列及与上游存在部分互补的不同的下游序列，经过PCR扩增得到双链DNA，利用体外转录的试剂盒得到，纯化提取的RNA于-80 度冻存备用。In the specific implementation of the present invention, the wild-type CasRx sequence is added to the nucleus localization sequence, and then the SP6 promoter sequence is added to the upstream of the entire sequence, and the capping is obtained by using an in vitro transcription kit. (capped) and polyA tailed mRNA, purified and extracted RNA is stored at -80 degrees for later use; design primers for exogenous fluorescent protein (GFP, BFP) DNA to synthesize DNA, and then synthesize mRNA through in vitro transcription, and purify the extracted RNA in -80 degrees cryopreserved for later use; different sgRNAs are synthesized by DNA synthesis upstream of fixed sequence with T7 promoter and different downstream sequences that are partially complementary to the upstream, and then double-stranded DNA is obtained by PCR amplification and obtained by in vitro transcription kit , The purified and extracted RNA is frozen and stored at -80 degrees for later use.
CRISPR/CasRx是一类新型的RNA编辑工具，CasRx是目前报道的效率最高的一个版本。虽然CRISPR/CasRx在细胞中的效率很好，但是目前还没有针对斑马鱼胚胎基因敲降的报道。CRISPR/CasRx is a new type of RNA editing tool, and CasRx is currently the most efficient version reported. Although CRISPR/CasRx is very efficient in cells, there are no reports of gene knockdown in zebrafish embryos.
有益效果Beneficial effect
本发明的优点在于：The advantages of the present invention are:
（1）、价格低廉，只需要更换一条短引物序列（＜50 bp）就能靶向一个目的基因的RNA；(1) Low price, only need to replace a short primer sequence (<50 bp) to target the RNA of a target gene;
相较与传统的斑马鱼胚胎Morpholino敲降技术，本发明设计方便，成本更低，在导向RNA的设计上采用引物配对扩增的方法，采用固定的正向引物，匹配特异靶向的反向序列，就能够扩增出导向RNA的模板DNA，提供纯化、转录能够快捷获得靶向特异的导向RNA。Compared with the traditional zebrafish embryo Morpholino knock-down technology, the present invention has convenient design and lower cost. In the design of guide RNA, the method of primer pairing amplification is adopted, and the fixed forward primer is used to match the reverse direction of specific targeting. The sequence can amplify the template DNA of the guide RNA, and provide purification and transcription to quickly obtain the target-specific guide RNA.
（2）、组分简单且全部为RNA，毒性小，操作简单；(2) The components are simple and all are RNA, with low toxicity and simple operation;
本组分只需要固定的CasRx mRNA，以及特异靶向的导向RNA，组分简单，剂量合适，毒性较小，可以减少样品毒性干扰。This component only requires fixed CasRx mRNA and specifically targeted guide RNA. The component is simple, the dosage is appropriate, and the toxicity is small, which can reduce the toxicity of the sample.
（3）、通过多条gRNA可以实现多基因同时敲降；(3) Multiple gRNAs can be used to knock down multiple genes at the same time;
导向RNA序列短，成本低，合成方便，毒性小，可以同时添加多条gRNA，实现多基因同时敲降。The guide RNA sequence is short, the cost is low, the synthesis is convenient, and the toxicity is low. Multiple gRNAs can be added at the same time to achieve simultaneous knockdown of multiple genes.
（4）、效率高，时效长；(4) High efficiency and long timeliness;
CasRx介导的RNA靶向切割具有非常高的序列特异性，因为CasRx mRNA采用了WPRE以及poly A加尾的双稳定措施，其在胚胎存在的时间长且稳定，足够保证胚胎发育过程中目标RNA的持续切割效果。CasRx-mediated RNA targeted cleavage has very high sequence specificity, because CasRx mRNA adopts WPRE and poly A tailed bi-stable measures, which can exist in the embryo for a long time and is stable, enough to ensure the target RNA during embryonic development The continuous cutting effect.
（5）、效率分析方法简单易行。(5) The efficiency analysis method is simple and easy to implement.
本研究提供了简便的靶向RNA切割效率的分析方法，采用real-timePCR的方法，通过提供合适的内参，能够快速分析出目的RNA序列的切割效率。This study provides a simple method for analyzing the efficiency of targeted RNA cleavage. Real-time PCR is used to quickly analyze the cleavage efficiency of the target RNA sequence by providing appropriate internal controls.
附图说明Description of the drawings
图1  CasRx介导的RNA编辑系统在斑马鱼胚胎中的运用简图。Figure 1 Schematic diagram of the application of CasRx-mediated RNA editing system in zebrafish embryos.
图2  CasRx介导的RNA编辑在斑马鱼胚胎中切割外源mRNA的效率验证；其中A 、B：CasRx在斑马鱼胚胎中切割EGFP mRNA的代表图和效率统计；C、D：CasRx在斑马鱼胚胎中切割BFP mRNA的代表图和效率统计。 Figure 2 The efficiency verification of CasRx-mediated RNA editing in cutting exogenous mRNA in zebrafish embryos; A and B: Representative images and efficiency statistics of CasRx cutting EGFP mRNA in zebrafish embryos; C and D: CasRx in zebrafish embryos Representative graph and efficiency statistics of cleavage of BFP mRNA in embryos.
