阅读说明：本技术 一种在鱼类胚胎中实现精确定点rna剪切的技术 (Technology for realizing precise fixed-point RNA shearing in fish embryo ) 是由 何小镇 钟丽丽 于 2019-10-18 设计创作，主要内容包括：本发明提供了一种在鱼类胚胎中实现精确定点RNA剪切的技术,利用CasRx介导的RNA编辑技术,只需要提供CasRx的mRNA,以及一条特异靶向导向RNA,能够在鱼胚胎中实现精确定点RNA敲降,具有高效、特异性高和方便且费用低等诸多优点。克服了目前已有的siRNA技术面临的效率偏低、Morpholino技术介导的基因敲降技术的代价高昂易脱靶等问题。(The invention provides a technology for realizing precise fixed-point RNA shearing in fish embryos, which can realize precise fixed-point RNA knock-down in fish embryos by using a CasRx-mediated RNA editing technology and only needing to provide mRNA of CasRx and a specific targeting guide RNA. The problems that the efficiency of the prior siRNA technology is low, the Morpholino technology mediated gene knock-down technology is high in cost and easy to miss, and the like are solved.)

1. A technology for realizing precise site-specific RNA shearing in fish embryos is characterized in that: and (3) realizing the precise fixed point RNA knockdown in the fish embryo by utilizing a CRISPR/CasRx mediated RNA editing technology and according to the fact that the guide RNA acts on the specific RNA in the zebra fish embryo at the precise fixed point.

2. The technique for achieving pinpoint RNA excision in fish embryos of claim 1, comprising the following steps:

A. designing and preparing a wild CasRx sequence, exogenous fluorescent protein mRNA, corresponding guide RNA and guide RNA of an endogenous gene;

B. determining the dosage of each component of RNA during microinjection;

C. cleavage of exogenous mRNA in zebrafish embryos;

D. cleavage of endogenous mRNA in zebrafish embryos;

E. endogenous multiple genes were knocked down simultaneously.

3. The technology for realizing the precise site-specific RNA cleavage in a fish embryo as claimed in claim 1, which specifically comprises the following steps:

(1) design and preparation of wild type CasRx sequence, exogenous fluorescent protein mRNA, corresponding guide RNA, guide RNA of endogenous gene: respectively adding a nuclear localization sequence into a CasRx sequence, then adding an SP6 promoter sequence into the upstream of the whole sequence, obtaining mRNA with a cap and a polyA tail by using an in vitro transcription kit, and purifying and storing the extracted RNA at-80 ℃ for later use; designing a primer of exogenous fluorescent protein DNA to synthesize DNA, synthesizing mRNA through in vitro transcription, and freezing and storing the purified and extracted RNA at-80 ℃ for later use; different sgRNAs are synthesized into a fixed sequence with a T7 promoter at the upstream and different downstream sequences partially complementary with the upstream through DNA, double-stranded DNA is obtained through PCR amplification, the double-stranded DNA is obtained by using an in vitro transcription kit, and the purified and extracted RNA is frozen at-80 ℃ for later use;

(2) determining the dosage of each component of RNA in the mixed solution during microinjection;

a. dosage of each component of RNA during microinjection of exogenous genes: the final mRNA concentration of each fluorescent protein in the mixed solution is 600 ng/ul; the final concentration of sgRNA is 100 ng/ul; the final concentration of CasRx mRNA was 200 ng/ul; 1nl per embryo;

b. dose of RNA components at microinjection for endogenous genes: the final concentration of sgRNA in the mixed solution is 100 ng/ul; the final concentration of CasRxmRNA is 200 ng/ul; 1nl per embryo;