图3  CasRx介导的RNA编辑在斑马鱼胚胎中切割内源mRNA的效率验证；其中A：24 h的表型图； B：qPCR分析图。Figure 3 The efficiency verification of CasRx-mediated RNA editing in cutting endogenous mRNA in zebrafish embryos; A: 24 h phenotype diagram; B: qPCR analysis diagram.
本发明的实施方式Embodiments of the present invention
 “靶位点”在本申请中是指，在目标核苷酸中任何一段欲加以敲降的RNA序列。靶位点附近的RNA序列，能够容纳外源序列在靶位点处的识别。在具体实施方式中，目标RNA序列是单链的RNA 序列，包括，但不限于，细胞的中的RNA序列、病毒等的RNA序列。"Target site" in this application refers to any RNA sequence that is to be knocked down in the target nucleotide. The RNA sequence near the target site can accommodate the recognition of the foreign sequence at the target site. In a specific embodiment, the target RNA sequence is a single-stranded RNA sequence, including, but not limited to, RNA sequences in cells and RNA sequences in viruses.
“外源RNA序列”在本申请中是指外源荧光蛋白加帽加尾RNA。"Exogenous RNA sequence" in this application refers to exogenous fluorescent protein capped and tailed RNA.
实施例 1野生型CasRx序列、外源荧光蛋白（EGFP、BFP）的mRNA以及sgRNA的制备 Example 1 Preparation of wild-type CasRx sequence, exogenous fluorescent protein (EGFP, BFP) mRNA and sgRNA
野生型CasRx序列分别加入核定位序列 (nucleus localization sequence)（序列为CCGCCACC），然后于整段序列之上游加入SP6启动子序列（序列为CATACGATTTAGGTGACACTATAGAA），利用体外转录的试剂盒得到加帽 (capped)及加polyA尾的mRNA，纯化提取的RNA于-80 度冻存备用；设计外源荧光蛋白（GFP、BFP）DNA的引物合成DNA,再经过体外转录合成mRNA，纯化提取的RNA于-80 度冻存备用 ；不同的sgRNA通过DNA合成上游带有T7启动子的固定序列及与上游存在部分互补的不同的下游序列，经过PCR扩增得到双链DNA，利用体外转录的试剂盒得到，纯化提取的RNA于-80 度冻存备用。The wild-type CasRx sequence was added to the nucleus localization sequence (sequence CCGCCACC), and then the SP6 promoter sequence (sequence CAPACGATTTAGGTGACACTATAGAA) was added to the upstream of the entire sequence, and capped using the in vitro transcription kit (capped) and polyA tailed mRNA, purified and extracted RNA is stored at -80 degrees for later use; design primers for exogenous fluorescent protein (GFP, BFP) DNA to synthesize DNA, and then synthesize mRNA through in vitro transcription, and purify the extracted RNA in -80 degrees cryopreserved for later use; different sgRNAs are synthesized by DNA synthesis upstream of fixed sequence with T7 promoter and different downstream sequences that are partially complementary to the upstream, and then double-stranded DNA is obtained by PCR amplification and obtained by in vitro transcription kit , The purified and extracted RNA is frozen and stored at -80 degrees for later use.
序列1： CasRx模板序列>PSKII-SP6-kozak-NLS-CasRx-NLS-HA-WPRE-PSKIISequence 1: CasRx template sequence>PSKII-SP6-kozak-NLS-CasRx-NLS-HA-WPRE-PSKII
GTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGCATACGATTTAGGTGACACTATAGAA（SP6启动子序列）CCGCCACC（核定位序列） atgagccccaagaagaagagaaaggtggaggccagcatcgaaaaaaaaaagtccttcgccaagggcatgggcgtgaagtccacactcgtgtccggctccaaagtgtacatgacaaccttcgccgaaggcagcgacgccaggctggaaaagatcgtggagggcgacagcatcaggagcgtgaatgagggcgaggccttcagcgctgaaatggccgataaaaacgccggctataagatcggcaacgccaaattcagccatcctaagggctacgccgtggtggctaacaaccctctgtatacaggacccgtccagcaggatatgctcggcctgaaggaaactctggaaaagaggtacttcggcgagagcgctgatggcaatgacaatatttgtatccaggtgatccataacatcctggacattgaaaaaatcctcgccgaatacattaccaacgccgcctacgccgtcaacaatatctccggcctggataaggacattattggattcggcaagttctccacagtgtatacctacgacgaattcaaagaccccgagcaccatagggccgctttcaacaataacgataagctcatcaacgccatcaaggcccagtatgacgagttcgacaacttcctcgataaccccagactcggctatttcggccaggcctttttcagcaaggagggcagaaattacatcatcaattacggcaacgaatgctatgacattctggccctcctgagcggactgaggcactgggtggtccataacaacgaagaagagtccaggatctccaggacctggctctacaacctcgataagaacctcgacaacgaatacatctccaccctcaactacctctacgacaggatcaccaatgagctgaccaactccttctccaagaactccgccgccaacgtgaactatattgccgaaactctgggaatcaaccctgccgaattcgccgaacaatatttcagattcagcattatgaaagagcagaaaaacctcggattcaatatcaccaagctcagggaagtgatgctggacaggaaggatatgtccgagatcaggaaaaatcataaggtgttcgactccatcaggaccaaggtctacaccatgatggactttgtgatttataggtattacatcgaagaggatgccaaggtggctgccgccaataagtccctccccgataatgagaagtccctgagcgagaaggatatctttgtgattaacctgaggggctccttcaacgacgaccagaaggatgccctctactacgatgaagctaatagaatttggagaaagctcgaaaatatcatgcacaacatcaaggaatttaggggaaacaagacaagagagtataagaagaaggacgcccctagactgcccagaatcctgcccgctggccgtgatgtttccgccttcagcaaactcatgtatgccctgaccatgttcctggatggcaaggagatcaacgacctcctgaccaccctgattaataaattcgataacatccagagcttcctgaaggtgatgcctctcatcggagtcaacgctaagttcgtggaggaatacgcctttttcaaagactccgccaagatcgccgatgagctgaggctgatcaagtccttcgctagaatgggagaacctattgccgatgccaggagggccatgtatatcgacgccatccgtattttaggaaccaacctgtcctatgatgagctcaaggccctcgccgacaccttttccctggacgagaacggaaacaagctcaagaaaggcaagcacggcatgagaaatttcattattaataacgtgatcagcaataaaaggttccactacctgatcagatacggtgatcctgcccacctccatgagatcgccaaaaacgaggccgtggtgaagttcgtgctcggcaggatcgctgacatccagaaaaaacagggccagaacggcaagaaccagatcgacaggtactacgaaacttgtatcggaaaggataagggcaagagcgtgagcgaaaaggtggacgctctcacaaagatcatcaccggaatgaactacgaccaattcgacaagaaaaggagcgtcattgaggacaccggcagggaaaacgccgagagggagaagtttaaaaagatcatcagcctgtacctcaccgtgatctaccacatcctcaagaatattgtcaatatcaacgccaggtacgtcatcggattccattgcgtcgagcgtgatgctcaactgtacaaggagaaaggctacgacatcaatctcaagaaactggaagagaagggattcagctccgtcaccaagctctgcgctggcattgatgaaactgcccccgataagagaaaggacgtggaaaaggagatggctgaaagagccaaggagagcattgacagcctcgagagcgccaaccccaagctgtatgccaattacatcaaatacagcgacgagaagaaagccgaggagttcaccaggcagattaacagggagaaggccaaaaccgccctgaacgcctacctgaggaacaccaagtggaatgtgatcatcagggaggacctcctgagaattgacaacaagacatgtaccctgttcagaaacaaggccgtccacctggaagtggccaggtatgtccacgcctatatcaacgacattgccgaggtcaattcctacttccaactgtaccattacatcatgcagagaattatcatgaatgagaggtacgagaaaagcagcggaaaggtgtccgagtacttcgacgctgtgaatgacgagaagaagtacaacgataggctcctgaaactgctgtgtgtgcctttcggctactgtatccccaggtttaagaacctgagcatcgaggccctgttcgataggaacgaggccgccaagttcgacaaggagaaaaagaaggtgtccggcaattccggatccggacctaagaaaaagaggaaggtggcggccgcttacccatacgatgttccagattacgctaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAAT GTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGCATACGATTTAGGTGACACTATAGAA (SP6 promoter sequence) CCGCCACC (nuclear localization sequence) atgagccccaagaagaagagaaaggtggaggccagcatcgaaaaaaaaaagtccttcgccaagggcatgggcgtgaagtccacactcgtgtccggctccaaagtgtacatgacaaccttcgccgaaggcagcgacgccaggctggaaaagatcgtggagggcgacagcatcaggagcgtgaatgagggcgaggccttcagcgctgaaatggccgataaaaacgccggctataagatcggcaacgccaaattcagccatcctaagggctacgccgtggtggctaacaaccctctgtatacaggacccgtccagcaggatatgctcggcctgaaggaaactctggaaaagaggtacttcggcgagagcgctgatggcaatgacaatatttgtatccaggtgatccataacatcctggacattgaaaaaatcctcgccgaatacattaccaacgccgcctacgccgtcaacaatatctccggcctggataaggacattattggattcggcaagttctccacagtgtatacctacgacgaattcaaagaccccgagcaccatagggccgctttcaacaataacgataagctcatcaacgccatcaaggcccagtatgacgagttcgacaacttcctcgataaccccagactcggctatttcggccaggcctttttcagcaaggagggcagaaattacatcatcaattacggcaacgaatgctatgacattctggccctcctgagcggactgaggcactgggtggtccataacaacgaagaagagtccaggatctccaggacctggctctacaacctcgataagaacctcgacaacgaatacatctccaccctcaactacctctacgacaggatcaccaatgagctgaccaactccttctccaagaactccgccgccaacgtgaactatattgccgaaactctgggaatcaaccctgccgaattcgccgaacaatatttcagattcagcattatgaaagagcagaaaaacctcggattcaatatcaccaagctcagggaagtgatgctggacaggaaggatatgtccgagatcaggaaaaatcataaggtgttcgactccatcaggaccaaggtctacaccatgatggactttgtgatttataggtattacatcgaagaggatgccaaggtggctgccgccaataagtccctccccgataatgagaagtccctgagcgagaaggatatctttgtgattaacctgaggggctccttcaacgacgaccagaaggatgccctctactacgatgaagctaatagaatttggagaaagctcgaaaatatcatgcacaacatcaaggaatttaggggaaacaagacaagagagtataagaagaaggacgcccctagactgcccagaatcctgcccgctggccgtgatgtttccgccttcagcaaactcatgtatgccctgaccatgttcctggatggcaaggagatcaacgacctcctgaccaccctgattaataaattcgataacatccagagcttcctgaaggtgatgcctctcatcggagtcaacgctaagttcgtggaggaatacgcctttttcaaagactccgccaagatcgccgatgagctgaggctgatcaagtccttcgctagaatgggagaacctattgccgatgccaggagggccatgtatatcgacgccatccgtattttaggaaccaacctgtcctatgatgagctcaaggccctcgccgacaccttttccctggacgagaacggaaacaagctcaagaaaggcaagcacggcatgagaaatttcattattaataacgtgatcagcaataaaaggttccactacctgatcagatacggtgatcctgcccacctccatgagatcgccaaaaacgaggccgtggtgaagttcgtgctcggcaggatcgctgacatccagaaaaaacagggccagaacggcaagaaccagatcgacaggtactacgaaacttgtatcggaaaggataagggcaagagcgtgagcgaaaaggtggacgctctcacaaagatcatcaccggaatgaactacgaccaattcgacaagaaaaggagcgtcattgaggacaccggcagggaaaacgccgagagggagaagtttaaaaagatcatcagcctgtacctcaccgtgatctaccacatcctcaagaatattgtcaatatcaacgccaggtacgtcatcggattccattgcgtcgagcgtgatgctcaactgtacaaggagaaaggctacgacatcaatctcaagaaactggaagagaagggattcagctccgtcaccaagctctgcgctggcattgatgaaactgcccccgataagagaaaggacgtggaaaaggagatggctgaaagagccaaggagagcattgacagcctcgagagcgccaaccccaagctgtatgccaattacatcaaatacagcgacgagaagaaagccgaggagttcaccaggcagattaacagggagaaggccaaaaccgccctgaacgcctacctgaggaacaccaagtggaatgtgatcatcagggaggacctcctgagaattgacaacaagacatgtaccctgttcagaaacaaggccgtccacctggaagtggccaggtatgtccacgcctatatcaacgacattgccgaggtcaattcctacttccaactgtaccattacatcatgcagagaattatcatgaatgagaggtacgagaaaagcagcggaaaggtgtccgagtacttcgacgctgtgaatgacgagaagaagtacaacgataggctcctgaaactgctgtgtgtgcctttcggctactgtatccccaggtttaagaacctgagcatcgaggccctgttcgataggaacgaggccgccaagttcgacaaggagaaaaagaaggtgtccggcaattccggatccggacctaagaaaaagaggaaggtg gcggccgcttacccatacgatgttccagattacgctaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAAT
序列2： EGFP模板序列> SP6-kozak-EGFPSequence 2: EGFP template sequence> SP6-kozak-EGFP
CATACGATTTAGGTGACACTATAGAA（SP6启动子序列）CCGCCACC（核定位序列）GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA。 CATACGATTTAGGTGACACTATAGAA (SP6 promoter sequence) CCGCCACC (nuclear localization sequence) GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA. To
序列3： BFP模板序列> SP6-kozak-EGFP> SP6-vertebrate kozak-TagBFP -stop Sequence 3: BFP template sequence>SP6-kozak-EGFP> SP6-vertebrate kozak-TagBFP -stop
CATACGATTTAGGTGACACTATAGAA（SP6启动子序列）CCGCCACC（核定位序列）atgagcgagctgattaaggagaacatgcacatgaagctgtacatggagggcaccgtggacaaccatcacttcaagtgcacatccgagggcgaaggcaagccctacgagggcacccagaccatgagaatcaaggtggtcgagggcggccctctccccttcgccttcgacatcctggctactagcttcctctacggcagcaagaccttcatcaaccacacccagggcatccccgacttcttcaagcagtccttccctgagggcttcacatgggagagagtcaccacatacgaagacgggggcgtgctgaccgctacccaggacaccagcctccaggacggctgcctcatctacaacgtcaagatcagaggggtgaacttcacatccaacggccctgtgatgcagaagaaaacactcggctgggaggccttcaccgagacgctgtaccccgctgacggcggcctggaaggcagaaacgacatggccctgaagctcgtgggcgggagccatctgatcgcaaacatcaagaccacatatagatccaagaaacccgctaagaacctcaagatgcctggcgtctactatgtggactacagactggaaagaatcaaggaggccaacaacgagacctacgtcgagcagcacgaggtggcagtggccagatactgcgacctccctagcaaactggggcacaagcttaatTAACATACGATTTAGGTGACACTATAGAA (SP6 promoter sequence) CCGCCACC (nuclear localization sequence) atgagcgagctgattaaggagaacatgcacatgaagctgtacatggagggcaccgtggacaaccatcacttcaagtgcacatccgagggcgaaggcaagccctacgagggcacccagaccatgagaatcaaggtggtcgagggcggccctctccccttcgccttcgacatcctggctactagcttcctctacggcagcaagaccttcatcaaccacacccagggcatccccgacttcttcaagcagtccttccctgagggcttcacatgggagagagtcaccacatacgaagacgggggcgtgctgaccgctacccaggacaccagcctccaggacggctgcctcatctacaacgtcaagatcagaggggtgaacttcacatccaacggccctgtgatgcagaagaaaacactcggctgggaggccttcaccgagacgctgtaccccgctgacggcggcctggaaggcagaaacgacatggccctgaagctcgtgggcgggagccatctgatcgcaaacatcaagaccacatatagatccaagaaacccgctaagaacctcaagatgcctggcgtctactatgtggactacagactggaaagaatcaaggaggccaacaacgagacctacgtcgagcagcacgaggtggcagtggccagatactgcgacctccctagcaaactggggcacaagcttaatTAA