(3) cleavage of exogenous mRNA in zebrafish embryos: after injecting corresponding experimental group and control group components in the zebra fish embryo unicellular stage, taking a plurality of fluorescence images of microinjected control group and each experimental group embryo by confocal shooting at 12 h, then respectively taking 30 embryos of the control group and each experimental group, subpackaging the control group and each experimental group into 2 tubes of EP tubes, adding 200ul Trizol into the EP tubes, freezing at-80 ℃, and then extracting RNA; carrying out reverse transcription on the RNA by using a reverse transcription kit, and carrying out real-time fluorescence quantitative PCR on the obtained DNA; correspondingly analyzing a fluorescence image shot by confocal and a quantitative PCR image by LightCycler 96 SW 1.1 and GraphPad Prism 5 by using corresponding software LAS AF Lite, GraphPad Prism 5 and ImageJ;

(4) Cleavage of endogenous mRNA in zebrafish embryos: after injecting corresponding experimental group and control group components in the zebra fish embryo single cell period, respectively carrying out phenotype photographing on microinjected and untreated WT by using a fluorescence inverted microscope at 24h and carrying out deformity statistics at 24h, subpackaging 30 malformed embryos of the control group and each experimental group into 3 tubes of EP tubes after 24h, adding 200ul Trizol into the EP tubes, freezing at-80 ℃, and then extracting RNA; carrying out reverse transcription on the RNA by using a reverse transcription kit, and carrying out real-time fluorescence quantitative PCR on the obtained DNA; carrying out corresponding analysis on a quantitative PCR diagram by using corresponding software LightCycler 96 SW 1.1 and GraphPad Prism 5;

(5) Endogenous multiple genes were knocked down simultaneously: preparing a mixed solution with the final concentration of CasRx mRNA of 200 ng/ul, the final concentration of A gene-oriented sgRNA of 100ng/ul, the final concentration of B gene-oriented sgRNA of 100ng/ul and the final concentration of C gene-oriented sgRNA of 100ng/ul, injecting the components into zebrafish embryos at the same time, and observing the development condition of the zebrafish embryos.

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a technology for realizing precise fixed-point RNA shearing in fish embryos.

Background

With the continuous development and improvement of whole genome sequencing technology and the implementation of large genome annotation projects, the research of biological science enters the post-genome era. In the post-genome era, the focus of genomic research is shifting to gene function, i.e., the study of biological functions from the molecular ensemble level by determining the DNA sequence of genes and interpreting all genetic information of life, and exploring the human health and disease at a molecular level (pelton and McKusick, 2001). Researchers have begun to try to bring the results of genome research into various fields of basic scientific research and personalized medicine (Chan and Ginsburg, 2011) as early as possible through various attempts. However, in the face of massive and boring genome information, a key to solving the problem is to develop an efficient and reliable method for helping researchers to research the influence of genotype on phenotype (phenotype) as soon as possible.

By technological advances, mapping of cellular functional and disease transcriptome changes has been translated from microarrays (Schena et al, 1995) to next generation sequencing and single cell studies (sheddere et al, 2017). However, interrogating the function of individual transcriptional kinetics and establishing causal relationships between observed transcriptional changes and cellular phenotype requires the ability to actively control or modulate the desired transcription. DNA engineering techniques such as CRISPR-Cas9 (Doudna Charpentier, 2014; Hsu, 2014) enable researchers to dissect the function of a particular genetic element or correct a pathogenic mutation. However, simple and scalable tools for studying and manipulating RNA are significantly behind their DNA counterparts. Existing RNA interference technologies are capable of breaking down or suppressing the desired transcript, have significant off-target effects (off-targeteffect) due to their critical role in the endogenous process, and remain a challenging goal (Birming-Ham et al, 2006; Jackson et al, 2003). Therefore, methods for directly investigating the function of RNA function are still limited.

One key limitation in RNA engineering is the lack of an RNA binding domain that can be easily relocated and introduced into target cells. For example, the ms2-RNA binding domain recognizes an invariant 21 nucleotide (nt) RNA sequence (diabody, 1993), and thus requires genomic modification to label the desired transcript. The Pumilio homeodomains have modular repeats, each protein module recognizing a single RNA base, but they can only target short 8nt RNA sequences (Cheong, Hall, 2006). While previously characterized 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 could be reprogrammed to recognize 20-30 nt RNAs, their large size (about 1200 aa) made it difficult to package into adeno-associated viruses (AAV) for primary cell and in vivo delivery. CasRx, one of the most compact single-cell effector Cas enzymes, can also be flexibly packaged into adeno-associated viruses.

Gene knock-down technology (gene knock-down) is needed for gene function screening, and because the siRNA technology has poor effect in zebra fish, the gene knock-down operation on zebra fish embryos mainly adopts antisense oligonucleotide (MO) technology (Nasevicius and Ekker, 2000). Although the MO technique has good effects, because of easy off-target, two specific MO sequences are generally required to be designed when performing knock down on each gene, and the current MO synthesis company is not in China, resulting in a long ordering period. Furthermore, MO technology has drawbacks such as high toxicity in addition to off-target (Stainier et al, 2017; Van Gils and Vanakker, 2019). Therefore, establishing a new RNA interference technology in the zebra fish has important significance for researching the gene function of the zebra fish.

Disclosure of Invention

The invention aims to provide a technology for realizing precise fixed-point RNA shearing in a fish embryo, the CasRx-mediated knockout has higher efficiency and specificity, the CasRx can be flexibly packaged into adeno-associated virus, the CasRx is a programmable RNA binding module, the cellular RNA can be effectively positioned, the CasRx can be used for shearing off the RNA corresponding to the exogenous fluorescent protein with high efficiency, and the visualized genetic marker is weakened; CasRx can also be used for efficiently knocking down some RNAs corresponding to genes with obvious phenotypes or key genes so as to lead the RNAs to have different phenotypes or die.

In order to achieve the purpose, the invention adopts the following technical scheme:

a technology for realizing precise site-specific RNA shearing in fish embryos comprises the following steps:

A. designing and preparing a wild type CasRx sequence, exogenous fluorescent protein (GFP and BFP) mRNA and corresponding guide RNA (sgRNA) and guide RNA (sgRNA) of an endogenous gene;

B. determining the dosage of each component of RNA during microinjection;

C. a method for detecting the cutting efficiency of exogenous mRNA in zebra fish embryos;

D. a method for detecting the cutting efficiency of endogenous mRNA in zebra fish embryos;

E. a method for knocking down multiple endogenous genes simultaneously.

The method specifically comprises the following steps:

(1) designing and preparing a wild type CasRx sequence, exogenous fluorescent protein (GFP and BFP) mRNA and corresponding guide RNA (sgRNA) and guide RNA (sgRNA) of an endogenous gene;

adding a nuclear localization sequence (nucleous localization sequence) into the CasRx sequence respectively, then adding an SP6 promoter sequence into the upstream of the whole sequence, obtaining mRNA with a capped (capped) and a polyA tail by using an in vitro transcription kit, purifying the extracted RNA, and freezing at-80 ℃ for later use; designing primers of exogenous fluorescent protein (GFP, BFP) DNA to synthesize DNA, synthesizing mRNA through in vitro transcription, purifying the extracted RNA, and freezing at-80 ℃ for later use; different sgRNAs are synthesized into a fixed sequence with a T7 promoter at the upstream and different downstream sequences partially complementary with the upstream through DNA, double-stranded DNA is obtained through PCR amplification, the double-stranded DNA is obtained by using an in vitro transcription kit, and the purified and extracted RNA is frozen at-80 ℃ for later use.

(2) Determining the dosage of each component of RNA in the mixed solution during microinjection;

a. the final mRNA concentration of each fluorescent protein in the mixed solution is 600ng/ul, the final concentration of sgRNA is 100ng/ul, the final concentration of CasRx mRNA is 200 ng/ul., and about ~ 1nl is injected into each embryo;

b. the final concentration of sgRNA in the mixture was 100ng/ul, and the final concentration of CasRxmRNA was 200 ng/ul., and about ~ 1nl per embryo was injected for the microinjection of endogenous genes.