序列4：sgRNA合成用的DNA模板序列引物合成序列Sequence 4: DNA template sequence for sgRNA synthesis primer synthesis sequence
sg-scaffold-Fsg-scaffold-F GAAATTAATACGACTCACTATAGGcactagtgcgaatttgcactagtctaaaacGAAATTAATACGACTCACTATAGGcactagtgcgaatttgcactagtctaaaac
sg-Rsg-R (22-26bp特异靶向序列插入处) gttttagactagtgcaaa(22-26bp specific targeting sequence insertion) gttttagactagtgcaaa
实施例Example 2. 2. 斑马鱼胚胎体内切割外源Zebrafish embryos cut exogenous in vivo EGFP mRNAEGFP mRNA 序列sequence
野生型CasRx序列分别加入核定位序列 (nucleus localization sequence)，然后于整段序列之上游加入SP6启动子序列，利用体外转录的试剂盒得到加帽 (capped)及加polyA尾的mRNA，以外源EGFP DNA序列利用体外转录的试剂盒得到外源EGFP mRNA序列,sgRNA通过DNA合成上游带有T7启动子序列及与上游存在部分互补的下游序列，经过PCR扩增得到双链DNA，利用体外转录的试剂盒得到；之后于-80℃冻存备用。显微注射时拿出以确定的比例混合注入单细胞胚胎，12 h用共聚焦显微镜拍摄荧光图及冻存胚胎待进行qPCR分析。The wild-type CasRx sequence was added to the nucleus localization sequence, and then the SP6 promoter sequence was added to the upstream of the entire sequence, and the in vitro transcription kit was used to obtain the capping (capped) and polyA-tailed mRNA, exogenous EGFP DNA sequence is obtained by in vitro transcription kits to obtain exogenous EGFP mRNA sequence, sgRNA has a T7 promoter sequence upstream of DNA synthesis and a downstream sequence that is partially complementary to the upstream. The double-stranded DNA obtained by PCR amplification was obtained by using an in vitro transcription kit; then, it was frozen at -80°C for use. During the microinjection, single-cell embryos were mixed and injected at a certain ratio. The fluorescence images were taken with a confocal microscope for 12 hours and the embryos were frozen for qPCR analysis.
EGFP sgRNA合成用的DNA模板序列引物合成序列DNA template sequence for EGFP sgRNA synthesis, primer synthesis sequence
sg-scaffold-Fsg-scaffold-F GAAATTAATACGACTCACTATAGGcactagtgcgaatttgcactagtctaaaacGAAATTAATACGACTCACTATAGGcactagtgcgaatttgcactagtctaaaac
EGFP-sg-R1EGFP-sg-R1 tgaagttcatctgcaccaccgggttttagactagtgcaaatgaagttcatctgcaccaccgggttttagactagtgcaaa
EGFP-sg-R2EGFP-sg-R2 CTTCAAGGACGACGGCAACTACgttttagactagtgcaaaCTTCAAGGACGACGGCAACTACgttttagactagtgcaaa
实施例Example 3. 3. 斑马鱼胚胎体内切割外源Zebrafish embryos cut exogenous in vivo BFP mRNABFP mRNA 序列sequence
野生型CasRx序列分别加入核定位序列 (nucleus localization sequence)，然后于整段序列之上游加入SP6启动子序列，利用体外转录的试剂盒得到加帽 (capped)及加polyA尾的mRNA，以外源BFP DNA序列利用体外转录的试剂盒得到外源BFP mRNA序列，sgRNA通过DNA合成上游带有T7启动子序列及与上游存在部分互补的下游序列，经过PCR扩增得到双链DNA，利用体外转录的试剂盒得到；之后于-80℃冻存备用。显微注射时拿出以确定的比例混合注入单细胞胚胎，12 h用共聚焦显微镜拍摄荧光图及冻存胚胎待进行qPCR分析。The wild-type CasRx sequence was added to the nucleus localization sequence, and then the SP6 promoter sequence was added upstream of the entire sequence. The in vitro transcription kit was used to obtain capped and polyA tailed mRNA, foreign BFP The DNA sequence uses the in vitro transcription kit to obtain the exogenous BFP mRNA sequence. The sgRNA is synthesized upstream with the T7 promoter sequence and the downstream sequence that is partially complementary to the upstream. After PCR amplification, double-stranded DNA is obtained. The in vitro transcription reagents are used. Obtained in a box; then freeze at -80°C for later use. During the microinjection, single-cell embryos were mixed and injected at a certain ratio. The fluorescence images were taken with a confocal microscope for 12 hours and the embryos were frozen for qPCR analysis.