(3) A method for detecting the cutting efficiency of exogenous mRNA in zebra fish embryos;

after injecting corresponding experimental group and control group components in the zebra fish embryo unicellular stage, taking a plurality of fluorescence images of microinjected control group and each experimental group embryo by confocal shooting at about 12 h, then respectively taking 30 embryos of the control group and each experimental group, subpackaging 2 tubes of EP tubes (15 embryos each), adding 200ul Trizol into the EP tubes, freezing at-80 ℃, and then extracting RNA. The RNA is used for reverse transcription by a reverse transcription kit, and the obtained DNA is used for real-time fluorescence quantitative PCR. The fluorescence images shot by confocal and the quantitative PCR images shot by LightCycler 96 SW 1.1 and GraphPad Prism 5 were analyzed by corresponding software LAS AFLite, GraphPad Prism 5 and ImageJ.

(4) A method for detecting the cutting efficiency of endogenous mRNA in zebra fish embryos;

after injecting corresponding experimental group and control group components in the zebra fish embryo unicellular stage, respectively carrying out phenotype photographing on microinjected WT and untreated WT by using a fluorescence inverted microscope at 24h and carrying out deformity statistics at 24h, taking 30 malformed embryos of the control group and each experimental group after 24h, subpackaging 3 tubes of EP (10 embryos) and adding 200ul Trizol into the EP tubes, freezing at-80 ℃, and then extracting RNA. The RNA is used for reverse transcription by a reverse transcription kit, and the obtained DNA is used for real-time fluorescence quantitative PCR. The quantitative PCR graphs were analyzed using the corresponding software LightCycler 96 SW 1.1, GraphPad Prism 5.

(5) A method for knocking down multiple endogenous genes simultaneously.

Preparing a mixed solution with the final concentration of CasRx mRNA of 200 ng/ul, the final concentration of A gene-oriented sgRNA of 100ng/ul, the final concentration of B gene-oriented sgRNA of 100ng/ul and the final concentration of C gene-oriented sgRNA of 100ng/ul, injecting the components into zebrafish embryos at the same time, and observing the development condition of the zebrafish embryos.

The invention utilizes CRISPR/CasRx mediated RNA editing technology capable of RNA targeted editing to act on zebra fish embryos, CasRx mediated knockout has higher efficiency and specificity, CasRx can be flexibly packaged into adeno-associated virus, CasRx is a programmable RNA binding module, cellular RNA can be effectively positioned, and the CasRx can be used for efficiently shearing off RNA corresponding to exogenous fluorescent protein to weaken visual genetic markers; CasRx can also be used to knock down RNA corresponding to genes with obvious or key phenotype to cause the RNA to have different phenotypes or die.

CasRx is one of Cas13 d. The Cas13 RNA editing system consists of a Cas13 protein and a strand of CRISPR RNA (crRNA) 64 to 66 nt in length that is capable of recognizing a 22 to 30 nt specific RNA sequence by a spacer. The Cas13 system has several major advantages as an RNA editing tool: 1. cas13 is able to recognize all target RNAs by changing the sequence of the crRNA spacer; 2. cas13 differs from Cas9 and Cpf1 in that no specific sequence elements (such as PAM sites) are required for the target sequence; 3. the effective complex of Cas13 is the simplest one of CRISPR-Cas systems, and a system consisting of Cas 13-crRNA is easier to handle and deliver than a trimeric multimerization system; 4. multiple crRNAs directed against different target sequences can be delivered simultaneously. High efficiency, easy operation, high specificity and the like, and it is noted that the Cas 13-mediated RNA editing technology can be highly distinctive in gene function studies (Kim, 2018).