BFP sgRNA合成用的DNA模板序列引物合成序列DNA template sequence for BFP sgRNA synthesis, primer synthesis sequence
sg-scaffold-Fsg-scaffold-F GAAATTAATACGACTCACTATAGGcactagtgcgaatttgcactagtctaaaacGAAATTAATACGACTCACTATAGGcactagtgcgaatttgcactagtctaaaac
BFP-sg-R1BFP-sg-R1 CACCGTGGACAACCATCACTTCgttttagactagtgcaaaCACCGTGGACAACCATCACTTCgttttagactagtgcaaa
BFP-sg-R2BFP-sg-R2 CTTCACATGGGAGAGAGTCACCgttttagactagtgcaaaCTTCACATGGGAGAGAGTCACCgttttagactagtgcaaa
实施例Example 4. 4. 斑马鱼胚胎体内切割内源Zebrafish embryos cut endogenous in vivo ActbActb mRNA mRNA 序列sequence
野生型CasRx序列分别加入核定位序列 (nucleus localization sequence)，然后于整段序列之上游加入SP6启动子序列，利用体外转录的试剂盒得到加帽 (capped)及加polyA尾的mRNA，sgRNA通过DNA合成上游带有T7启动子序列及与上游存在部分互补的下游序列，经过PCR扩增得到双链DNA，利用体外转录的试剂盒得到；之后于-80℃冻存备用。显微注射时拿出以确定的比例混合注入单细胞胚胎，24 h倒置显微镜拍摄表型图及冻存胚胎待进行qPCR分析。The wild-type CasRx sequence was added to the nucleus localization sequence, and then the SP6 promoter sequence was added to the upstream of the entire sequence, and the in vitro transcription kit was used to obtain the capping (capped) and polyA tailed mRNA, sgRNA through DNA synthesis upstream with a T7 promoter sequence and a downstream sequence partially complementary to the upstream, after PCR amplification to obtain double-stranded DNA, using in vitro transcription kit to obtain; Freeze at -80℃ for later use. During the microinjection, single-cell embryos were mixed and injected at a certain ratio. The phenotype map was taken by a 24-hour inverted microscope and the embryos were frozen for qPCR analysis.
Actb sgRNA合成用的DNA模板序列引物合成序列Actb sgRNA synthesis DNA template sequence primer synthesis sequence
sg-scaffold-Fsg-scaffold-F GAAATTAATACGACTCACTATAGGcactagtgcgaatttgcactagtctaaaacGAAATTAATACGACTCACTATAGGcactagtgcgaatttgcactagtctaaaac
Actb-sg-RActb-sg-R ccagacatcagggagtgatggtgttttagactagtgcaaaccagacatcagggagtgatggtgttttagactagtgcaaa
实施例Example 5. 5. 斑马鱼胚胎体内切割内源Zebrafish embryos cut endogenous in vivo Neurog1 mRNANeurog1 mRNA 序列sequence
野生型CasRx分别加入核定位序列 (nucleus localization sequence)，然后于整段序列之上游加入SP6启动子序列，利用体外转录的试剂盒得到加帽 (capped)及加polyA尾的mRNA，sgRNA通过DNA合成上游带有T7启动子序列及与上游存在部分互补的下游序列，经过PCR扩增得到双链DNA，利用体外转录的试剂盒得到；之后于-80℃冻存备用。显微注射时拿出以确定的比例混合注入单细胞胚胎，24 h倒置显微镜拍摄表型图及冻存胚胎待进行qPCR分析。Wild-type CasRx was added to the nuclear localization sequence (nucleus localization sequence), and then the SP6 promoter sequence was added to the upstream of the entire sequence, and the in vitro transcription kit was used to obtain the capping (capped) and polyA tailed mRNA, sgRNA through DNA synthesis upstream with a T7 promoter sequence and a downstream sequence partially complementary to the upstream, after PCR amplification to obtain double-stranded DNA, using in vitro transcription kit to obtain; Freeze at -80℃ for later use. During the microinjection, single-cell embryos were mixed and injected at a certain ratio. The phenotype map was taken by a 24-hour inverted microscope and the embryos were frozen for qPCR analysis.
Neurog1 sgRNA合成用的DNA模板序列引物合成序列Neurog1 sgRNA synthesis DNA template sequence primer synthesis sequence
sg-scaffold-Fsg-scaffold-F GAAATTAATACGACTCACTATAGGcactagtgcgaatttgcactagtctaaaacGAAATTAATACGACTCACTATAGGcactagtgcgaatttgcactagtctaaaac
Neurog1-sg-RNeurog1-sg-R AACCTCAAGCTGTGACTACTCCgttttagactagtgcaaaAACCTCAAGCTGTGACTACTCCgttttagactagtgcaaa
为方便起见，本实施例采用显微注射将本发明的mRNA组合物导入斑马鱼受精卵中。组合物中各荧光蛋白终mRNA浓度为600 ng/ul；sgRNA终浓度为100ng/ul；CasRx mRNA200 ng/ul。注射时每个胚胎注射大约~1nl。For convenience, microinjection is used in this example to introduce the mRNA composition of the present invention into zebrafish fertilized eggs. The final mRNA concentration of each fluorescent protein in the composition is 600 ng/ul; the final concentration of sgRNA is 100 ng/ul; CasRx mRNA 200 ng/ul. Each embryo is injected approximately ~1nl at the time of injection.
实施例Example 6. 6. 制备转基因斑马鱼品系Preparation of genetically modified zebrafish strains
收集单细胞期斑马鱼胚胎，按上述方案进行注射，将其从胚胎养成成鱼。Collect single-cell stage zebrafish embryos, inject them according to the above scheme, and grow them from embryos into fish.
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。The foregoing descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made in accordance with the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims (3)

  1. 一种在鱼类胚胎中实现精确定点RNA剪切的技术，其特征在于：利用CRISPR /CasRx介导的RNA编辑技术，根据导向RNA精确定点作用于斑马鱼胚胎中的特定RNA，在鱼胚胎中实现精确定点RNA敲降。A technology for realizing precise point RNA shearing in fish embryos, which is characterized by: using CRISPR/CasRx-mediated RNA editing technology to precisely point specific RNAs that act on zebrafish embryos according to the guide RNA, in fish embryos Achieve precise point RNA knockdown.
  2. 根据权利要求1所述的一种在鱼类胚胎中实现精确定点RNA剪切的技术，其特征在于，包括以下方法：The technology for realizing precise point RNA shearing in fish embryos according to claim 1, characterized in that it comprises the following methods:
    A、野生型CasRx序列、外源荧光蛋白mRNA、相应的导向RNA、内源基因的导向RNA的设计和制备；A. Design and preparation of wild-type CasRx sequence, exogenous fluorescent protein mRNA, corresponding guide RNA, guide RNA of endogenous gene;
    B、显微注射时RNA各组分的剂量确定；B. Determine the dosage of each component of RNA during microinjection;
    C、外源mRNA在斑马鱼胚胎中的切割；C. Exogenous mRNA cutting in zebrafish embryos;
    D、内源mRNA在斑马鱼胚胎中的切割；D. Cutting of endogenous mRNA in zebrafish embryos;
    E、内源多个基因同时进行敲降。E. Multiple endogenous genes are knocked down at the same time.
  3. 根据权利要求1所述的一种在鱼类胚胎中实现精确定点剪切RNA的技术，其特征在于，具体包括以下方法：The technique for realizing precise point shearing of RNA in fish embryos according to claim 1, characterized in that it specifically comprises the following methods:
    （1）野生型CasRx序列、外源荧光蛋白mRNA、相应的导向RNA、内源基因的导向RNA的设计和制备：CasRx序列分别加入核定位序列，然后于整段序列之上游加入SP6启动子序列，利用体外转录的试剂盒得到加帽及加polyA尾的mRNA，纯化提取的RNA于-80 度冻存备用；设计外源荧光蛋白DNA的引物合成DNA,再经过体外转录合成mRNA，纯化提取的RNA于-80 度冻存备用；不同的sgRNA通过DNA合成上游带有T7启动子的固定序列及与上游存在部分互补的不同的下游序列，经过PCR扩增得到双链DNA，利用体外转录的试剂盒得到，纯化提取的RNA于-80 度冻存备用；(1) Design and preparation of wild-type CasRx sequence, exogenous fluorescent protein mRNA, corresponding guide RNA, guide RNA of endogenous gene: CasRx sequence was added to nuclear localization sequence, and then SP6 promoter sequence was added upstream of the entire sequence Use an in vitro transcription kit to obtain capped and polyA tailed mRNA. The purified RNA is frozen and stored at -80 degrees for later use; the primers for exogenous fluorescent protein DNA are designed to synthesize DNA, and then the mRNA is synthesized through in vitro transcription and the extracted RNA is purified. RNA is frozen at -80°C for use; different sgRNAs are synthesized by DNA synthesis upstream of fixed sequence with T7 promoter and different downstream sequences that are partially complementary to the upstream, and then double-stranded DNA is obtained by PCR amplification, using in vitro transcription reagents Obtained in the box, and the purified and extracted RNA is frozen and stored at -80 degrees for later use;