In the specific implementation of the invention, a nuclear localization sequence (nuclear localization sequence) is respectively added into a wild type CasRx sequence, then an SP6 promoter sequence is added into the upstream of the whole sequence, mRNA with a capped (capped) and a poly A tail is obtained by using an in vitro transcription kit, and the extracted RNA is purified and frozen at-80 ℃ for later use; designing primers of exogenous fluorescent protein (GFP, BFP) DNA to synthesize DNA, synthesizing mRNA through in vitro transcription, purifying the extracted RNA, and freezing at-80 ℃ for later use; different sgRNAs are synthesized into a fixed sequence with a T7 promoter at the upstream and different downstream sequences partially complementary with the upstream through DNA, double-stranded DNA is obtained through PCR amplification, the double-stranded DNA is obtained by using an in vitro transcription kit, and the purified and extracted RNA is frozen at-80 ℃ for later use.

CRISPR/CasRx is a novel class of RNA editing tools, and CasRx is the most efficient version reported to date. Although the efficiency of CRISPR/CasRx in cells is good, no report on gene knockdown of zebrafish embryos exists at present.

The invention has the advantages that:

(1) the price is low, and only one short primer sequence (less than 50 bp) needs to be replaced to target the RNA of one target gene;

compared with the traditional zebra fish embryo Morpholino knock-down technology, the invention has the advantages of convenient design and lower cost, adopts a primer pairing amplification method in the design of the guide RNA, adopts a fixed forward primer, matches a specific targeted reverse sequence, can amplify the template DNA of the guide RNA, provides purification and transcription, and can quickly obtain the targeted specific guide RNA.

(2) The components are simple and are all RNA, the toxicity is low, and the operation is simple;

the component only needs fixed CasRx mRNA and specific targeted guide RNA, has simple components, proper dosage and less toxicity, and can reduce the toxic interference of samples.

(3) Multiple gRNAs can realize simultaneous knockdown of multiple genes;

the guide RNA sequence is short, the cost is low, the synthesis is convenient, the toxicity is low, a plurality of gRNAs can be added simultaneously, and the simultaneous knock-down of multiple genes is realized.

(4) The efficiency is high, and the aging is long;

the CasRx-mediated RNA targeted cleavage has very high sequence specificity, because CasRx mRNA adopts WPRE and poly A-tailed bistable measures, and the CasRx mRNA has long and stable existence time of an embryo and is enough to ensure the continuous cleavage effect of target RNA in the process of embryo development.

(5) The efficiency analysis method is simple and easy to implement.

The research provides a simple and convenient analysis method of the cutting efficiency of the target RNA, and the cutting efficiency of the target RNA sequence can be quickly analyzed by adopting a real-timePCR method and providing a proper internal reference.

Drawings

FIG. 1 is a schematic diagram of the use of the CasRx mediated RNA editing system in zebrafish embryos.

FIG. 2 validation of the efficiency of CasRx mediated RNA editing to cleave exogenous mRNA in zebrafish embryos; wherein A, B: representation and efficiency statistics of CasRx cleavage of EGFP mRNA in zebrafish embryos; C. d: representation and efficiency statistics of CasRx cleavage of BFP mRNA in zebrafish embryos.

FIG. 3 validation of the efficiency of CasRx mediated RNA editing to cleave endogenous mRNA in zebrafish embryos; wherein A: a 24h tabular chart; b: graph of qPCR analysis.

Detailed Description

"target site" in this application refers to any segment of the RNA sequence to be knocked down in the target nucleotide. An RNA sequence in the vicinity of the target site that is capable of accommodating recognition of the exogenous sequence at the target site. In particular embodiments, the target RNA sequence is a single-stranded RNA sequence, including, but not limited to, RNA sequences in cells, RNA sequences of viruses, and the like.

By "exogenous RNA sequence" is meant in this application exogenous fluorescent protein-capped tailed RNA.