    （2）显微注射时混合液中RNA各组分的剂量确定；(2) Determine the dosage of each component of RNA in the mixed solution during microinjection;
    a、外源基因时显微注射时RNA各组分的剂量：混合液中各荧光蛋白终mRNA浓度为600 ng/ul；sgRNA终浓度为100ng/ul；CasRx mRNA 终浓度为200 ng/ul；每个胚胎注射1nl;a. The dosage of each component of RNA during microinjection of exogenous genes: the final mRNA concentration of each fluorescent protein in the mixture is 600 ng/ul; the final concentration of sgRNA is 100 ng/ul; the final concentration of CasRx mRNA is 200 ng/ul; Inject 1nl per embryo;
    b、内源基因时显微注射时RNA各组分的剂量：混合液中sgRNA终浓度为100ng/ul；CasRx mRNA终浓度为200 ng/ul；每个胚胎注射1nl；b. The dose of each component of RNA during microinjection of endogenous genes: the final concentration of sgRNA in the mixture is 100ng/ul; the final concentration of CasRx mRNA is 200 ng/ul; each embryo is injected 1nl;
    （3）外源mRNA在斑马鱼胚胎中的切割：斑马鱼胚胎单胞期注射相应实验组和对照组组分后，12 h时运用共聚焦拍摄显微注射的对照组及各实验组胚胎的多张荧光图，之后分别取对照组及各实验组30颗胚胎分装2管EP管，并往EP管中加200ul Trizol后冻于-80 ℃，之后提RNA；用所提的RNA利用逆转录试剂盒进行逆转录，得到的DNA进行实时荧光定量PCR；利用相应的软件LAS AF Lite、GraphPad Prism 5、ImageJ对共聚焦拍摄的荧光图和LightCycler® 96 SW 1.1、GraphPad Prism 5对定量PCR的图进行相应的分析；(3) Exogenous mRNA cleavage in zebrafish embryos: After the zebrafish embryos were injected with the corresponding experimental group and control group at the single cell stage, confocal photography was used at 12 h to capture the microinjected control group and the embryos of each experimental group. Take multiple fluorescence images, and then separately take 30 embryos from the control group and each experimental group into 2 tubes of EP tubes, add 200ul Trizol to the EP tubes and freeze at -80 ℃, then extract RNA; use the extracted RNA to reverse Reverse transcription with the recording kit, and the DNA obtained for real-time fluorescent quantitative PCR; use the corresponding software LAS AF Lite, GraphPad Prism 5, ImageJ to take the fluorescence images of confocal and LightCycler® 96 SW 1.1, GraphPad Prism 5 to quantitative PCR Figure for corresponding analysis;
    ( 4 )内源mRNA在斑马鱼胚胎中的切割：斑马鱼胚胎单胞期注射相应实验组和对照组组分后，在24h时利用荧光倒置显微镜对显微注射及未处理的WT分别进行表型拍照并在24 h时进行畸形统计，24 h之后取对照组及各实验组的畸形胚胎30颗胚胎分装3管EP管，并往EP管中加200ul Trizol后冻于-80 ℃，之后提RNA；用所提的RNA利用逆转录试剂盒进行逆转录，得到的DNA进行实时荧光定量PCR；利用相应的软件LightCycler® 96 SW 1.1、GraphPad Prism 5对定量PCR的图进行相应的分析；(4) Cleavage of endogenous mRNA in zebrafish embryos: After zebrafish embryo single cell stage injection of the corresponding experimental group and control group components, at 24h, the microinjected and untreated WT were analyzed by a fluorescent inverted microscope. After 24 hours, take pictures of abnormalities and make statistics of abnormalities. After 24 hours, take 30 embryos from the control group and each experimental group into 3 tubes of EP tubes, add 200ul Trizol to the EP tubes and freeze them at -80 ℃, then Extract RNA; use reverse transcription kit to perform reverse transcription with the extracted RNA, and perform real-time fluorescent quantitative PCR with the obtained DNA; use the corresponding software LightCycler® 96 SW 1.1, GraphPad Prism 5 to analyze the quantitative PCR diagram accordingly;
    ( 5 )内源多个基因同时进行敲降：配置CasRx mRNA 终浓度为200 ng/ul，A 基因导向sgRNA终浓度为100ng/ul，B 基因导向sgRNA终浓度为100ng/ul, C 基因导向sgRNA终浓度为100ng/ul的混合液，将该组分同时注射到斑马鱼胚胎中，观察斑马鱼胚胎的发育情况。(5) Simultaneous knockdown of multiple endogenous genes: configure the final concentration of CasRx mRNA to 200 ng/ul, the final concentration of A gene targeting sgRNA to 100ng/ul, the final concentration of B gene targeting sgRNA to 100ng/ul, and the C gene targeting sgRNA The final concentration of the mixture is 100ng/ul, and the components are injected into zebrafish embryos at the same time to observe the development of zebrafish embryos.
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